Multi Photon

Quarterly Reviews of Biophysics 38, 2 (2005), pp. 97166. f 2006 Cambridge University Press doi:10.
1017/S0033583505004129 Printed in the United Kingdom First published online 15 February 2006
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Two-photon fluorescence excitation and related techniques in biological microscopy

Alberto Diaspro1,2*, Giuseppe Chirico3,4 and Maddalena Collini3,4
1 Laboratory for Advanced Microscopy, Bioimaging and Spectroscopy (LAMBS), MicroScoBiO Research Center, University of Genoa, Italy 2 National Institute for Matter Physics (INFM) and Department of Physics, University of Genoa, Italy 3 Laboratory for Advanced Biospectroscopy (LABS), University of Milano Bicocca, Milano, Italy 4 National Institute for Matter Physics National Council for Research (INFM-CNR), and Department of Physics, University of Milano Bicocca, Milano, Italy
Abstract. This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely : choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.
1. Introduction
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2. Historical background of two-photon effects 99 2.1 2PE 100 2.2 Harmonic generation 100 2.3 Fluorescence correlation spectroscopy 100 * Author for correspondence : Professor A. Diaspro, LAMBS-MicroScoBiO, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy. Tel. : +39 10 3536426 ; Fax : +39 10 314218 ; E-mail : diaspro@sica.unige.it
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A. Diaspro, G. Chirico and M. Collini
3. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 101 3.1 General considerations 101 3.2 Fluorescence intensity under the 2PE condition 103 3.3 Optical consequences of 2PE 104 3.4 Saturation effects in 2PE 108 3.5 Fluorescence correlation spectroscopy 109 3.5.1 Autocorrelation analysis 110 3.5.2 Photon-counting histogram analysis 112 4. Two-photon-excited probes 115 119
5. Design considerations for a 2PE fluorescence microscope 5.1 General aspects 119 5.2 Descanned and non-descanned 2PE imaging 121 5.3 Lens objectives and pulse broadening 122 5.4 Laser sources 125 5.5 Example of a practical realization 127 6. Applications 134 6.1 Biological applications of 2PE 134 6.1.1 Brain images 134 6.1.2 Applications on the kidney 139 6.1.3 Mammalian embryos 139 6.1.4 Applications to immuno-response 141 6.1.5 Myocytes 141 6.1.6 Retina 142 6.1.7 DNA imaging 143 6.1.8 FISH applications 144 6.2 2PE imaging of single molecules 144 6.3 FCS applications 148 6.4 Signals from nonlinear interactions 151 7. Conclusions 153 154
8. Acknowledgements 9. References 155
1. Introduction Optical microscopy is the only means of seeing structure in living cells at submicron resolution (Amos, 2000 ; Hell, 2003 ; Bastiaens & Hell, 2004). In recent decades, the method has gained great chemical specicity from the development of uorescent probes (Arndt-Jovin et al. 1985 ; Beltrame et al. 1985 ; Kasten, 1989 ; Shotton, 1989 ; Wang & Herman, 1996 ; Tsien, 1998, 1999 ; Periasamy, 2001 ; Haughland, 2002 ; Zhang et al. 2002). Imaging three-dimensional (3D) structure has been aided by the introduction of confocal optical methods (Sheppard & Choudhury, 1977 ; Brakenho et al. 1979, 1985 ; Sheppard & Wilson, 1980; Wilson & Sheppard, 1984 ; White & Amos, 1987 ; White et al. 1987; Minsky, 1988 ; Carlsson et al. 1989 ; Wilson, 1990 ; Pawley, 1995 ; Webb, 1996 ; Masters, 1996 ; Amos, 1998 ; Benedetti, 1998 ; Masters, 2002 ; Diaspro,
2PE in biological microscopy
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2002 ; Amos & White, 2003 ; Periasamy & Diaspro, 2003). This review is concerned with twophoton excited uorescence microscopy (2PE) (Denk et al. 1990 ; Pennisi, 1997 ; Girkin, 2003 ; Zipfel et al. 2003b), which is probably the most important advance in optical microscopy since the introduction of confocal imaging in the 1980s, and related nonlinear optical methods. 2PE requires scanning optics similar to those used for confocal imaging, but also ultra-fast pulsed laser sources (Curley et al. 1992 ; Gratton & Van de Vende, 1995 ; Wokosin et al. 1997 ; Svelto, 1998). The advantages of 2PE over confocal imaging are still being evaluated (Gu & Sheppard, 1995 ; Brakenho et al. 1996 ; Sako et al. 1997). In order to collect 3D data, a substantial volume of the specimen has to absorb light, with an inevitable concomitant photobleaching and phototoxicity, which may be particularly severe when ultraviolet (UV)-excited uorochromes are used (Stelzer et al. 1994). In principle, 2PE is superior to conventional single-photon scanned imaging (e.g. confocal), in that absorption can be limited to a very small volume at the focus of the objective lens at any one time. It can be the most ecient way of collecting 3D information in the sense of using the lowest time-integrated dose of radiation to the volume of interest. Besides this eciency, 2PE has other advantages in imaging. It uses longer wavelengths [often infrared (IR)] which are not only less scattered by the specimen but may also fail to excite background autouorescence. This is particularly important in the imaging of single molecules (Mertz et al. 1995 ; Xie & Lu, 1999 ; Sonnleitner et al. 1999 ; Chirico et al. 2001a, 2003a ; Cannone et al. 2003a). Finally, because of their broader and overlapping two-photon excitation (2PE) spectra, several uorochromes can be excited simultaneously by the same IR beam (Denk, 1996 ; Potter, 1996 ; Piston, 1999 ; Squirrel et al. 1999 ; Straub et al. 2000 ; White & Errington, 2000 ; Stosiek et al. 2003). However, it has been the experience of many investigators that the intensity of the beam in 2PE has to be controlled carefully to gain the often-claimed advantage in eciency compared with confocal laser scanning microscopy. A possible reason for this is that 2PE photobleaching in the focal plane greatly depends (according to about the third power) on the excitation intensity (Patterson & Piston, 2000 ; Chirico et al. 2003b). Although the introduction of 2PE appeared incremental in the sense of providing similar optical sections to those obtained with confocal optics, and using similar scanning apparatus, it was also revolutionary, in that it represented a novel application of quantum physics (Loudon, 1983 ; Feynman, 1985 ; Shih et al. 1998). 2PE has stimulated the application of other nonlinear optical processes (see below). Three-photon uorescence excitation was rst recognized in imaging in Whites laboratory in Wisconsin (Wokosin et al. 1996 ; Hell et al. 1996 ; Maiti et al. 1997). The sharply dened and tiny focal volume has proved ideal for uorescence correlation spectroscopy (FCS) (Wiseman et al. 2000). The femtosecond pulse obtained with newly developed laser systems has been used to improve the time-resolution of lifetime imaging (Konig et al. 1996a ; French et al. 1997 ; Sytsma et al. 1998 ; Straub & Hell, 1998a ; Peter & Ameer-Beg, 2004). The connement of absorption to the focus in 2PE has also been exploited in photodynamic therapy (Bhawalkar et al. 1997). Moreover, one of the most recent applications of 2PE is given by the photoactivation in conned volumes of phoactivable proteins (Post et al. 2005 ; Schneider et al. 2005). Therefore, 2PE has become important not only in imaging but also in medicine and biology. 2. Historical background of two-photon effects The rst prediction of two-photon absorption was by Go ppert-Mayer (1931, see also Axe, 1964). She proposed, as a consequence of the Heisenberg uncertainty principle, that one atom
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or molecule should be capable of absorbing two photons in the same quantum event within 10x1610x17 s. Because it is a rare event at ordinary light intensities (Denk & Svoboda, 1997), it was only in the 1960s, after the development of laser sources (Svelto, 1998), that the prediction could be veried. 2.1 2PE Kaiser & Garret (1961) reported 2PE of uorescence in CaF2 :Eu2+. Soon after, Singh & Bradley (1964) were able to estimate the three-photon absorption cross-section for naphthalene crystals. Rentzepis and colleagues (1970) observed three-photon excited uorescence from organic dyes. In 1976, Berns observed microscopically what was probably a two-photon-excited uorescence as a result of focusing an intense pulsed laser beam on to chromosomes of living cells (Berns, 1976). The rst applications of 2PE to uorescence microscopy were presented at the beginning of the 1990s by Denk and colleagues (1990), who demonstrated that images with excellent optical sectioning, could be obtained without killing cells. The development of commercially available mode-locked lasers, with high peak-power femtosecond pulses and repetition rates around 100 MHz was then the trigger for a fast uptake of the multi-photon method in biology (Spence et al. 1991 ; Gosnell & Taylor, 1991 ; Curley et al. 1992 ; Fisher et al. 1997 ; Wise, 1999 ; Girkin, 2001a ; Courjaud et al. 2001). 2.2 Harmonic generation For many years the only practical application of two-photon absorption was in spectroscopy (Lee, 1975; Friedrich & McClain, 1980 ; Friedrich, 1982 ; Birge, 1986 ; Callis, 1997). Franken et al. (1961) described second-harmonic generation (SHG) in a quartz crystal using a ruby laser. SHG is the production of a scattered beam at twice the frequency of the input beam, which is propagated in phase with the input and in the original direction. Hellwarth & Christensen (1974) collected SHG signals from ZnSe polycrystals at the microscopic level. The original idea of generating 3D microscopic images by means of nonlinear optical eects was rst suggested and successfully demonstrated by second-harmonic imaging in the 1970s by Sheppard and colleagues of the Oxford group (Gannaway & Sheppard, 1978 ; Sheppard & Kompfner, 1978). SHG has now been applied to biological specimens (Gauderon et al. 1999 ; Campagnola et al. 1999, 2002; Cox et al. 2003), as Gannaway and Sheppard predicted it would be. Third-harmonic generation (Barad et al. 1997 ; Mueller et al. 1998 ; Squier et al. 1998) has also shown promise. 2.3 Fluorescence correlation spectroscopy The same optics and lasers as used in imaging, led to improvements in FCS (Magde et al. 1972 ; Ehrenberg & Rigler, 1974 ; Koppel, 1974 ; Aragon & Pecora, 1975). Thus far, FCS has been used to determine translational and rotational diusion (Ehrenberg & Rigler, 1974 ; Fahey et al. 1977 ; Borejdo, 1979), chemical reactions (Thompson & Axelrod, 1983 ; Icenogle & Elson, 1983 ; Bonnet et al. 1998 ; Haupts et al. 1998 ; Starr & Thompson, 2001), ow rates (Magde et al. 1978 ; Gosch et al. 2000), aggregate formation (Palmer & Thompson, 1989 ; Qian & Elson, 1990 ; Petersen et al. 1993 ; Berland et al. 1996), and triplet state parameters (Widengren et al. 1994).

S2 Internal conversion 1012 s
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S1 Absorption 1015 s
Inter-system Fluorescence crossing 1010 s 109 s Radiation less Decay <109 s Phosphorescence 103 s
S0
Fig. 1. Fluorescence pathways and transition times in a general PerrinJablonsky scheme for conventional excitation of uorescence. (Courtesy of Dave Jameson, University of Hawaii.)
FCS is at present a very powerful tool for the characterization of biomolecules in vitro and is now developing into an adjunct to microscopy in which it is possible to probe the molecular aggregation and dynamics inside cellular compartments (Schwille et al. 1999b ; Schwille, 2001 ; Schwille & Heinze, 2001). The technological advances which contributed to the development of 2PE microscopy occurred in several elds, including laser scanning microscopy, ultra-fast laser sources, more sensitive detectors, fast acquisition devices, and digital electronic tools (Shotton, 1993 ; Piston, 1999 ; Tan et al. 1999; Robinson, 2001; Gratton et al. 2001 ; Periasamy & Diaspro, 2003 ; Esposito et al. 2004 ; Masters & So, 2004). 3. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 3.1 General considerations Conventional one-photon excited uorescence microscopy (1PE) techniques for uorescence excitation make use of a UV or visible photon that is able to match the energy gap (Fig. 1) between the ground and the excited state (Lakowicz, 1999). 2PE of uorescent molecules is a nonlinear process related to the simultaneous absorption of two, typically IR, photons whose total energy is sucient to produce a molecular transition to an excited electronic state (Birks, 1970 ; Denk et al. 1995 ; Callis, 1997 ; Nakamura, 1999). In general, one can use photons of wavelengths l1 and l2 within the constraint that 1 1 x1 l1 P + , (1) l1 l2 where l1P is the wavelength needed to prime uorescence emission in a one-photon absorption event. For practical reasons one has l1=l2 and the starting experimental choice is l1B2l1P (Denk et al. 1990 ; Diaspro, 2001a ; Girkin & Wokosin, 2001a), as shown in Fig. 2, where l1P=340 nm. 2PE of uorescent molecules provides an immediate practical advantage over confocal microscopy (Denk et al. 1990 ; Potter, 1996 ; Centonze & White, 1998 ; Piston, 1999; Squirrel et al.
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S* 1020 nm 680 nm 340 nm 1020 nm 420 nm
680 nm
1020 nm
S0
Fig. 2. PerrinJablonsky simplied scheme for one-, two- and three-photon excitation. When the uorescent molecule is brought to the excited state it relaxes back emitting the very same uorescence as in the one-photon excitation case. In this case conventional uorescence could be primed at 340 nm producing 420 nm emission. The rule of thumb is that one can use multiple wavelengths with respect to the one-photon excitation condition accordingly with the nth photon excitation order. Optimization can be obtained at dierent wavelengths.
1999 ; Diaspro & Robello, 2000 ; Wilson, 2001) since out-of-focus excitation is avoided, thereby reducing the overall photobleaching and phototoxicity of thick samples (Brakenho et al. 1996 ; Patterson & Piston, 2000 ; Tauer, 2002 ; Drummond et al. 2002). However, the observation time for a uorophore in 2PE decreases with the third power of the excitation intensity due to photobleaching in the focal plane (Chirico et al. 2003b). These data led the authors to conclude that the uorophore is initially excited by simultaneous two-photon absorption, and then one or more photons interact with the excited molecule to increase the probability of photobleaching similarly to a sequential absorption model previously suggested in a photobleaching study of Rhodamine 6G (Widengren & Rigler, 1996) and Rhodamine B (Sanchez et al. 1997). A simple extra photon model is not sucient to describe the phenomenon, and possibly higher-order resonance absorption is involved. At the lowest excitation intensities, photobleaching rates between one- and two-photon excitation are comparable, but at power levels that are typically used in biological imaging (35 mW at the sample for 150 fs laser pulses) this dierence can easily be a factor of 10. This means that the two-photon method is substantially detrimental in a subset of experiments, particularly for thin samples (<5 mm) and single-molecule studies, and cells for a particular treatment to achieve the highest collection eciency in 2PE, thereby using the lowest possible light exposures on live cells, and minimizing photobleaching. The main advantage of 2PE lies in the high localization of the excitation and in the fact that emitted light is detected, even if strongly scattered, because the full sensitive area of the detector is used as opposed to confocal microscopy. This allows the application of 2PE to thick, turbid samples and simplies the collection optics scheme. Further considerations are needed here to characterize 2PE in biological applications. Since, molecular cross-sections for the two-photon absorption d2, are typically very small [of the order of 10x58 (m 4 sx1)], high light uxes%1030 photons sx1 mx2 are required.
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This can be reached either by means of high-power continuous wave (CW) lasers or by means of short-pulse (%10x13 s) lasers that allow reduction of the thermal load of the sample and selectively excite a single electronic transition. In fact the time-scale of the 2PE process is t2PE =10x16 x10x17 s (Louisell, 1973) as determined by the Heisenberg uncertainty principle : t2PE l1 P 10x16 s, 4 pc (3) (2)
for a typical visible wavelength l1P=500 nm. However, picosecond or CW IR excitation can be eectively applied to prime uorescence (Hanninen et al. 1994 ; Hell, 1996 ; Bewersdorf & Hell, 1998 ; Booth & Hell, 1998 ; Hell et al. 1998 ; Leica Microsystems, 1999 ; Konig et al. 1999a), although with higher thermal loads on the sample. In this case, as well as direct 2PE from ground state to excited states, transitions from higher excited states also have time to occur and this brings a broadening of the 2PE cross-section. Another consideration involves the 2PE selection rules. Onephoton-allowed transitions are due to opposite parity of the initial and nal states, while in the two-photon case the two states have the same parity (So et al. 2000). This is due to the fact that the state reached by TPE is obtained from a two-step process through vibronic coupling. These dierent selection rules are responsible for the fact that, even when we divide the 2PE wavelengths by 2, a considerable shift between the 1PE and the 2PE spectra is found (Blab et al. 2001). Moreover, we must also consider that the two-photon crosssection, d2, may depend on the laser pulse width due to dierent vibronic coupling (He et al. 2002). 3.2 Fluorescence intensity under the 2PE condition The 2PE uorescence intensity, If(t), is proportional to the square of the excitation intensity, I(t), to the molecular cross-section d2=d2(lexc), and to the quantum yield g=g(lem). Here, lexc and lem, are the excitation and emission wavelengths respectively. The excitation intensity in photons cm2 sx1 is I(t)=lP(t)/(hcA), where P(t) is the instantaneous power on the illuminated area, A%p[lexc/(2 NA)]2 (Born & Wolf, 1993), NA is the numerical aperture of the microscope objective, h and c are Plancks constant and the speed of light in vacuum respectively. The 2PE collectable uorescence intensity is given by : 2 2 2 2 (NA) If (t )=kd2 gI (t ) =kpd2 gP (t ) , (4) hc lexc where k is a factor that takes into account the collection eciency of the set up. The time-averaged two-photon uorescence intensity per molecule within an arbitrary time interval T for a CW excitation is : 2 NA2 nIf , cw m kpd2 gPave : hc lexc
2
(5)
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For femtosecond lasers with pulse repetition rate fP=1/T, and pulse width, tP, the average power depends on the peak power as, Pave=tP fPPpeak(t) and Eq. (5) becomes (So et al. 2001) : 2 2 Pave NA2 nIf , P m d2 g : (6) tP fP hc lexc The conclusion here is that CW and pulsed lasers have the very same excitation eciency in terms of collectable uorescence intensity when the average power of the CW laser is kept p higher by a factor of 1= tP fP and the TPE cross-section is not changed with the laser pulse width (Xu, 2001 ; Konig et al. 1996b). This means that, for exciting uorescence, 300 mW delivered by a CW laser are nearly equivalent to 1 mW of a pulsed laser with repetition rate %80 MHz and pulse width%100 fs. As an example, a 1 mW average power laser source (l=1000 nm, 80 MHz repetition rate, 100 fs pulse width) focused by a 14 NA objective on molecules endowed with a typical two-photon cross-section of d2 %10 GM, gives [Eq. (6)] a uorescence count rate %105 Hz. It must also be considered that the TPE cross-sections for picosends and CW excitations are larger than the TPE femtosecond cross-sections by at least one order of magnitude (Kirkpatrick et al. 2001). This allows for picosecond 2PE of biological samples (Bewersdorf & Hell, 1998), although local heating eects of the sample can be relevant in this case. 3.3 Optical consequences of 2PE The use of near-infrared (NIR) radiation in 2PE would imply an optical resolution lower than in conventional 1PE (Born & Wolf, 1993 ; Cox & Sheppard, 2004). However, since under 2PE uorescence is exclusively excited in a sub-femtolitre () volume (Vexc%0105 , Fig. 3), 3D optical sectioning (Weinstein & Castleman, 1971; Agard, 1984 ; Diaspro et al. 1990) can then be achieved with the signal-to-background ratio noticeably increased. Figure 4 shows an optical sectioning scheme along with the excitation beam shape (Born & Wolf, 1993 ; Bianco & Diaspro, 1989). One should consider that the laser excitation intensity spatial prole is given by the intensity point spread function (PSF), S(r/l1,exc, z/l1,exc) (Born & Wolf, 1993), where l1,exc is the wavelength that characterizes the one-photon excitation. Here we indicate positions in space with the radial [r=(x, y)] and axial (z) distances from the origin that is placed on the focal plane on the maximum laser intensity position. The PSF can be approximated with GaussianLorentzian (GL) spatial proles whose widths are w0 and z 0 in the radial and axial dimensions, as shown in Fig. 4. A detailed discussion of a GL propagating beam through a nite aperture is complex and can be found in Dickson (1970) and Xu (2001). Radial and axial beam waist values can be determined using the relations w0 0 61 l NA and z0
2 pw0 0 37pl = , NA2 l
where NA=n sin(a), n is the refractive index of the medium between the lens and the sample and a is the half-aperture angle of the lens (Fig. 4). The uorescence emission If(r, z) depends on the distribution of uorophores in the excitation volume, C(r, z) and is given by If(r, z)=C(r, z)S(r/l1,exc, z/l1,exc) for 1PE, and to If(r, z)=C(r, z)S2(2r/l1,exc, 2z/l1,exc) for 2PE. The second relation is due to the second-order

(a) From source
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n1 photons (constant) 1
Microscope
two axis
x y
x,y,z
z Important parameters Resolution: x, y, z
Position: x, y, z 2 Quantity: n2 n2 photons Probability of two-photon absorption
To detector: n2 counts
(b)
Fig. 3. (a) Optical resolution parameters for determining the extent of the volume of two-photon excitation (TPE) event and TPE probability along the optical axis (adapted from Pawley, 1995). (b) Fluorescence emission obtained from a solution of uorescent molecules by means of one- (double cone, above) and two-photon (bright dot, pointed by the arrow) excitation. From this illustration the spatial connement of TPE is extraordinarly clear. The immediate implication is that one does not need any computation or pinhole for removing out-of-focus contributions. Out-of-focus contributions are simply absent. (Courtesy of Brad Amos, MRC, UK.)
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(a)
z axis
(b)
W0 0
2W0 z
2z0
Fig. 4. Optical sectioning. (a) A real 3D structured object showing a dierent detail plane by plane (left) can be optically sectioned (centre) to get images of the distinct features along the z-axis (right). (b) Beam waist along the xyz directions determining spatial resolution (see text).
power dependence of the excitation probability for 2PE and to the fact that the 2PE excitation wavelength is generally taken as l1,exc=l2,exc/2. The collection of the uorescence signal, F(r, z), with emission wavelength lem, is given by a convolution between the emission intensity PSF, S(r/lem, z/lem), and the uorescence emission, If(r, z) (Weinstein & Castleman, 1971 ; Agard, 1984 ; Bianco & Diaspro, 1989 ; Diaspro et al. 1990) plus a random noise that takes into account the image collection statistics. In confocal 1PE the use of a pinhole imposes a restriction on the integration of F(r, z) over the detector area. This leads to a collection eciency function [=the fraction of the uorescence signal emitted from the point of coordinates (r, z) and that is actually collected by the detector] proportional to S(r/lexc, z/lexc) S(r/lem, z/lem). By comparison, assuming a 2PE scheme, all the uorescence photons are usually collected since the pinhole is usually excluded, F(r, z) has to be averaged over the whole detector area

z/z0 0 100 1 2 3
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Output fluorescence rate (kHz)
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0 0 2 z (m) 4 6
Fig. 5. Connement property of the two-photon excitation (TPE) spectroscopy. The simulation of the uorescence output from a molecule that is moved along the optical path is performed according to Eq. (16). The simulation parameters are : Pave=1 mW, average laser power, fP=80 MHz repetition rate and tP=100 fs pulse width, l=1000 nm. Two-photon cross-section, d2 at 10 GM, NA=14. The solid line is a t to an equation of the type A/(B2+z2/C2)2. The depth of focus is 16 mm.
and this leads to a 2PE collection eciency function : S2(2r/lexc, 2z/lexc). In confocal 1PE and in 2PE the resolution enhancement is given by the quadratic form of S due to dierent physical reasons. In fact, in confocal microscopy the resolution is enhanced because of a combined action of the excitation and detection PSFs. A plane of thickness z0 centred around z=0 (the focal plane as determined by the position of the objective) predominates over the other planes. In 2PE the resolution enhancement is obtained only through the excitation PSF which is squared because of the nonlinear eects of the excitation due to two statistically independent events that occur, i.e. simultaneous absorption of two photons (Andrews, 1985). It must be noted that the eect of the excitation and emission wavelengths is dierent in the two cases, but in both cases the uorescence signal depends quadratically on the intensity PSF. The microscope intensity PSF which is the response of the optical system to a Dirac distribution of uorescence, can be experimentally determined by imaging subresolution uorescent objects, i.e. microspheres having dimensions <100 nm or thin uorescent layers (van der Voort & Brakenho, 1988 ; Schrader et al. 1998 ; Diaspro et al. 1999a). The 2PE PSF is axially conned (Nakamura, 1993 ; Gu & Sheppard, 1995 ; Jonkman & Stelzer, 2001) since the integral of S(r/l1,exc, z/l1,exc)2 over r has a half-bell shape with a zx4 behaviour for z4z0=pw)2 0/l (Fig. 5). 4 2PE excitation volumes can then be estimated as Vexc%pw2 z = p w / l . As an example, 0 0 0 3 for l=800 nm, NA=14, the diraction limited volume is%006 mm (see also Table 1 for values of the half-width of the 3D intensity point spread). The axial connement of the excitation is demonstrated in Fig. 6. In 2PE over 80% of the total intensity of uorescence
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Table 1. Values of the half-width of the 3D intensity point spread function in the transverse and axial directions, v and u respectively (values from Gu & Sheppard, 1995)
1 2 1 2
1P v u
1 2 1 2
2P 234 802
C1P 117 401
C2P 134 462
162 556
(a)
(b)
(c)
(d)
Fig. 6. One- (a, b) and two-photon (c, d) photobleaching induced within a uorescent microsphere by means of 488 nm and 720 nm wavelengths respectively. As in Fig. 13, the better connement of two- in respect to one-photon excitation under point-like laser scanning conditions is evident. (a, c) are xy views ; (b, d) are axial views obtained from optical sectioning collections. (Adapted from Diaspro, 2001b.)
comes from a 7001000 nm thick region about the focal point for objectives with NAs in the range of 1214 (Brakenho et al. 1979 ; Wilson & Sheppard, 1984 ; Jonkman & Stelzer, 2001 ; Torok & Sheppard, 2001 ; Wilson, 2001). As a comparison, typical axial extension of the confocal volumes are of the order of 500700 nm, although these values are reached at much lower collection eciency than in the 2PE case. Finally, it is worth noting that, when considering in-depth imaging of thick samples, optical aberrations should be properly considered (de Grauw & Gerritsen, 2001 ; Diaspro et al. 2002b ; Neil et al. 2002 ; Marsh et al. 2003). 3.4 Saturation effects in 2PE Besides photobleaching, an important factor is given by the saturation of the uorescence emission. For excitation intensities, I, higher than a threshold value, Isat, the uorescence
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emission increases less rapidly than%I for confocal 1PE, or%I 2 for 2PE. This is due to excitation rates that are larger than the uorescence de-excitation rates (ground state depletion) or, in the case of pulsed 2PE, to excitation to higher excited states from the rst excited state due to the high photon ux. As for the rst possibility, we estimate that the number of photon pairs, na, that a uorescent molecule simultaneously absorbs during a single pulse is (Denk et al. 1990) : 2 2 NA2 (2) d2 lexc Pave na / g , (7) tP fP2 2 hc lexc where Pave is the time-averaged power of the beam, lexc is the excitation wavelength, NA is the objective NA, tP is the pulse duration and fP is the pulse train repetition rate. The g(2) factor is 10, 066 and 059 for the Rectangular, Gaussian and Hyperbolic-secant-squared pulse shape (Svelto, 1998). Ground state depletion occurs when nafPB1/t, i.e. when natfPB1, where t is the uorescence lifetime of the uorescent molecule. Now, if we want at least one 2PE per pulse (i.e. naB1) while keeping the intensity below the saturation threshold, the pulse repetition rate should be of the order of 1/t or less, which, for commonly used uorescent molecules for which tB10 ns means fPf100 MHz. The typical repetition rate of titanium:sapphire lasers used for 2PE (y80 MHz), just happened to be in the right range for cavity requirements (Girkin, 2003). Now, by taking the uorescein uorescent molecule as an example, for lexc=780 nm we 2 obtain na%67r10x3 Pave , where Pave is expressed in mW. We have assumed d2=38 GM at 780 nm [1 GM (GoppertMayer)=10x58 (m 4s)] (Denk et al. 1990 ; Xu, 2001), NA=14, fP=100 MHz and tP=100 fs. Since for uorescein t=4r10x9 s, saturation begins to occur at 20 mW (Koester et al. 1999 ; So et al. 2001). Besides the saturation eect, in 2PE higher-order transitions usually occur even before the saturation becomes evident. Several brilliant and eective comparisons between 1PE and 2PE on simple dyes have been previously reported (Mertz, 1998 ; Eggeling et al. 2005). It must be noted that these eects increase at reasonably low excitation powers. As an example, rhodamine, even at average powers (%4 mW of 2PE) does not show the uorescence rate increasing quadratically with the excitation intensity (Mertz, 1998). 3.5 Fluorescence correlation spectroscopy Information on the internal dynamics of the sample can be obtained from the analysis of the uctuation of the TPE uorescence signal. Fluorescence spectroscopy combined with the unique spatial resolution features of TPE allows the localization of rare uorescent components and the dynamics of chemical reactions in cellular compartments to be followed (Eigen & Rigler, 1994 ; Brock et al. 1998 ; Schwille et al. 1999a ; Schwille, 2001 ; Harms et al. 2001 ; Chen, 2002 ; Chen et al. 2002). The analysis of the uorescence uctuations is usually performed by means of FCS. In this technique, the autocorrelation of the uorescence signal is performed online by fast digital correlator boards. FCS is a rapidly developing eld in its own right, and it is already recognized as an essential tool for the in vivo characterization of absolute concentrations, molecular interactions, and kinetic processes, such as diusion and chemical reactions (Haustein & Schwille, 2004). In FCS we want to measure the light emitted from a few (%110) molecules with microsecondsecond time resolution, much larger than the excited state lifetime (%1 ns). On this time-scale, which is typical of molecular diusion to internal photodynamics and chemical kinetics, the uorescence emission can be thought of as an instantaneous relaxation
110
process and the laser excitation is actually seen by the sample as a continuum wave due to the high repetition rates (%80 MHz). For this reason the time dependence of the excitation intensity is not usually taken into account. Moreover, since uorescence emission is measured, the measured light intensity is the sum of the individual molecular contributions which are proportional to the local laser beam intensity (Thompson, 1991). In order to understand the origin of the uorescence uctuations due to molecular diusion, let us assume that the laser prole in the focal plane (z=0), has a top-hat prole with radius w0. The uorescence would then simply be proportional to the number N of molecules in the excitation volume and the uctuations are related to the change of N, i.e. ndF2m/ nF2m=ndN2m/nN2m. For more realistic laser beam shapes, such as the GL shape (Born & Wolf, 1993) a complex mathematical treatment leads to a proportionality of the type ndF2m/nF2m=cndN2m/nN2m, where c is a geometric factor that depends on the shape of the collection eciency function (Berland & Shen, 2003). Now, can we determine the number uctuations in the observation volume ? The mean number of molecules per excitation volume is nNm=CVexc, where C is the number concentration (molecules/litres of solution) of the polymer in the sample. For the case Vexc=10x15 l (=) and C=6r10x14 molecules/l, which corresponds to a concentration of 1 nM, we obtain nNm%1. The probability P(N) of nding N molecules in Vexc is proportional (Eigen & Rigler, 1994) to the number of ways N molecules can be arranged in Vexc : P (N )= nN mN xnN m e : N! (8)
According to this distribution, the number uctuation is ndN2m/nNm2%1/nNm=1/(CVexc) and, therefore, the uorescence uctuations ndF2m/nFm2 scale as 1/C. The more diluted the polymer solution is, the higher the signal-to-noise ratio will be. Obviously we cannot increase the uctuation amplitudes at will due to the eect of the background that reduces the measured uctuations. However, a compromise must always be found between the average uorescence signal, which scales as nNm, and the signal-to-noise ratio, S/N=ndF2m/nFm2%(nNmVexc)x1 (Koppel, 1974 ; Quian, 1990 ; Kask et al. 1997). Obviously S/N also depends on the total averaging 0 5 time, Tm, and better S/N ratios can be reached by increasing Tm, since S =N Tm (Kask et al. 1997). Moreover, S/N is dependent on the molecular brightness, dened as e=d2(lexc)g(lem). This is especially true for the fast time range (%1 ms) of the autocorrelation function (ACF) (Koppel, 1974) that is related to internal photodynamics and chemical kinetics. For the diusion dynamics the uorescence uctuations are slower (%100300 ms), the dependence of S/N on the molecular brightness is less severe (Koppel, 1974), and lower excitation power and less bright dyes can be employed. In 2PE experiments, however, the saturation of the uorescence output due to transitions to higher excited states (Mertz, 1998 ; Eggeling et al. 2005) may severely limit the applications to dim dyes. 3.5.1 Autocorrelation analysis Now, when the average number of molecules is nNmf1 (Magde et al. 1972 ; Eigen & Rigler, 1994), the signal is at the background level for most of the observation time and occasionally we may detect a uorescence burst corresponding to a molecule diusing through the volume, such as the case reported in Fig. 7 : since the duration of a uorescence burst is related to

100000 C = 10 f M (a) 10000 190 191 1000 100
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100000 C = 100 f M 10000 1000 Rate (Hz) 100 C = 1 pM
(b)
(c)
100000 10000 1000
100000 10000 1000 100
(d)
100
C = 10 pM
0 200 400 Time (s) 600 800
Fig. 7. Fluorescence photon counts versus time collected through a two-photon excitation (TPE) from a solution of uoresceine-labelled microspheres 20 nm in diameter at concentrations : C=10 fM (a) ; C=100 fM (b) ; C=1 pM (c) ; and C=10 pM (d). Excitation wavelength=770 nm. The inset in panel (a) is a blow-up of the rst peak in the trace of panel (a).
the translational diusion properties of the molecules, one can get estimates of the translational diusion of the molecules similarly to photon correlation spectroscopy by means of the ACF : gF (t )= ndF (s +t )dF (s )ms : nF (s )m2 s (9)
However, conversely to the case of photon correlation spectroscopy, much more information can be gained from higher-order correlation functions of the uorescence signal (Thompson, 1991) or the analysis of the distribution of photon counts (Chen et al. 1999 ; Kask et al. 1999). The exact 2PE gF(t) cannot be analytically derived (Berland et al. 1995 ; Xu, 2001). However, it can be tted by a function of the type : gF (t )=gF , 0 (1+t =txy )x1 (1+t =tz )x05 =gF , 0 GD (t , txy , tz ), (10)
2 2 where w0 =8D and the axial diusion time tz z0 =8D is larger than eld txy. Since for 2PE xy 2 pt w0 z0 = 2p =lexc , for l=800 nm and NA=09 we get w0%054 mm and z 0%092 mm, while for
112
NA=14 we nd w0/z 0%1. It is of note that, in spite of the complexity of the exact relationship of the 2PE ACF derived by Berland et al. (1995), the time behaviour of the correlation functions in the TPE mode can still be analysed by means of a simple hyperbolic equation [e.g. Eq. (10)] for most of the microscopy-spectroscopy applications. As expected from the analysis performed of the number uctuations, the zero lag-time correlation function, gF,0, is related to the concentration number of uorophores nNm within the excitation volume : gF , 0 =c=nN m, (11)
where the contrast factor c=0076 for TPE. Since nFm=nNme, an estimate of the molecular brightness can be obtained from the product : e%nFmgF,0/c. In a case where the background contribution aects the measured count rate, nFmeasm=nFm+nFBm, the molecular brightness must be corrected (Koppel, 1974 ; Thompson, 1991) as e=emeasnFm2/(nFmeasm nFBm)2. This eect would lead to a decrease of the signal-to-noise ratio at extremely low sample concentrations. Besides translational diusion, any molecular brightness uctuation is reported by the ACF. In the case of unimolecular chemical reactions, or of internal photodynamics, in which a change of uorescence yield is involved, the uorescence signal uctuates according to the process kinetics. Superimposed on these uctuations we nd those relative to the molecular diusion through the excitation volume. The uorescence ACF is then given by a superposition of an exponential relaxation and a diusion term (Schwille, 2001) : gF (t )=gF , 0 (1xT +T exp [ xt =tT ]) GD (t , txy , tz ): 1xT (12)
In this equation the function GD(t, txy, tz) represents the diusion contribution to the ACF [Eq. (10)], tF is the characteristic time of the brightness uctuation and T is the fraction of the molecules that are undergoing this uctuation. We have assumed that (Widengren et al. 1994) the internal kinetics is completely decoupled from the diusion process and that the relaxation time of the kinetics, tT, is much shorter than tx,y. Excited triplet-singlet transitions and molecular-binding kinetics are treated by means of Eq. (12) in order to get information on the rate constants of the process. In general for a uni-molecular reaction of the type B , D , where B is the bright species and D the dark species (quantum yield=0), tT and T are related to the kinetics constants kon and ko of the reaction as (Schwille, 2001) : 9 tT =(kon +koff )x1 , > = (13) kon > T= , ; kon +koff
3.5.2 Photon-counting histogram analysis Statistical requirements for the collection of the full-time decay of the ACF are quite severe. On the other hand for most applications we are only interested in the measurement of gF(0) in order to obtain an estimate of the number of molecules per excitation volume. Such information can also be achieved by data analyses that are complementary to FCS (Chen et al.
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1999 ; Kask et al. 1999). For sampling times much smaller than the diusion time tx,y, the photon-counting distribution, P(k), depends on the average number of molecules nNm in the excitation volume and the molecular brightness e, dened as the product of the excitation intensity, the sampling time, the molecular absorption cross-section and the uorescence quantum yield. In the case of a single uorescent species nNm and e can be measured from the rst two moments, nkm and nk2m, of the photon-counting distribution (Chen et al. 1999 ; Chirico et al. 2000 ; Malengo et al. 2004) : nkm2 xnkm2 e= x1 c nkm (14) nN m=nkm=e In the case of a multiple-species sample, one should apply a nonlinear tting of the full photon-counting histogram (PCH) according to the numerical expressions given by Gratton and co-workers (Chen et al. 1999). The average nkm and nk2m, are simply given by : ! 9 M X > > nkm= ki M, > > > = i =1 (15) ! > M > X > > k2 nk2 m= M, > ; i
i =1
where M is the total number of samplings and ki is the number of photons collected per sampling time at the ith sampling of the experiment. FCS can be performed also in a point-by-point fashion on cells thereby reconstructing an image in terms of molecular brightness, and local molecular concentration collected on wellresolved excitation volumes. This technique, called image correlation spectroscopy (ICS) and introduced by Petersen et al. (1993), conjugates the counting feature of FCS to high spatial resolution of microscopy. The basis of the application of ICS is a spatial autocorrelation analysis, at a xed time, of uorescence images on which single uorescence centres are visible all over the eld of view (Wiseman & Petersen, 1999). This allows measurement of the number N of uorescent spots under observation according to the relation: g F ( j , g) = ndF (x +j, y+g)dF (x , y)mx , y nF (x , y)m2 x, y = 2 j +g 2 c exp x , 2 N w0 (16)
where w0 is the beam waist of the laser on the focal plane and the symbol n_mx,y indicates an average over all the image pixels. Finally, recent image applications of PCH analysis have been presented (Malengo et al. 2004). If the uorescence counts k(t, i, j) are acquired versus time (t=Ts, 2Ts, _, NTs) over each pixel (x, y) with a sampling time Ts, the pixel content on the conventional uorescence image is given by : I ( i , j )=
NTs 1 X k(t , i , j ), M t =T s
(17)
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(a)

(b) 52 42 0 02 04 05 07 09 11 12 14 52 42 y (m) 31 21 10 10 21 31 x (m) 42 52 0 275 550 825 1100 1375 1650 1925 2200
y (m)
31 21 10 10 21 31 x (m) (c) 52 42 y (m) 31 21 10 10 21 42 52
31 x (m)
42
52
0 002 005 007 010 012 015 017 020
Fig. 8. PCH imaging of a nanocapsule stained with FITC (uorescein) (Malengo et al. 2004). The eld of view is 52 mmr52 mm, acquisition rate 10 kHz, total acquisition time per pixel=30 s, x and y resolution=300 nm, excitation power at 100 mW. (a) Conventional image, I(i, j) ; (b) number image, nNm(i, i) ; (c) brightness image e(i, j).
where i=1, _ , m and j=1, _ , n spans the pixels of the image and M is the length of the acquisition buer. On the other hand the second moment of the photon count distribution can be computed on each image pixel as : I (2) (i , j )=
NTs 1 X k2 ( t , i , j ) : M t =T s
(18)
By applying the algorithm given by Chen et al. (1999), the average numbers nNm, and average brightness e, can be computed pixel-by-pixel by the following set of equations : 9 I (2) (i , j )xI (i , j )2 > e(i , j )c= x1, = I (i , j ) (19) > ; nN m(i , j )=I (i , j )=e(i , j ): As an example we report the images of uorescent nanocapsules (Diaspro et al. 2002d ; Diaspro, 2004b ; Malengo et al. 2004) acquired with a spatial resolution of 300 nm. The PCH image [Eq. (19)] of one nanocapsule stained with uorescein (see Fig. 8), indicates that the origin of uorescence spatial changes in the sample are mainly due to dierences in
2PE in biological microscopy Table 2. Properties of some common uorescent molecules under two-photon excitation
Extrinsic uorophores Bis-MSB Bodipy Calcium Green Calcouor Cascade Blue Coumarin 307 CY2 CY3 CY5 DAPI (free) Dansyl Dansyl hydrazine Dil Filipin FITC Fluorescein (pHy11) Fura-2 (free) Fura-2 (high Ca) Hoechst Indo-1 (free) Indo-1 (high Ca) Lucifer Yellow Nile Red Oregon Green Bapta 1 Rhodamine B Rhodamine 123 Syto 13 Texas Red Triple probe (DAPI, FITC, and Rhodamine) TRITC (Rhodamine) l (nm) 691/700 920 740990 780/820 750800 776, 700800 780/800 780 780/820 700/720 700 700 700 720 740820 780 700 700 780/820 700 590/700 840860 810 800 840 780860 810 780 720/740 800840 gd2 6018 1749 2106 1955 016005 1 07202 9528 11 12 4513 1204 09503 d2
115
6318 y80 y3 y20 y35* y2538* 3897 124 2106 y2 21055
brightness. In the example shown in Fig. 8, the brightness image [e(i, j) ; Fig. 8c] shows an almost constant value over the region expected for the shape of the nanocapsule as observed from the conventional image [I(i, j) ; Fig. 8a]. The number image nNm(i, j), instead shows (Fig. 8b) that at least in two distinct regions the number of molecules is larger than the average. The combination of uorescence uctuation spectroscopy and microscopy therefore allows us to obtain important direct information on the heterogeneity of the samples.
4. Two-photon-excited probes The probability of a nonlinear interaction between the excitation light beam and a uorescent molecule depends on its two- or multi-photon cross-section. In the specic case of TPE, crosssections are given in GM units (1 GM=10x50 cm4 sx1 photon). The successful application of 2PE to biological samples is partly due to the fact that commonly used uorescent molecules could be eciently excited by 2PE (Xu, 2001). 2PE cross-sections have been measured for a large number of popular uorescent molecules (Xu & Webb, 1996). Table 2 summarizes the properties of some common uorescent molecules under TPE (So et al. 2000 ; Xu, 2001 ;
116

(a) 1000
Bodipy xs DAPI xs
100
DIL xs FITC xs
10
Coumarin xs Cascade Blue xs Lucifer Yellow xs Rhodamine xs
1 690 01
740
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940
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1090
001
0001 (b) 1000

Ca-Crimson xs Ca-Green xs
100 10
Ca-Orange wl Fluo3 xs Fura Ca xs
1 690 01 001 0001
Indo+Ca xs
740
790
840
890
940
990
1040
Indo free xs Fura free mean xs
Fig. 9. Sample of measured cross-sections (GM) of common cellular uorescent molecules (a) and calcium markers (b) within the wavelength range of the most popular ultra-fast lasers, i.e. 6901040 nm. (Data from http://microscopy.bio-rad.com/products/multiphoton/Radiance2100MP/mpspectra.htm).
Dickinson et al. 2003). Figure 9 shows a sample of measured cross-sections within the wavelength range of the most popular ultra-fast lasers (Girkin & Wokosin, 2001a). Moreover, newly engineered organic molecules, endowed with large two-photon absorption cross-sections have been recently developed (Albota et al. 1998 ; Abbotto et al. 2003 ; DAlfonso et al. 2003). A practical rule of thumb , essentially valid for symmetrical uorescent molecules, states that, as a function of the excitation wavelength, one may expect to have a TPE cross-section relative maximum at a wavelength twice that needed for one-photon excitation for a certain uorescent molecule. However, this rule of thumb is not fullled by more complex structures, for example most of the Green Fluorescence Protein (GFP) mutants (Blab et al. 2001 ; Fig. 10).

10 8
OPE (1015 cm2) 08 2PE (GM) 2PE (GM) 380 400 420 440 460 480 500 10
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50 40
380 400 420 440 460 480 500 520 540 eGFP
eCFP
20 15
OPE (1015 cm2) OPE (1015 cm2)
6 4 2 0 750
06 04 02 00 1000
30 20 10 0 750 10 05 00 1000 1050 1000
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Wavelength (nm) 380 400 420 440 460 480 500 520 540 eYFP 20
Wavelength (nm) 380 400 420 440 460 480 500 520 540 560 DsRed
30
35 30 OPE (1015 cm2) 2PE (GM) 25 20 15
30
08 20 06 04 10 03 0 750
00 950 1000 1050 1100 1150
2PE (GM)
10
10 05
0 750 800 850 900 950
00 1000 1050 1100
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Wavelength (nm)
Wavelength (nm)
Fig. 10. Absolute two-photon excitation (2PE) spectra times the quantum yield (action cross-section, sTPE, left axes) of eCFP, eGFP, eYFP, and DsRed in phosphate buer (wavelength scale at bottom). The one photon cross-section (sOPE, right axes) are shown for comparison (solid lines, wavelength scale at top). (After Blab et al. 2001.)
In addition to extrinsic uorescent molecules, 2PE uorescence from NAD(P)H, avoproteins (Piston et al. 1995 ; So et al. 2000), tryptophan and tyrosine in proteins have been measured. Special mention is also warranted on the imaging of GFP, which is an important molecular marker for gene expression (Potter, 1996; Chale & Kain, 1998). GFP cross-sections are y6 GM (800 nm) and 7 GM (960 nm) in the case of wild and S65T type respectively. As a comparison one should consider that the cross-section for NADH, at the excitation maximum of 700 nm, is y002 GM (So et al. 2000). This extension to autouorescent molecules that can be eectively exploited without the need of UV excitation opens new relevant perspectives in 3D imaging of biological systems and also in connection to other types of nonlinear interactions, i.e. second- and third-harmonic generation (Herman & Tanke, 1998 ; Lakowicz, 1999 ; Zoumi et al. 2002 ; Zipfel et al. 2003). Table 3 summarizes, even if in a less than rigorous way due to the continuing work in progress on it, 2PE cross-section data from classes of intrinsically uorescent molecules. By comparison we also report recent data about uorescent semiconductor nanocrystals quantum dots (Qds) (Hines & Guyot-Sionnest, 1996 ; Mattoussi et al. 2000 ; Peng & Peng, 2001) that allow multi-colour imaging in demanding biological environments such as living tissue, i.e. living animals or organs and within cellular cytoplasm (Sokolov et al. 2003 ; Larson et al. 2003 ; Jaiswal & Simon, 2004). These uorescent probes exhibit two-photon crosssections of the order of thousands of GM and a wide range of emission wavelengths. They are indicated as good candidates for deep and multi-colour imaging (Chan et al. 2002 ; Parak et al. 2002 ; Akerman et al. 2002 ; Dubertret et al. 2002; Sokolov et al. 2003 ; Voura et al. 2004), despite some problems related to intermittent uorescent behaviour and to a high propensity for
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Table 3. Two-photon excitation cross-section data from classes of intrinsically uorescent molecules
Intrinsic emitters GFP wt GFP S65T BFP CFP YFP EGFP DsRed Coral Red Citrine Coral Yellow Phycoerythrin Flavins NADH Retinol Pyridoxine Folic acid Lipofuscin Collagen, elastin Qdots l (nm) 800850 y960 780/820 780/840 860/900 9401000 960990 950 1064 y700730 y690730 700830 690710 700770 700850 700740 7001000 d2 y6 y7 y250 y20110 y70 y300 y0108 y002009 y007 y0008 y0007 y200047000
aggregation. These facts promoted research activities in nano-sized uorescent probes (Tokumasu & Dvorak, 2003 ; Gao et al. 2004 ; Jaiswal & Simon, 2004). As can be seen from 2PE cross-section tables and gures there is a very interesting and useful variety of relative peaks . The practical consequence is that unlike one-photon excitation when using 2PE one can nd a wavelength that can be used for exciting several dierent molecules. The relevance of this fact is obvious, for example with respect to co-localization problems. One can try to nd an optimal excitation wavelength for simultaneously priming uorescence of dierent uorochromes. This means that a real and eective multiple uorescence co-localization of biological molecules, macromolecules, organelles, etc. can be obtained. Figure 11 shows examples of uorescence molecular cross-sections demonstrating multiple uorescence excitation in the case of 2PE (red line) with respect to the 1PE (green) case. Reduced photobleaching is an essential requirement for the uorescence microscopy application of a uorescent molecule (see also discussion in Section 3.2). While the 2PE scheme reduces overall photobleaching of the sample by limiting excitation to a small volume element, photobleaching within the excited area is not reduced (Brakenho et al. 1996). In fact, accelerated rates of photobleaching have been observed in bulk by using 2PE compared to conventional one-photon excitation (Patterson & Piston, 2000). Recent studies reported on the eect of TPE on the total amount of uorescence that can be collected from a single immobilized molecule (Chirico et al. 2003b ; Federici et al. unpublished data). For the four dyes considered in this study, namely : Indo-1, Rhodamine 6G, Fluorescein and Pyrene, the 2PE bleaching rate increases by a power of 3 of the excitation intensity. This brings the conclusion that a trade-o must be considered in order to limit bleaching, while gaining in emission by increasing the excitation intensity (Chirico et al. 2003a). However, the earlier fear of destroying the sensitive biological sample by direct absorption of the NIR light has also been shown to be exaggerated, although it always requires checking (Schonle & Hell, 1998 ; Hopt & Neher, 1999 ; Tirri et al. 2003).

(a) 1103 (b) 1102 1101 110 1101 1101 1102 600 (c) 1102 700 800 900 1000 1100 (d) 1101 110 1101 1101 110 1102 1103 600 700 800 900 1000 1100 650 700 750 800 850 900 600 700 800 900
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1102
110
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Fig. 11. Rhodamine B (a), Coumarine (b), Fluoresceine (c) and Cascade Blue (d) one- (solid line) and twophoton (dotted line) excitation cross-sections (GM) (Xu, 2001). The wavelength scale has to be divided by 2 when considering the one-photon excitation case.
5. Design considerations for a 2PE fluorescence microscope 5.1 General aspects The original 2PE systems were mainly based around conventional confocal scanning systems and all of these demonstrated the principles of the technique and produced excellent results (Girkin, 2003). In principle, any laser scanning microscope can be modied to perform 2PE or multi-photon excitation microscopy by replacing the CW laser source with a pulse laser and optimizing some optical elements (Diaspro, 2001b ; Centonze, 2002). Nowadays, turn-key 2PE or multi-photon excitation microscopes are commercially accessible at a price ranging from 250 000 to 700 000 E/US$. The main parts for realizing a 2PE architecture are the following : a high peak-power laser delivering pulsed (fs or ps) IR or NIR photons at 100 MHz repetition rate and mW average power, a laser beam or sample scanning device, a high NA objective (>1), a high-throughput microscope pathway, and a high-sensitivity detection system (Denk et al. 1995 ; Konig et al. 1996c ; Potter et al. 1996 ; So et al. 1996; Soeller & Cannell, 1996, 1999 ; Wokosin & White, 1997 ; Centonze & White, 1998 ; Wolleschensky et al. 1998 ; Diaspro et al. 1999b ; Tan et al. 1999 ; Hopt & Neher, 1999 ; Fan et al. 1999 ; Wier et al. 2000 ; McCarthy, 1999 ; Mainen et al. 1999 ; Majewska et al. 2000 ; Nguyen et al. 2001 ; Diaspro, 2001b ; Girkin & Wokosin, 2001a ; Centonze, 2002 ; Iyer et al. 2002 ; Sanderson & Parker, 2003; Girkin, 2003 ; Muller et al. 2003 ; Malengo et al. 2004 ; Ridsdale et al. 2004). In a general scheme for a 2PE microscope, conventional excitation ability for confocal or wide-eld uorescence microscopy should be maintained. Within the general view, let us consider image formation modalities. Typically, 2PE or confocal microscopy images are built by raster scanning obtained by xy mirrors of a
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galvanometric-driven mechanical scanner (Ploem, 1987 ; Webb, 1996). This fact implies that the image acquisition rate is mainly determined by the mechanical properties of the scanner. Fast beam-scanning schemes can be realized that exploit the localized uorescence by using multielement lenses, thin beam splitters or, in general, a number of 2PE beams scanned through the sample simultaneously (Buist et al. 1998 ; Straub & Hell, 1998b ; Fan et al. 1999 ; Tan et al. 1999 ; Straub et al. 2000 ; Hell & Anderson, 2001 ; Nielsen et al. 2001). In this case one should accurately consider the compromise between sensitivity, spatial resolution, temporal resolution and photobleaching (Girkin & Wokosin, 2001a ; Girkin, 2003). However, in the most popular case of the utilization of scanning mirrors, enhanced silver coatings are prevalently used to optimize reectivity of the IR excitation wavelengths. Then, the excitation light should reach the microscope objective passing through the minimum number of optical components and possibly along the shortest path. Typically, high NA objectives, with high IR transmission (7080 %), are used to maximize 2PE eciency (Benham, 2002 ; Benham & Schwartz, 2002). While the xy scanners provide lateral focal-point scanning, axial scanning can be achieved by means of dierent positioning devices, the most popular being: belt-driven systems using a DC motor and objective piezo nano-positioners (Diaspro, 2001b). Acquisition and visualization are typically computer controlled by dedicated software, that allows the control of dierent key parameters such as : light detector gain, spectral separation, acquisition channel, scanning speed, excitation laser beam, eld of view, image size, z-axis positioning (Webb, 1996). Detection system is another critical issue mentioned here only in terms of general aspects (Wokosin et al. 1998 ; So et al. 2000; Girkin & Wokosin, 2001a). Photodetectors that can be used include photomultiplier tubes, avalanche photodiodes, and charge-coupled device (CCD) cameras (Denk et al. 1995 ; Murphy, 2001). Photomultiplier tubes are the most commonly used. This is due to their low cost, good sensitivity in the blue-green spectral region, high dynamic range, large size of the sensitive area, and single-photon counting mode availability (Hamamatsu Photonics, 1999). They usually provide a quantum eciency around 2040% in the blue-green spectral region that drops to <1 % moving to the red region. This is a favourable condition under the 2PE regime since one wants to reject as much as possible those wavelengths above 680 nm, mainly used for excitation. Moreover, the large size of the sensitive area of photomultiplier tubes allows ecient collection of signal in the non-descanned mode (see Section 52). On the other hand, avalanche photodiodes (APD) are excellent in terms of sensitivity exhibiting quantum eciency close to 7080 % in the visible spectral range. Unfortunately, they are expensive and the small active photosensitive area introduces some drawbacks in the detection scheme and requires special descanning optics (Farrer et al. 1999). Moreover, CCD cameras can also be used, especially in video rate multi-focal imaging (Buist et al. 1998 ; Straub & Hell, 1998b ; Fuijta & Takamatsu, 2001 ; Girkin & Wokosin, 2001a). Another general aspect is related to the possibility given by the 2PE process of priming uorescence in dierent molecules using a unique radiation wavelength (see also Section 4). This fact suggests that a very precise spectral separation is much to be desired (Farkas, 2001 ; Dickinson et al. 2001 ; Haraguchi et al. 2002 ; Zimmerman et al. 2003 ; Berg, 2004). Usually, the emitted uorescence is collected by an objective and transferred to the detection system through a dichroic mirror along the emission path that realizes the rst stage of spectral ltering. This device should also block reected excitation. Then optical lters are inserted before emission can reach the detectors in order to select the emitted wavelength for a specic acquisition channel. In 2PE, due to the high peak excitation intensity, an additional barrier lter is needed to avoid mixing of the excitation and emission light at the detection system, which is positioned
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dierently depending on the acquisition scheme being used. Recently, the introduction of newgeneration scanning heads was accompanied by ecient spectral detection systems (Wright & Wright, 2002). Nikon invested in a separate lter box with the new model C1 (Ridsdale et al. 2004). This allows better spectral control than in the classical schemes completely integrated into the scanning head and the adaptation of monochromator or similar devices in a very simple and eective customized way. Zeiss developed on its LSM510 a spectral separation scheme based on multiple detectors. A highly ecient optical grating provides an innovative way of separating the uorescence emissions in the incorporated Hamamatsu detectors, META (Dickinson et al. 2003). Here, the entire uorescence signal is projected onto the 32 channels of the META detector acting as a spectral discriminator. Thus, a spectral signature can be acquired for each pixel of the scanned image and subsequently used for the digital spectral separation into component dyes (Dickinson et al. 2001). Unfortunately, some spectral gaps are in the red detection portion of the signal due to the characteristics of the detector itself. Also FluoView 1000 by Olympus has a spectral head made by mixing dichroics and grating-slit systems. Another comparatively new system is the Leica TCS SP2 AOBS. This is the natural evolution of the spectral confocal microscope SP1, designed to be a lter-free spectral confocal and multi-photon microscope. Spectral detection is performed by a prism spectrophotometer detector (Seyfried et al. 2003). This allows performance of continuous spectral detection without the need of changing optical lters. Leica Microsystems was the rst company that introduced the new concept of a spectral scanning head (Calloway, 1999). In any case highoptical-density lters able to block the IR high-peak-power excitation radiation should be used (Diaspro, 2001b ; Stanley, 2001). As a nal general aspect, once the best quality image possible has been obtained then sophisticated mathematical algorithms can be applied to enhance those ne details and features of interest to the biological researcher and to improve the quality of data to be used for 3D modelling (Weinstein & Castleman, 1971; Diaspro et al. 1990, 2000, 2004c ; van der Voort & Strasters, 1995 ; Boccacci & Bertero, 2001 ; Vermolen et al. 2004). Recently, an image restoration web service has been established to obtain the best quality 3D dataset from wide-eld, confocal or 2PE optically sectioned samples (Bonetto et al. 2004). Now, let us focus on four more specic aspects, namely : descanned and non-descanned imaging, lens objectives, laser sources, and an example of a practical realization. 5.2 Descanned and non-descanned 2PE imaging Two dierent approaches are currently utilized to perform 2PE, namely : descanned and nondescanned modes (Sandison & Webb, 1994 ; Cannel & Soellet, 1997). The former uses the very same optical pathway and mechanism employed in confocal laser scanning microscopy. The latter essentially optimizes the optical pathway by minimizing the number of optical elements encountered on the way from the sample to detectors, and increases the detection area. The non-descanned approach is possible in 2PE since the signal generated by the sample is exclusively originated from the focal volume (Centonze, 2002). The major implication of this behaviour is that is no longer necessary to re-image the diraction-limited spot of emitted light to impose an imaging aperture able to cut o out-of-focus information. Figure 12 illustrates these two approaches. The non-descanned detection scheme is in tune with the need for collecting every excited photon possible (Pawley, 1995), with the rule that The best optics are no optics ! (Girkin & Wokosin, 2001a). When working with point-scanning laser excitation
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systems short pixel dwell times (microseconds) are often used, which necessitate very high source intensities for sucient signal-to-noise imaging. High laser intensities have a correspondingly high risk of photobleaching and saturation potential (Hopt & Neher, 1999). The best solution is one that improves the signal-to-noise ratio within the detection scheme. The descanned detection scheme allows increasing resolution at the cost of a drop in detection eciency when pinhole diameter is reduced (Sheppard & Gu, 1990 ; Sandison & Webb, 1994 ; Gauderon et al. 1999 ; Torok & Sheppard, 2001). Conversely, the non-descanned mode requires that the confocal architecture is modied : the emitted radiation is collected using dichroic mirrors on the emission path or external detectors without passing through the galvanometric scanning mirrors or the scanning device being employed for point-like excitation (Piston et al. 1994 ; Williams et al. 1994). A photomultiplier tube can be placed immediately beneath the objective or above the condenser to collect all the uorescence captured by the lens. The resulting shorter emission pathway, possibly with the use of demagnication lenses, ameliorates the collection of emitted photons from a greater solid angle (Centonze, 2002 ; Koester et al. 1999). This permits collection of more scattered photons emitted by uorescent molecules, thereby increasing the signal-to-noise ratio especially when imaging very deeply in thick lightscattering samples (Centonze & White, 1998). Thus far, non-descanned imaging results in a signicant increase in detection sensitivity (Wokosin & White, 1997 ; Centonze & White, 1998 ; Koester et al. 1999), although it makes the detector sensitive to ambient room light. All recent confocal microscopes allow adaptation of collection in both modes. 5.3 Lens objectives and pulse broadening For a uorescence microscope, special criteria have to be satised (Benham, 2002 ; Benham & Schwartz, 2002) for the choice of the objective lens besides those common to any microscope (Keller, 1995). For 2PE, an adequate transmission in the IR regime has to be coupled with good collection eciency towards the UV region. Moreover, the number of components, their shape and the type of glass should be optimized without aecting resolution properties in order to reduce pulse-width distortions due to group velocity dispersion (Wokosin et al. 1998 ; Wolleschensky et al. 1998, 2001 ; Netz et al. 2000). Although the collection eciency of the timeaveraged photon ux is dependent on the NA of the collecting lens, it was demonstrated that the total uorescence generation is independent of the NA of the focusing lens when imaging thick and uniformly uorescent samples (Xu, 2001). This is due to the fact that the increase of intensity, obtained by a sharper focusing (high NA), is counterbalanced by the shrinking of the excitation volume. Thus, the integral of uorescence over the entire space remains constant. The very relevant practical consequence of this fact is that in 2PE measurements from thick samples, assuming no aberrations, the generated uorescence is insensitive to the size of the focal spot. As a positive consequence, a moderate variation of the laser beam size would not aect the measurements. This depends on the fact that it is possible to collect all the generated uorescence by using an appropriate (non-descanned) acquisition scheme. For pointscanning systems, the acceptance angle of the objective lens is related to the NA. Girkin & Wokosin (2001a) reported on the light collection performances of a variety of lenses. The 14 NA 60r oil and 12 NA 60r water lenses have the best collection potential, the 13 NA oil lenses can collect 85 % compared to the best two lenses, while the 075 NA air lenses and 10 NA water-dipping lens only collect 66% as much light. As usual, a compromise must be reached, as the higher NA lenses (recently 165 by Olympus and 145 by Nikon and Zeiss have become

Excitation light Scanning system A Detector DM Pinhole xy Scanner Non-descanned detector z
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Detector DM Fluorescence Objective Lens Focal Plane Scattering specimen A Multiphoton (descanned) B Multiphoton (direct detection) B
Fig. 12. Simplied optical schemes for de-scanned (A) and non-descanned (B) detection. Confocal pinhole can be used or fully opened. Images along the z-axis under (B) condition have a better signal-to-noise ratio than under (A) one. (Courtesy of Mark Cannel ; adapted from Soeller & Cannell, 1999 ; courtesy of Ammasi Periasamy, image inset).
available) gain the light collection power at the loss of working distance. Typical working distances for oil immersion lenses with a NA of 14 are y250 mm (which include the thickness of a cover slip that most commonly is 017 mm). This gives a practical working distance of <100 mm, making them unsuitable for really deep imaging of intact tissue. As a general rule one selects a lens with as large a NA as possible giving the working distance required for the preparation under investigation. The 14 NA lenses provide a good starting point for 2PE lens selection. In addition NA has a signicant contribution in determining the signal-tobackground and signal-to-noise ratios. These factors play a large role in determining whether a particular sample can be successfully imaged, and in particular on the maximum depth that can be visualized (Girkin & Wokosin, 2001a ; Jonkman & Stelzer, 2001 ; de Grauw & Gerritsen, 2001). Table 4 shows a collection of transmission data for Nikon CFI60 series of objectives (Girkin & Wokosin, 2001a ; Benham & Schwartz, 2002). Objective lenses and optical components play a role in pulse broadening and this inuences the eciency of the 2PE process. The short pulses typically used for 2PE or multi-photon imaging microscopy have bandwidths of the order of 15 nm (corresponding to%100 fs pulse time duration), and the passage through microscope optics produces a dispersion that results in an eective stretching of the pulse in time and reduction of the peak power (Muller et al. 1998 ; Netz et al. 2000 ; Girkin & Wokosin, 2001a). This is similar to an avalanche eect. Once the pulse has started to be stretched, the eect continues as it is due to the spectral bandwidth and not to the actual pulse length in the optical train (Girkin & Wokosin, 2001a). Now, considering an ideal chirp-free pulse at the entrance of an optical system having a pulse width tin, the pulse broadening B can be calculated as tout/tin where tout is the pulse width at
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Table 4. Collection of transmission data for Nikon CFI60 series of objectives

Objective CFI Plan Apochromat CFI Plan Fluor CFI Fluor Water dipping CFI Plan Fluor CFI Fluor Water dipping CFI Super (S) Fluor CFI Super (S) Fluor CFI Fluor Water dipping CFI Super (S) Fluor MAG/NA 100rOil IR 140 100rOil 130 60r100 60rOil 125 40r080 40rOil 130 20r075 10r030 10r050 Working distance (mm) 013 020 200 020 200 022 100 200 120 T % at 350 nm <15 5170 5170 3150 5170 5170 5170 5170 5170 T % at 550 nm >71 >71 >71 >71 >71 >71 >71 >71 >71 T % at 900 nm 5170 >71 >71 5170 >71 >71 >71 5170 >71
Table 5. Optical dispersion parameters for dierent glass types

D at 800 nm (fs2 cmx1) +251 +300 +389 +445 +1030 +1600 Glass type CaF2 quartz FK-3 BK7 SF2 SF10
the focal plane (Konig, 2000) : q 2 B =tout =tin = 1+7 68(DL =t2 in ) ,
(20)
Typical values for the optical dispersion parameter D for glass objectives at 800 nm are given in Table 5. Considering the optical path length factor L, a typical DL value of the whole microscope, including oil immersion high-NA objective is 5000 fs2 (Konig, 2000). Wolleschensky et al. (2001) reported a summary of the dispersion parameter for Zeiss microscope objective lenses measured at 800 nm. DL values (in fs2) are 1714, 1494, 2398, and 1531 (within an error of y10%) for 40r/08 water IR Achroplan, 63r/09 water IR Achroplan, 40r/13 oil Plan Neouar, and 20r/075 Plan Apochromat respectively. Pulse broadening factor for a 100 fs pulse was estimated to be between 114 and 123 (Wolleshensky et al. 2001). In order to minimize dispersion problems one should use pulses of around 150200 fs (Konig, 2000). Operatively, a typical optical system, considering the lens objective eect as predominant, will stretch a 200 fs pulse to 220 fs at y850 nm producing a peak power loss of 10 %. Moving to picosecond pulses it should be borne in mind that 30 % more average power is required for the same signal when compared with 100 fs pulses (Girkin & Wokosin, 2001a).
2PE in biological microscopy 5.4 Laser sources
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Laser sources can be considered as a sort of key step for the development and dissemination of 2PE. Within the non-resonant 2PE framework, owing to the comparatively low cross-sections of uorescent molecules, high photon ux densities are required, >1024 photons cmx2 sx1 (Konig, 2000). 2PE or multi-photon imaging typically requires y500 times more average power delivered at the sample than conventional confocal laser scanning microscopy in order to achieve a comparable number of excitation events within the focal volume (Girkin & Wokosin, 2001a). Using radiation in the spectral range of 6001100 nm, excitation intensities in the MW-GW cmx2 are required. Colliding pulse dye lasers used in the early development of 2PE imaging (Denk et al. 1990 ; Valdemains et al. 1995) could not really be considered as practical systems for the long term (Girkin, 2003), due to the limited tunability, moderate output average power 15 mW and the considerable maintenance needed. However, a similar source with high-peak powers operating at around 600630 nm, an unsual range for solid-state-based lasers (see below), has been recently developed and used for multi-photon imaging applications (McConnell et al. 2004). Solid-state-based lasers were introduced in 1992 with the development of femtosecond titanium:sapphire lasers (Curley et al. 1992 ; Fisher et al. 1997). Even if such systems are not perfect for the application they have become the laser of choice for most users (van de Vende & Gratton, 1995 ; Centonze, 2002 ; Girkin, 2003). This is especially due to the fact that all solid-state sources requiring standard mains electrical power supply and minimal cooling systems favoured the dissemination of 2PE and multi-photon imaging systems (Wokosin et al. 1997 ; Diaspro, 2004a). More recently a number of advances have been made that, when available commercially, may change the practical application of multi-photon microscopy (Girkin & Wokosin, 2001b ; Girkin, 2003 ; Tirri et al. 2003). 2PE imaging has been achieved with signicantly longer pulses than the most commonly used 100200 fs case : picosecond (Hanninen et al. 1995) and CW (Hanninen et al. 1994) 2PE has been accomplished successfully. However, this would not be the preferred choice for most applications due to linear absorption and hence sample heating (Konig et al. 1995). Nowadays, most commercial systems operate around 100200 fs and 80 MHz for historical reasons (see Table 6). The original synchronously pumped short-pulse sources were excited by large-frame ion lasers which happened to have a cavity length equivalent to an 80 MHz pulse repetition rate (Girkin, 2003), a fact particularly useful for uorescence excitation (see Section 34 ; Denk et al. 1995). Such a repetition rate, is high enough to allow rapid scanning approximately 1 frame/s in a conventional system but low enough that the uorescence lifetime of the uorophores does not cause excitation events to be wasted because the uorescence has not decayed before the next excitation pulse arrives. Considering this, the ideal repetition rate would be around 80200 MHz and an average power output around 200 mW. This means, considering the optical eciency of the microscope, an average power at the sample of a few tens of mW. Table 6 also includes data about a new promising ytterbium-based source. This novel laser source, emitting high energy (20 nJ) femtosecond pulses, in a broad spectrum (250 nm), can be tuned from 950 to 1200 nm, without any laser adjustment, and delivers sub-300 fs pulses with a 10 nm spectral width (http://www.amplitude-systemes.com ; Courjaud et al. 2001 ; Deguil et al. 2004). As recently reported by Hanninens group, low-cost lasers can be used to utilize the positive properties of TPE with certain compromises and well-designed experiments (Tirri et al. 2003).
126 A. Diaspro, G. Chirico and M. Collini
Table 6. Laser sources for 2PE

Laser material Ti :Sapphire Company, model Coherent, Mira Spectra Physics, Tsunami Coherent, Chameleon XR Spectra Physics, Mai Tai Time Bandwidth, Pallas Time Bandwidth, Tiger Femtosource MicroLase/Coherent Scotland, BioLite Time Bandwidth, GLX200 Amplitude Systems Highqlasers Coherent and Spectra Physics Wavelength (nm) 700980 7001000 705980 710990 780860 780860 750850 1047 1058 1030 850 3501200 Pulse length <200 fs <100 fs (or 2 ps as option) <140 fs 120 fs <100 fs <100 fs <12 fs 200 fs <250 fs <200 fs 100 fs 100 fs Repetition rate (MHz) 76 80 90 80 75 100 75 120 100 50 50 Power 07 W 13 W 08 W 14 W 17 W 1 5 W 500 mW 400 mW 400 mW 600 mW 500 mW >400 mW 1W >1 mW y200 mW Demonstrated on dyes inc Texas Red and GFP ; optional 9501150 selectable Exact parameters depends crystal selected. Requires titanium:sapphire as pump source Comments May require mirror change for full tuning May require mirror change for full tuning Hands free operation, computer controlled, compact design, single box Hands free operation, computer controlled, compact design, single box
Nd :YLF Nd :Glass Ytterbium Cr :LiSAF OPO
Not presently available
The pulse length given is the average pulse length through over the full tuning range of the system. Power is the average power at the peak of the tuning curve, typically 800 nm, or at the operating wavelength. Both Coherent Mira and Spectra Physics Tsunami systems can be pumped with a 5, 8 or 10 W green laser source. When two powers are given these are for 5 and 10 W pumped. Higher pump power may be needed for full tuning range. Between 925 and 960 nm titanium:sapphire lasers need to be purged to ensure that no water vapour is present within the laser cavity. Computer tunable systems are sealed to prevent this complication and are endowed of a pointing stability system that reduces the need of new alignment following tuning across a wide range of wavelengths.
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Fig. 13. Two-photon architectures at LAMBS (Laboratory for Advanced Microscopy, Bioimaging and Spectroscopy) incorporated in the new Research Centre of the University of Genoa for Correlative Microscopy (MicroScoBio) funded by IFOM (Italian Molecular Oncology Foundation). Under the auspices and grants of the National Institute for the Physics of Matter (INFM, Istituto Nazionale per la Fisica della Materia) this was the rst Italian 2PE architecture (Diaspro, 2001b). Leica TCS SP2 AOBS (front, centre) and PCM2000 Nikon (back, right) confocal laser scanning systems. They are operating sharing the very same Titanium Sapphire laser source. (Photograph taken at LAMBS.)
Once the laser source has been selected, one has to couple it with microscope optics. Wolleschensky and colleagues (2001) reported accurate data, and analysis of benets and problems in using an optical bre or direct coupling to launch the excitation within a confocal scanning head. Direct coupling allows the maximum exibility, together with the minimum level of extra problems at the excitation stage. In fact, even if the bre coupling solution seems the best in terms of practicality and safety at a rst glance, some practical problems can arise related to the bre coupling solution itself. For example, due to high-order nonlinearities in the bre, there is a limit regarding the maximum deliverable power, typically 60100 mW. Moreover, because the bre coupler needs some devices for maintaining pulse width and beam shape, it is not totally turn-key demanding further control and alignment procedures especially when wavelength changes are required or laser output beam changes occur, due, for example to room temperature variations. 5.5 Example of a practical realization This section deals with an example of a practical realization of a 2PE microscope based on a commercial confocal laser scanning microscope (CLSM), Nikon PCM2000 (Nikon Instruments, Florence, Italy), as previously reported in detail (Diaspro et al. 1999b ; Diaspro, 2001b). Figure 13 illustrates the 2PE laboratory at the University of Genoa showing the recent addition of a spectral CLSM, Leica SP2/AOBS (Leica Microsystems, Heidelberg GmbH, Germany). A core component of the 2PE microscope is the laser source. In this case, two mode-locked titanium:sapphire IR pulsed lasers are available, namely: a Tsunami 3960 (Spectra Physics Inc., Mountain View, CA, USA), pumped by a (5 W at 532 nm) solid-state laser Millennia V (Spectra
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Physics Inc.), and a Chameleon-XR (Coherent Inc., Santa Clara, CA, USA), a widely tunable, compact and hands-free laser diode, pumped by an incorporated high-power Verdi (Coherent Inc.) solid-state 532 nm laser (see also Table 6). This allows 2PE in the 680980 nm range. Two excitation ranges are covered by the Tsunami, e.g. 680830 and 730900 nm, depending on the set of the mirrors actually mounted into the laser cavity, and a third range from 710 to 980 nm is achieved by Chameleon. This exible conguration permits 2PE of a variety of uorescent molecules normally excited by visible and UV radiation, including the GFP family, according to the cross-section behaviours given in Tables 2 and 3. Power and wavelength measurements are carried out using an RE201 model ultra-fast laser spectrum analyser (Ist-Rees, UK) and an AN2/10A-P model thermopile detector power meter (Ophir, Israel) that constitute the beam diagnostics module of the architecture. Recently we have added a home-made compact optical autocorrelator, which allows measurement of the width of laser pulses on the microscope objective plane (Cannone et al. 2003b). A special dichroic mirror set (CVI, USA), optimized for high-power ultra-short IR pulsed laser beams, is employed to bring the laser excitation directly into the confocal laser scanning head. Before entering into the PCM2000 or Leica SP2 scanning head, beam average power is brought to desired values using a neutral-density rotating wheel (Melles Griot, USA). We refer to the PCM2000 scanning head unless specied dierently. An average power of 30 mW at the entrance of the scanning head produces an average power before the microscope objective of 915 mW, depending on the wavelength and on the alignment. The PCM2000 Nikon scanning head has a comparatively low number of optical components along the excitation pathway, and the resulting average power at the sample, i.e. at the focal plane of the objective, is estimated to be between 5 and 12 mW. This further loss of power is due to the transmission property of the lenses in the IR wavelength region (see Table 4). In terms of pulse width, considering a pulse duration in the range of 90150 fs at the exit of the laser output window, after passing driving mirrors, neutral density lter and the scanning lens, a 1518 times broadening was found when using a high NA objective (Diaspro, 2001b). This is in agreement with earlier reports (Hanninen & Hell, 1994; Soeller & Cannell, 1996). For the optical system described at this point, pulse widths in the range of 220265 fs have been measured at the sample focal plane (Cannone et al. 2003b). However, in order to check the pulsing status of the laser source during the measurement sessions, one should continuously display the pulse condition. This can be easily done by monitoring the wavelength spectrum by means of an oscilloscope connected to the output of the spectrum analyser, as shown in Fig. 14. The pulsing condition of the laser can also be veried using a simple and inexpensive reective grating. In this case the reected image on a screen will be sharp for quasi-continuous emission and blurred for pulsed emission. This is due to the fact that the output of the pulsed laser beam is more spectrally broadened in the case of pulsed emission. Unfortunately, the Tsunami requires periodical re-alignment due to beam displacement following wavelength changes. This problem is completely overcome when using Chameleon since it is endowed with a beam-pointing system, named PowerTrack, allowing active alignment for long-term stability. Moreover, the utilization of Chameleon is currently preferred due to the fact that the laser output pulse width is stable around 120140 fs. This fact keeps the broadening of pulse width limited. In fact, considering a 2PE microscope with acousto-optical modulator having a b value [group velocity dispersion ; DL in Eq. (20)] of y13 500 fs2 at 800 nm, 6080 fs pulses will broaden to 540 fs at the sample, while 120 fs pulses will broaden to 340 fs. Figure 15 shows calculated broadening of pulses due to dispersion in a 2PE microscope (www.coherentinc.com, 2002). More broadening will produce a considerable loss in 2PE eciency according to Eq. (7). The other core component of the 2PE

Beam diagnostic 2P laser
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Tsunami Variable N.D. filter 1P laser module
Z PCM2000
PCM2000 controller EZ 2000
Fig. 14. Overall scheme of the Nikon PCM2000-based two-photon microscope described in the text (Diaspro, 2001b).
architecture is the scanning and imaging system. As anticipated, the scanning and acquisition system for microscopic imaging is based on a commercial single-pinhole scanning head Nikon PCM2000 (Nikon Instruments, Florence, Italy) mounted on the lateral port of an inverted microscope, Nikon Eclipse TE300. The Nikon PCM2000 has a simple, ecient and compact light path that makes it very appropriate for conversion to a 2PE microscope (Diaspro, 2001b). Figure 16 illustrates the optical pathways in the confocal laser scanning head. The optical resolution performances of the PCM2000 confocal laser scanning microscope when operating in conventional confocal mode, and using a 100r/13 NA oil immersion objective, have been reported in detail elsewhere and are 18020 nm laterally and 51050 nm axially (Diaspro, 1999a). Better performances can be obtained by means of digital deconvolution methods (Diaspro et al. 1997, 2000). Under the TPE regime the scanning head operates in the open pinhole condition; i.e. a wide-eld descanned detection scheme is used (Diaspro, 2001b). Figure 17 gives an insight of the open scanning head. A dichroic mirror (1st) has been substituted in the original scanning head to allow excitation from 680 to 1050 nm (Chroma Inc., Brattleboro, VT, USA). The substituted dichroic mirror reects very eciently (>95 %) from 680 to 1050 nm. The 50 % cut-o is at y640 nm. This dichroic mirror transmits more than 90 % radiation from 410 to 620 nm. The neutral density lter at the open-pinhole location of the original PCM2000 scanning head has been removed. Galvanometric mirrors are metal-coated (silver) on fused silica. The minimum pixel residence time is 15 ms. A series of emission custom-made lters (Stanley, 2001), blocking IR radiation with dierent thresholds >650, >700, >720 nm to an optical
2P optical pump
Specimen
TE 300 Millenia
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(a) 700 = 17,500 fs 600 Broadened pulse width (fs) 500 400 300 200 100 0 0 100 200 Pulse width from laser (fs) 300 400 = 13.500 fs
~ 6080 fs typ. Chameleon ~ 120 fs
(b)
Fig. 15. (a) Calculated broadening of pulses due to dispersion in a 2PE typical microscope (see text, for a range of laser pulse widths). (b) The microscope, coupled with an ultra-fast laser spectrum analyser RE201 (Ist-Rees, UK), allows pulse control. (Courtesy of Mario Arace who purchased the oscilloscope in 1978.)
density of 67 within 50 mW of beam power incident on the lters themselves. This allows the use of those uorophores emitting in the red region of the spectrum and of growing interest. The optical lters more commonly utilized are the following : E650P, HQ 460/50, HQ 535/50, HQ 485/30 and HQ 405/30 (Chroma Inc.). More specically, the E650P lter constitutes the base for the other HQ lters. This means that for some applications a kind of sandwich made by optical lters can be easily realized thanks to the optically simple and accessible PCM2000 scanning head. 1PE mode is preserved by the switching of two optical pathways: the singlemode optical bre used for 1PE and the 2PE direct coupling (Diaspro, 2001b). Switching from conventional to TPE allows retention of the focus and position on the sample as demonstrated
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PCM2000
PMT
TE 300
xy 3
Fig. 16. Details of the optical pathway of the confocal scanning head Nikon PCM2000. The excitation beam enters into the PCM2000 scanning head through an optical coupler (1) in order to reach the sample on the xyz stage (5). The beam passes through the pinhole holder (2) kept in open position, the galvanometric mirrors (3) and the scanning lens (4). Fluorescence generated from the sample (5) is delivered to the PMT through acquisition channels directed by two selectable mirrors (6, 8) via optical bre (7, 9) coupling. One-photon and two-photon mode can be accomplished simply by switching from the single-mode optical bre (one-photon), coupled to a module containing conventional laser sources (ArIon, HeNe green), to the two-photon optical coupler (TPOC) shown in Fig. 38, allowing Tsunami laser beam delivery (twophoton). Axial scanning for confocal and two-photon excitation (TPE) 3D imaging is actuated by means of two dierent positioning devices depending on the experimental circumstances and axial accuracy needed, namely : a belt-driven system using a DC motor (RFZ-A, Nikon, Japan) and a single objective piezo nanopositioner (PIFOC P-721-17, Physik Instrumente, Germany). The piezoelectric axial positioner allows an axial resolution of 10 nm within a motion range of 1000 nm in 100 nm steps and utilizes a linear variable dierential transformer (LVDT) integrated feedback sensor (Diaspro, 2001b).
in Figs 18 and 19. At the end of the collection pathway, a high throughput optical bre delivers the emitted uorescence from the scanning head to the PCM2000 control unit where photomultiplier tubes (R928, Hamamatsu, Japan) are physically plugged. Axial scanning for confocal and TPE 3D imaging is actuated by means of two dierent positioning devices depending on the experimental circumstances and axial accuracy needed, namely : a belt-driven system using a DC motor (RFZ-A, Nikon, Japan) and a single objective piezo nano-positioner (PIFOC P-721-17 ; Physik Instrumente, Germany). The piezoelectric axial positioner allows an axial resolution of 10 nm within a motion range of 1000 nm at 100 nm steps and utilizes a linear variable dierential transformer (LVDT) integrated feedback sensor. Acquisition and visualization is completely computer controlled by dedicated open software (EZ2000 ; Coord, The Netherlands; http:// www.coord.nl). Image processing based on deconvolution algorithms is performed by means of a web-based package designed and implemented at the Laboratory for Advanced Microscopy, Bioimaging and Spectroscopy (LAMBS) (Diaspro et al. 2002b ; Bonetto et al. 2004). Now, in order to evaluate the performances of the microscope some basic trials have to be performed, namely : evaluation of the uorescence quadratic behaviour and of the PSF. As a rst step, the quadratic relationship of the uorescence intensity versus excitation power has to be demonstrated before speculating on the uorescence signal. This can be simply accomplished by measuring uorescence intensity from a standard or from the actual sample as a function
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Fig. 17. Nikon PCM 2000 confocal laser scanning head modied for two-photon excitation at LAMBS. Main modications are: pinhole and neutral density lter removal in the large pinhole position ; D1 dichroic optimized for IR transmission ; free-space coupling input ; additional barrier lter (Chroma Inc.) for avoiding collection of high-power peaks of the excitation beam (Diaspro, 2001b).
of the excitation power. Special attention has to be given to the acquisition conditions. They should be kept constant during the acquisition session or calibrated for the intensity range. Figure 20 shows the 2PE trend obtained from a solution of uoresceine (Diaspro, 2001b). The evaluation of the PSF can be performed using uorescent microspheres of dimensions below optical resolution, i.e. 5200 nm or thin uorescent layers. In the rst case one determines the 3D PSF (Diaspro et al. 1999b). In the second case, one measures the axial behaviour of the optical system (Schrader et al. 1998). PSF measurements reported here are referred to a planachromatic Nikon 100r, 14 NA immersion oil objectives with enhanced transmission in the IR region. Lateral and axial resolutions were evaluated as 21040 and 70050 nm respectively (Diaspro, 2001b). The 3D imaging capabilities of the 2PE microscope is also shown in Fig. 21. A DNA sample marked with DAPI was imaged using the intrinsic optical sectioning ability of the TPE of uorescent molecules. The 3D assembly of the sample is clearly evident even in the case of the thick sample being imaged, i.e. a sea urchin egg. Optical sections exhibit dierent structural details at high signal-to-noise ratio without any image processing. Figure 22 reports autouorescence optical slices of another thick biological sample, e.g. the cistis of Colpoda. This very same architecture is also used for single-molecule detection and spectroscopic measurements (Diaspro et al. 2001 ; Chirico et al. 2001a ; Cannone et al. 2003a). Recently at LAMBS a new 2PE microscope based on a Leica SP2 AOBS confocal laser scanning microscope has been established. Laser coupling is realized utilizing an extra laser port available at the scanning head. Since this confocal laser scanning microscope is endowed with a high-performance spectral detection unit, it is ideal for spectral analysis of autouorescence signals and for other nonlinear interactions like SHG. Figure 23 shows an image of a protozoan exhibiting

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Fig. 18. Optical sectioning of uorescent microspheres demonstrated in confocal and two-photon excitation (TPE) mode after switching from (a) one- to (b) two-photon mode using the TPOC developed at LAMBS. It is worth noting that after switching sample positioning is retained.
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Fig. 19. Same biological cell to demonstrate switching from (a) one- to (b) two-photon excitation mode. In two-photon excitation mode the internal structure of the nucleus is clearly visible, i.e. chromatin DNA marked by DAPI. This gure clearly shows the control of positioning after mode switching.
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autouorescence and a second-harmonic signal on the backscattering pathway when excited at 720 nm (Campagnola et al. 1999 ; Diaspro et al. 2002c).
6. Applications This section, for obvious reasons, is far from being exhaustive. It reports some of the most relevant aspects, in the authors opinion, in relation to TPE and related methods. Interested readers can nd other applications and stimuli in some books and special issues on this subject (Pawley, 1995 ; Hell, 1996 ; Lakowicz, 1999 ; Diaspro, 1998, 1999a, b, 2001a, 2004a ; Periasamy, 2001 ; Matsumoto, 2002 ; Masters, 2002 ; Ferguson, 2003 ; Periasamy & Diaspro, 2003 ; Feijo & Moreno, 2004). 6.1 Biological applications of 2PE 2PE is growing tremendously in terms of applications in many areas of biology, physics, medicine, and engineering. Its capability of deep imaging makes it extremely valuable for in vivo imaging. The related technology is still developing rapidly and its range of utilization is broadening. The rst 2PE applications to biological cells were presented by Denk and co-workers (1990) within the neurosciences, and most of the applications presented up to now are related to calcium mapping in cells, the intact brain and brain slices (Denk et al. 1994 ; Oheim et al. 2001 ; Helmchen & Denk, 2002 ; Tsai et al. 2003 ; Theer et al. 2003). Recently Taki et al. (2004) have presented developments in ratiometric dyes for the detection of zinc ions by means of 2PE that are also promising for biological applications. 6.1.1 Brain images Signalling mechanisms at individual synapses can be monitored in brain slices. The ability of 2PE to provide high-resolution images within scattering turbid media has a key role for these

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Fig. 21. Three-dimensional optical slices from a sea urchin egg marked by DAPI, two-photon excitation at 720 nm. The heterochromatin distribution within the female pronucleus is visible. The whole egg has a diameter of 80 mm while the nucleus is 10 mm (as reference this is the maximum visible diameter). In conventional wide-eld microscopy one could see only a bright spot of confusion from the nucleus. (Sample by Carla Falugi, Department of Biology, University of Genoa.)
studies (Beaurepaire et al. 2001). Oertner and colleagues (2002) found that the amount of glutamate released per action potential changes during short-term facilitation, suggesting a multivescicular release of glutamate. Moreover, Sabatini et al. (2002) investigated calcium buering capacity in spines of pyramidal neurons using 2PE. Examples of these applications are given in Fig. 24, showing a calcium-related image of rat granule cerebrellar cells loaded with Indo-1 acetoxymethyl (AM). The cellular network has been optically sectioned at a moderate average power on the focal plane, 2 mW, at 720 nm excitation wavelength and by focusing with a 100r/13 NA objective. The complex and intricate organization in Purkinje cells are shown in Fig. 25. 2PE calcium imaging of isolated cells can be used to monitor the activity of distinct neurons in brain tissue in vivo. Recently, Stosiek and colleagues (2003) introduced a versatile approach for loading membrane-permeant uorescent indicator dyes in large populations of cells. They
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Fig. 22. Optical sections from Colpodas cistis. From these images was possible to detect the dierent layers of membrane composition by means of the autouorescence signal, primed at 780 nm, as well as small nuclei membranes visible as small dots in round-shaped structures. (Sample by Paola Ramoino, DIPTERIS, University of Genoa.)
established a pressure ejection-based local dye-delivery protocol that can be used for a large spectrum of membrane-permeant indicator dyes, including Calcium Green-1 AM-ester, Fura-2 AM, Fluo-4 AM, and Indo-1 AM. This dye-loading protocol was successfully applied in mouse brain tissue in vivo and in vitro at any developmental stage from newborn to adult. Thus, these results demonstrated the suitability of 2PE imaging for real-time analyses of intact neuronal circuits with the resolution of individual cells. Another outstanding application is given by a study of temporal and spatial dynamics of Ca2+ signalling in mouse cortical neurons (Stutzmann et al. 2003). Dynamic processes in the brain require long-term imaging that a 2PE approach can uniquely provide while limiting the bleaching of out-of-focus planes in the sample. It is possible to study structural reorganization in the brain on a slow time-scale compared to neural activity. This can be done by utilizing cranial windows similar to the approach used in cancer research (Jain et al. 2002) where the skull is removed over a small area and permanently replaced with a cover glass. TPE imaging is also possible through the thinned skull of living animals (Christie et al. 2001 ; Yoder & Kleinfeld, 2002). The overall approach is minimally invasive and allows stable imaging over a period of weeks. Christie and colleagues (2001) provided the rst demonstration of growth and stability of senile plaques in a mouse model of Alzheimers disease using in vivo multi-photon microscopy. Figure 26 shows a top-down projection of senile plaques in the brain. In a related recent study Nitsch et al. (2004) have shown, for the rst time, that within the complex cellular network of living brain tissue, specic T cells contact neurons in which they induce calcium
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Fig. 23. Three-dimensional view of a second-harmonic generation and autouorescence from Paramecium primaurelia multimodal image obtained by means of two-photon excitation at 810 nm. As a rst check, also used for two-photon excitation autouorescence, the laser was taken out of mode locking and the signals vanished. This fact indicates that the signals origin was due to nonlinear processes, which were also veried by a quadratic dependence on the laser power. Moreover, the laser was scanned between 750 and 830 nm with the 405 nm emission lter kept xed. No signal was detected at 405 nm within a range of y5 nm at y810 nm. Finally, the potential second-harmonic generation image appeared bleach resistant. Diaspros group is indebted to Colin Sheppard, Tony Wilson, Bruce Tromberg and Guy Cox for critical and useful discussions about the still unclear origin of such a signal on the back-scattering pathway (Diaspro et al. 2002c). (Sample prepared by Paola Ramoino, DIPTERIS, University of Genoa.)
oscillations resulting in a lethal increase in neuronal calcium levels. This in vivo study, which reveals important details for the understanding of multiple sclerosis, has employed TPE ion-sensitive dyes. Wang and colleagues (2003) produced a very impressive work combining the 2PE method with the mapping of the activity of a large number of olfactory neurones in the y brain by means of a genetically targeted endogenous calcium probe used in conjunction with a diverse sensory input to the olfactory receptors. Very recently Vest et al. (2004) have tried to validate, also by means of 2PE, the hypothesis that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaet of the cell membrane. Changes in membrane order were assessed with steady-state uorescence spectroscopy and 2PE with an environment-sensitive probe, laurdan.
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Fig. 24. Granule rat cerebrallar cell loaded with Indo-1 AM, a calcium-binding dye. This UV-excitable uorescent molecule has been excited at 720 nm at a moderate average power at the focal plane of 2 mW. (Courtesy of Alessandro Esposito, LAMBS.)
Fig. 25. Purkinje cell labelled with Oregon Green. Calcium ion concentration is mapped by means of a colour scale from blue (low concentration level) to red (maximum concentration level). (Courtesy of Professor Cesare Usai, Institute of Biophysics, National Research Council, Genoa, Italy.)
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Fig. 26. Top-down projection of senile plaques in the brain of a living transgenic mouse (Tg2576). This image is from an xyz volume of 500r615r200 mm3 (Christie et al. 2001). (Image by B. J. Bacskai, bacskai@helix.mgh.harvard.edu ; downloaded from Biorad site : http://microscopy.bio-rad.com/ gallery7.htm).
6.1.2 Applications on the kidney 2PE has been used to capture high-quality images and study the function of otherwise inaccessible cell types and complex cell structures of the juxtaglomerular apparatus (JGA) in living preparations of the kidney (Peti-Peterdi et al. 2002). This structure has multiple cell types that exhibit a complex array of functions, which regulate the process of ltrate formation and renal haemodynamics. For the rst time, it was possible to report on highresolution 3D morphology of isolated, perfused kidney glomeruli, tubules, and JGA. Calcium imaging of the glomerulus and JGA (Fig. 27), demonstrated the utility of 2PE in capturing the complexity of events and eects that are exerted by the specic hypertensive autacoid angiotensin II. 2PE in vivo has been used to study the renal excretion of sulfoneuorescein (SF) in normal and cystic rat kidneys (Tanner et al. 2004). Figure 28a shows a low power view of the kidney surface of a normal rat infused with SF. SF intensities in proximal tubules are not uniform, but are higher in late proximal tubules than in early proximal tubules. Figure 28b shows a representative high-power view from a non-pathological kidney. Proximal tubule cell SF uorescence intensity clearly exceeds that of the plasma and the tubule lumen. This brilliant work by Tanner and colleagues (2003) clearly demonstrates the ability of TPE microscopy to study quantitatively the renal excretion of uorescent probes in vivo. 6.1.3 Mammalian embryos Squirrel and co-workers (1999) studied mammalian embryos over 24 h without compromising viability. Two-photon microscopy permitted long-term uorescence observations of the dynamics of 3D cyto-architecture in highly photosensitive specimens such as mammalian
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Fig. 27. Visualization of various glomerular and juxtaglomerular apparatus (JGA) structures in situ. Glomerulus is perfused through the aerent arteriole (AA) with attached cortical thick ascending limb (cTAL) and macula densa (MD). The image shows the Ca2+ map of the double-perfused AA and cTAL containing the MD. Tissue was loaded with Indo-1. G, glomerulus ; EA, eerent ateriole (bar=10 mm).
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Fig. 28. (a) Low power (20r objective) view of the surface of the kidney of an anaesthetized rat kidney during constant intravenous infusion of a solution of sulfone uorescein (SF). SF is an organic anion secreted by proximal tubules, and it accumulates in proximal tubule cells, especially in late proximal segments. Distal tubules (arrowheads) do not secrete SF (scale=90 mm). (b) Higher power (60r objective) view of normal kidney. Proximal tubule cell SF uorescence intensity averages that of peritubullar capillary blood plasma (ne arrows) and is also higher than in the proximal tubule lumen. The distal tubule cells do not take up SF, and their nuclei stain brightly (blue) with the Hoechst dye 33342. SF is concentrated in the distal tubule lumen due to glomerula ltration of SF, secretion by upstream proximal tubule segments, and water reabsorption along the nephron (scale=30 mm). (Image courtesy of George Tanner, Indiana University School of Medicine.)
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embryos (White et al. 2001). Recently, Isogai et al. (2003) have used time-lapse multi-photon microscopy to image living Tg(i1 :EGFP)y1 zebrash embryos and to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Since protein expression patterns are a primary determinant of tissue function, a method to access macroscopic distances at subcellular levels of detail is of great interest. Jacobs and colleagues (2003) have shown how to combine two-photon microscopy with a novel image montage method (fast-beam blanking coupled with mathematical alignment tools) in order to extend the limited eld of view of laser scanning microscopes. These authors have shown the eectiveness of the combined approach to the analysis of the distribution of connexin-46, visualized across equatorial sections of the rat mammalian lens. Figure 29 shows an overview taken from the equatorial lens radius where gross changes in cell morphology with increasing depth into the lens are evident (Jacobs et al. 2003). 2PE has also allowed visualization of the lateral organization of cellular membranes. Gratton and colleagues (Gaus et al. 2003) have directly visualized the membrane lipid structure of living macrophages, providing evidence that the membrane coverage by lipid rafts and their uidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis. A ourishing of work (Arnulphi et al. 2005 ; Benninger et al. 2005 ; Sanchez & Gratton, 2005 ; Wang et al. 2005 ; Zoumi et al. 2005) in this direction makes it dicult to track all recent developments achieved by 2PE in this eld. A recent comprehensive review on the methods for detecting microdomains in intact cell membranes has also appeared (Lagerholm et al. 2005). 6.1.4 Applications to immuno-response New preparations, uorescent probes and imaging techniques are providing the means to observe the behaviour of cells in the tissue environment of lymphoid organs. When combined with two-photon laser microscopy, intravital imaging of surgically exposed lymph nodes provides a unique view of lymphocyte migration and antigen presentation as it occurs within the living animal. Cahalan et al. (2002, 2003), have reached relevant results that indicate that lymphocytes migrate randomly within lymphoid organs, and that lymphocyte contact with antigenpresenting cells may be a stochastic process rather than guided by chemokine gradients. These results have been conrmed and extended in a recent 2PE study of the interaction of the dendritic cells with T cells within intact lymph nodes in the absence of relevant antigen (Miller et al. 2004). In general the main advantage of TPE microscopy in the study of the immunological response lies in the possibility of performing in vivo observations as documented in recent experiments on the tracking of pathogenic myelin basic protein-specic CD4(+) eector T cells in early CNS lesions of experimental autoimmune encephalomyelitis (Kawakami et al. 2005). 6.1.5 Myocytes Time uctuations of the concentration of the reduced form of the nicotine adenine dinucleotide (NADH) have been studied by time-series of 2PE images of resting myocytes. These data (Blinova et al. 2004) suggest for the rst time that the cellular NADH uorescence is dominated by the sampling noise of the instruments and not signicantly modied by the biological processes. More conclusive studies on myocytes by 2PE have investigated the spatial distribution of intracellular sodium concentration in rat ventricular myocytes, showing that sodium diusion
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Fig. 29. Demonstration of the range of spatial scales encompassed by the composite image data. The sample is rat mammalian lens. (a) Overview taken from the equatorial lens radius, showing gross changes in cell morphology with increasing depth into the lens. (a) Five-fold magnication of the boxed region in (a) showing local cell morphology. (a) Five-fold magnication of the boxed region in (a) showing subcellular morphological detail. Images have been also taken (not shown) at distances of 500 mm and 700 mm into the lens respectively. Note that at this resolution the change in the expression pattern of labelled gap junctions (red) can be clearly identied. The arrow (c) indicates a small gap junction plaque on the narrow side of hexagonal bre cells. The extent of the displayed data in the horizontal direction is indicated above each image. All images were extracted from the same original data. (Adapted from Jacobs et al. 2003.)
in cardiac myocytes is slow with respect to trans-sarcolemmal sodium transport rates, although the mechanisms responsible are unclear (Despa et al. 2004). Moreover, Lindegger & Niggli (2005) compared UV and diraction-limited two-photon photolysis of caged Ca2+ in the investigation of Ca2+ release channels (ryanodine receptors, RyRs). These authors found opposite results when using UV or TPE photolysis inferring that changes of RyR open probabilities after b-adrenergic stimulation, enhancing local regenerativity and reliability of Ca2+ signalling. 6.1.6 Retina Imanishi et al. (2004) have recently established, by means of 2PE imaging, the presence of previously uncharacterized structures, very distinct from other cellular organelles in the retina. Besides this use of 2PE microscopy that has allowed in vivo experiments with unique insights in the function of these structures, Das et al. (2003) used TPE microscopy to reconstruct
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Fig. 30. Three-dimensional view of the mature sperm head of the octopus Eledone cirrhosa, loaded with DAPI, from 12 optical sections (Diaspro et al. 1997). (Courtesy of Silvia Scaglione ; image processing and visualization by Fabio Mazzone, Francesco Di Fato and Silvia Scaglione at LAMBS and BioLab, University of Genoa, Italy.)
cell divisions in living zebrash embryonic retinas. Even more recently, Niell & Smith (2005) showed how to provide controlled visual stimuli to larval zebrash, while performing twophoton imaging of tectal neurons loaded with a uorescent calcium indicator, allowing the determination of visual response properties in intact sh. 6.1.7 DNA imaging DNA imaging is possible by using UV excitable dyes whose uorescence can also be primed by 2PE. Figure 30 shows images of the helical structure of sperm heads of the octopus Eledone cirrhosa obtained by TPE of UV-excitable uorescent molecules. A 3D representation of the helical structure shown in Fig. 30, is realized by assembling 2D images from a stack of 64, 512r512r8 bits, taken 200 nm apart along the optical axis. The mature helical sperm head has a pitch of 06075 mm, an outer radius of 02503 mm, and is 43 mm long. From single 2D images it is not possible to get information about the spatial organization of the sperm head : the handiness, as in the confocal case, can be determined only using the completely assembled dataset (Diaspro et al. 1997; Difato et al. 2004). Of greater interest is the capability of TPE microscopy to track DNA in living cells. By the combination of two-photon and confocal microscopy, three main apoptosis parameters (the change of nuclear morphology, collapse of mitochondrial membrane potential, and increase of intracellular calcium) were recorded simultaneously for single As2O3-induced Molt-4 cells (Wang et al. 2004). In a recent review Errington et al. (2005) have discussed how to perform DNA imaging or tracking in living cells (for anti-cancer research purposes) by means of two-photon laser scanning microscopy. Tracking drug delivery in subcellular compartments, with the mapping of sites of critical
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drugDNA interactions was particularly addressed (Errington et al. 2005). Recently, dual-colour two-photon uorescence cross-correlation spectroscopy was used to detect single-stranded gamma tubulin DNA (Berland, 2004). Even if this work was carried out in solution, it constitutes the starting point for measurements and imaging in more complex environments such as within living cells. 6.1.8 FISH applications Konigs group has applied 2PE to multi-colour uorescence in situ hybridization (FISH) (Levsky & Singer, 2003). They performed 3D FISH to detect specic sequences within single DNA molecules in cells and tissues. They simultaneously excited the visible uorescence of a wide range of FISH uorochromes, such as FITC, DAC, Cy3, Cy5, Cy5.5, Rhodamine, Spectrum Aqua, Spectrum Green, Spectrum Orange, Jenuor, and Texas Red as well as of DNA/ chromosome stains, for example Hoechst 33342, DAPI, SYBR green, propidium iodide, ethidium homodimer, and Giemsa. In addition to the advantage of using only one excitation wavelength for a variety of uorochromes, multi-photon excitation provided the possibility of 3D uorescence imaging. The technology is of great impact in human genetics for the diagnosis of numerical chromosome aberrations and micro-deletions. In particular, Konig et al. (2000a) have obtained the intra-nuclear localization of FISH-labelled chromosome territories in interphase nuclei of amniotic uid cells by multi-colour 3D images. In the same work Konig and co-workers obtained optical sectioning of Hoechst 33342labelled DNA within living culture cells and within tissue of living tumour-bearing mice (Konig et al. 2000a) by using the high-light penetration depth at 770 nm. Figure 31 shows imaging of centrometric multiple uorescent probes in human biopsies (Konig et al. 2000b). 6.2 2PE imaging of single molecules 2PE has typically 02 mm radial resolution. As such it does not provide the resolution to image single chromophores and uorescent or labelled biomolecules embedded in entrapping matrices. However, when imaging sparse samples, one gets isolated uorescent spots that have the size of the PSF. We can ascribe those spots to single-molecule emission by performing a number of statistical checks on the amount of photons emitted, on their polarization (Ha et al. 1996 ; Bopp et al. 1998) and on the emission dynamics (Chirico et al. 2001a). Single-molecule optical spectroscopy by optical methods has recently acquired major interest for its unique ability to identify and characterize the properties of single molecular entities (Magde et al. 1972 ; Xie & Lu, 1999 ; Schwille, 2001). Here, we focus on single-molecule visualization (far-eld) using TPE-primed uorescence emission (Mertz et al. 1995 ; Sonneleitner et al. 1999 ; Diaspro et al. 2001). Following the pioneering work by Sanchez et al. (1997) on two-photon imaging of single rhodamine B glass immobilized molecules, spatially resolved applications of ultra-sensitive 2PE uorescence have shown promising results (Sonneleitner et al. 2000 ; Chirico et al. 2001a). Two basic issues of any type of single-molecule imaging are related to diminish the background signal, either residual scattering or uorescence, and to discriminate between the signals arising from single molecules and those that correspond to small molecular aggregates. The latter issue can only be tackled by following the time evolution of the uorescence emission of molecular aggregates, which may be degraded by the prolonged exposure to the exciting radiation as shown in Fig. 32. Moreover, one can discriminate single molecules from aggregates considering that

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Fig. 31. Imaging of centromeric uorescent probes in human biopsies by 3D two-photon multicolour FISH (Konig et al. 2000a). The localization of Spectrum Green (green colour) labelled C-4 probes, Spectrum Orange (red colour) labelled C-X probes and Spectrum Aqua (blue colour) labelled C-Y probes are depicted in a 10 mm thick kidney cryosection. The nuclear area was imaged after counterstaining with ethidium bromide (adapted from Konig et al. 2000b).
on an image the distributions of the pixel content show discrete peaks at levels that are all multiples of that corresponding to the dimmest spot revealed in the substrates (Fig. 33) (Cannone et al. 2003a ; Chirico et al. 2003a). TPE may also lead to an increase of the signalto-noise ratio for single-molecule detection, due to the tiny volumes (%01 ) obtained without the need of spatially ltering the emission signal as in confocal microscopy. A systematic study of single-molecule detection by 2PE has been reported on dyes (Pyrene, Rhodamine 6G, Fluoresceine and Indo-1) of increasing complexity. They are embedded in silica gels (Mozzarelli & Bettati, 2001) and illuminated by 770 nm radiation of a 100 fs, 80 MHz pulsed titanium:sapphire (Chirico et al. 2003b) through a scanning head. The uorescence output, collected with a 225 ms resolution, scales as the square of the exciting power (see lower inset of Fig. 34) and it is apparently constant until a sudden drop in the background level (Fig. 34). Due to a remarkable temperature dependence of this bleaching behaviour, the phenomenon has been interpreted as a temperature-induced structural change of the dyes due to the IR absorption that is not re-radiated into uorescence. Moreover, the bleaching rate computed on the single molecules scales as a power law of the excitation power with an exponent >2 (in the range 2126) depending on the complexity of the chemical structure (see upper inset in Fig. 34). Similar results were also found in solution by Patterson & Piston (2000). Since the uorescence output is always scaling with P2, the total number of photons that can be collected by 2PE uorescence spectroscopy is decreasing with the excitation power as Px05. This result indicates that the best observation conditions are obtained with very sensitive detectors and low excitation intensities. A second example that we report is the detection of the TPE uorescence of a single GFP mutant, called E2GFP (Cinelli et al. 2001) trapped in wet silica gels (Chirico et al. 2004). The typical image of GFP loaded gels, collected at 785 nm, is shown in the inset of Fig. 35. The uorescence kinetics are followed for%110 s and show a bleaching of the E2GFP molecule
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Fig. 34. Typical uorescence signal versus time for single molecules of (a) uorescein ( .), (b) Rhodamine 6G ($), (c) Indo-1 (%), and (d) Pyrene (m) at 67 mW of excitation power. Panel (a) (inset) : typical distribution of the bleaching time for uorescein. Panel (d) (inset) : uorescence signal (arbitrary units) per single molecule versus the excitation power. The average has been computed on at least 80 molecules. The symbols are as in the main panel. The data are tted to a law of the type AexcrP2. Panel (a) (inset) : average bleaching rate, CB, of Rhodamine 6G ($), uorescein ( .), Indo-1 (%) and Pyrene (m), versus the excitation power. The solid lines are ts to a power law, BrPC, with C varying from 21 to 26.
after%85 s. After 10 s of bleaching, the sample is scanned 40 times at 720 nm (open squares in Fig. 35) nding that the E2GFP has recovered uorescence (last lled square in Fig. 35). Imaging of individual molecules in a phospholipid membrane has also been obtained by TPE (Sonnleitner et al. 1999). Two-photon laser scanning microscopy at 700 nm was also used for detecting single Cy3-labelled lipids on the plasma membrane of living human coronary artery smooth muscle cells (Sonnleitner et al. 2000). Fluorescence images of cells, taken with a mean power of 6 mW and a dwell time of 50 ms per pixel, showed peaks from single Cy3-labelled lipids diusing on the cell membrane. This work clearly demonstrates that single-molecule detection with 2PE is also possible on cells in vivo. This has opened further perspectives to utilize the advantages of 2PE, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multi-colour experiments, for single-molecule imaging in living biological cells (Harms et al. 2001, Heinze et al. 2004). Finally we cite a recent TPE investigation of the unfolding of a GFP mutant (GFPmut2) performed at the single-molecule level (Cannone et al. 2005). In this case the TPE emission has been discriminated in two acquisition channels that correspond to the emission of the neutral and the anionic form of the GFP chromophore. The complex interplay of these two
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Fig. 35. Fluorescence kinetics of a single E2GFP molecule embedded in a silica gel. The illumination is at 785 nm, average power=4 mW for the rst 95 s. Then the image is scanned at 720 nm, average power 4 mW, for 10 s (open squares). The image is then scanned once more at 785 nm, average power 4 mW (last lled square). Inset : typical uorescence image of a 15r15 mm2 eld of E2GFP silica gels.
conformational/protonation states in the unfolding and refolding kinetics has been investigated in detail, showing that subtle memory eects are taking place in the unfolding of GFP. Even more recently a high time resolution (10 ms) investigation of the TPE-primed emission of the same mutant has shown oscillations in the GFP emission a few tens of milliseconds before the unfolding event (Baldini et al. 2005). The two emission levels correspond to the two protonation states and the characteristic frequencies of the oscillatory behaviour lie in the 4001000 Hz range. More notably the oscillations can be driven by both electric and acoustic elds (Baldini et al. 2005). 6.3 FCS applications The possibility of coupling FCS to scanning microscopy is extremely attractive as a sensitive method of probing local diusion in cells and in vivo, and to distinguish Brownian diusion from active transport phenomena (Schwille, 2001), which are common in cells. The 2PE version of FCS takes further advantage of the low background of the IR excitation in the cell and from the possibility of simultaneously exciting widely dierent chromophores. This peculiarity of TPE makes it ideal for applications in uorescence cross-correlation experiments as documented by the eort of Schwilles group (Heinze et al. 2000 ; Haustein & Schwille, 2004 ; Kim et al. 2005 ; Jahnz & Schwille, 2005). It must be noted that FCS is in itself a highly sensitive technique not free of artefacts and particular care must be taken in the choice of the uorescent probe, which could display dierent photophysics when excited by 1PE or 2PE, or could interact with immobile cellular structures (Schwille et al. 1999a, b). The actual extent of the advantages of 2PE in FCS, for example in limiting the sample photodamage in the cellular environments, have been investigated by Gratton and co-workers among the rst groups (Berland et al. 1995, 1996). In particular, Berland et al. (1995) applied these techniques to characterize the intracellular environment by measuring the diusion coecients of uorescent beads, 7 and 15 nm in diameter, injected into the cytoplasm of

08 Buffer Cytosol Membrane IgE receptor D = 3 1010 cm2/s 04
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Fig. 36. Various autocorrelation curves demonstrating the enormous dierence in motility between buer solution and cytosol. (Courtesy of Petra Schwille and Elke Haustein, MPI Gottingen, Germany.)
mouse broblast cells. Figure 36, taken from Schwille (2001), clearly shows the dierent motility of uorophores in dierent cellular compartments. The decay times of the ACFs vary by several orders of magnitude, with diusion coecients that span a range 3r10x6 cmx2 sx1 <D<3r10x10 cmx2 sx1, when rhodamine derivatives are studied in buer solutions, cytoplasm and cell membrane. Thus far, one of the most interesting potentialities of intracellular FCS is given by the possibility of discriminating between the random motion and the active transport. An example of this type of study that took great advantage of 2PE, dealt with the plastid stroma in vascular plants. GFP was targeted to the plastid stroma and revealed tubular structures that are able to interconnect separate plastids (Ko hler et al. 1997). In order to better elucidate the role of these structures, it was essential to quantify the velocity of the proteins moving therein and to assess the type of motion, either random or active. In fact, a combination of TPE FCS and microscopy revealed that the GFP motion was indeed an active transport that did not involve single GFPs, but rather larger patches with vesicular structure (Ko hler et al. 2000). This study has taken advantage of 2PE due to better background suppression as found in other applications to plant cells (Schwille et al. 1999a, b). Schwille and co-workers have recently presented a method to determine the ow parameters, velocity and direction, in microstructured channels by TPE FCS (Dittrich & Schwille, 2002). The highly restricted detection volumes and low scattering background, typical of TPE, are shown to be particularly valuable for measurements in tiny channel systems. Moreover, these authors employed a dual-beam cross-correlation technique to discriminate between isotropic and anisotropic dynamics. The study of the heterogeneity of diusion inside microbial biolms (Gulot et al. 2002) also takes advantage of the potentiality of 2PE FCS on conned cellular environments. In this case Gulot et al. have studied the penetration and diusion capabilities of uorescent probes of dierent size and electrical charge in models of mono-microbial biolms. FCS and, in particular its application to TPE, can also be a valuable tool for studying aggregation as demonstrated by Grattons group on the Crotalus atrox venom phospholipase A2. In this case the advantage of FCS lies in the possibility of investigating low concentrations, thereby allowing measurement of tight bindings. Sanchez et al. (2001) determined a dimer dissociation constant Kdf001 nm for the Crotalus phospholipase A2.
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Grattons laboratory has also recently presented a detailed study on the possibility of applying PCH analysis to the study of the motility of proteins in cellular compartments (Chen et al. 2002). These authors have characterized the molecular properties of autouorescence and transiently expressed enhanced green uorescence protein (EGFP) in the nucleus and in the cytoplasm of HeLa cells both by FCS and PCH analysis. Because the concept of molecular brightness is crucial for PCH analysis, they have determined the statistical accuracy of its measurement under in vivo conditions, nding a molecular brightness of EGFP that is a factor of 10 higher than the brightness of the autouorescence. The molecular brightness of EGFP measured in the nucleus, the cytoplasm, and in vitro are identical and one of the main conclusions of this study is that molecular brightness is a very stable and predictable quantity for cellular measurements. Further recent applications of 2PE FCS to the study of molecular diusion in cellular and solution environments have appeared. Ruan et al. (2002) fused cytosolic adenylate kinase (AK1, a ubiquitous enzyme) and its isoform (AK1b) with EGFP and expressed the chimera proteins in HeLa cells. By using TPE scanning uorescence imaging, Ruan et al. were able to directly visualize the localization of AK1-EGFP and AK1b-EGFP in live cells and to detect dierent diusion properties of the two isoforms depending on their location within the cytoplasm and on the plasma membrane (Ruan et al. 2002). Besides the possibility of investigating molecular motion, FCS is also capable of detecting the internal photodynamics of the uorophores and assess how it is aected by the dierent environments. The Webb group has thoroughly investigated the photodynamic properties of mutants of GFP in solution (Haupts et al. 1998 ; Heikal et al. 2001) and also in cells (Schwille et al. 1999a, b). The internal photodynamics of GFP was investigated at dierent pH, with one- and two-photon excitation, nding two fast blinking processes. Only the fastest one was sensible to the pH and was ascribed to the protonation of the chromophoric moiety of GFP, while the second was attributed to a cis-trans conformational change as also conrmed by theoretical investigations (Voityuk et al. 1998). Further characterizations of the heterogeneity of the uorescence emission of proteins by means of two-photon correlation spectroscopy have appeared for mutants of GFP, Coral Red protein (DsRed) (Heikal et al. 2000) and O-acetylserine sulfhydrylase (Chirico et al. 2001b). The FCS method has also been used recently to measure the very slow diusion of receptor clusters in cultured cells (Srivastava & Petersen, 1998). Additionally, a two-colour image crosscorrelation spectroscopy (ICCS) variant has been used for co-localization studies at the plasma membrane of inuenza virus (Brown et al. 1999). A pioneering work on TPE ICS in cells appeared in 2000, where the possibility of TPE co-localizing and simultaneously exciting two dierent uorophores has been exploited to perform image cross-correlation (Wiseman et al. 2000). Finally, a recent application of 2PE related to molecular diusion consists of an interesting blend of imaging and un-caging properties of the phenomenon. Reported for the rst time, is the experimental evidence of 2PE photolysis of a caged proton compound, 2-nitrobenzaldehyde (o-NBA), monitored using a new sensor system that utilizes uorescent-labelled nanocapsules, i.e. a uorescent nanostructured shell of micrometric size and nanometric thickness (Diaspro et al. 2003). The photo-labile compound undergoes one-photon absorption in the UV range (200380 nm), and the mechanism that leads to proton release is based on the well-known 2-nitrobenzyl photochemistry, which has been used for many photoactivable-caged compounds. Because the use of UV excitation can cause biological damage, the multi-photon un-caging

(a) (b)
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Fig. 37. Two-photon excitation images of FITC-doped polyelectrolyte nanocapsules [(PAH/PSS)3, 3rd and 5th layer FITC-labelled]. Fluorescein emission was excited at a wavelength of 720 nm, in the presence of 2-nitrobenzaldehyde, before (a) and after (b) 2-nitrobenzaldehyde photolysis that was induced by highenergy scanning with multi-photon excitation at 720 nm. The decrease of uorescence intensity monitors the uncaging eect (Diaspro et al. 2003).
process is preferable. Using a femtosecond titanium:sapphire laser operating at 720 nm, with a pulse width of 200 fs at the sample, delivered through the 2PE microscope (described in Section 5), and a 1-min exposure time with high power (4550 mW), an appreciable photolysis of 2-nitrobenzaldehyde was obtained (see Fig. 37). One of the relevant aspects in this work is the combined utilization of 2PE both for imaging and for an active role like that of the highly localized un-caging agent. 6.4 Signals from nonlinear interactions SHG is a second-order nonlinear optical process that is conned to regions that lacks a centre of symmetry. Interested readers can see the brilliant recent review by Campagnola & Loew (2003) about this important nonlinear technique. The origin of SHG is the dissipation of power in the sample due to a quadratic response of the microscopic currents to the electric eld. It is a coherent process reminiscent of the single-photon Rayleigh scattering and, therefore, intrinsically dierent from 2PE uorescence where a quantum absorption occurs, rapidly followed by emission of radiation. Since SHG is a nonlinear optical process, the microscopy application of this technique retains the 3D capabilities of 2PE. Several additional aspects make SHG microscopy very powerful. Since the excitation uses NIR wavelengths, this method is well suited for studying intact tissue as with 2PE. Moreover the SHG signals have well-dened polarizations, and SHG polarization anisotropy can be used to determine the absolute orientation and degree of organization of proteins in tissues, and to enhance the image contrast. Since SHG does not arise from absorption, in-plane photodamage is greatly reduced. In addition, 2PE uorescence images can be collected in a separate data channel simultaneously with SHG. Despite these attractive properties, SHG has only recently been used for biological imaging applications (Campagnola et al. 1999 ; Moreaux et al. 2000 ; Zoumi et al. 2002 ; Diaspro et al. 2002c ; Zipfel et al. 2003). A powerful advance is that obtained in coupling 2PE and SHG imaging on the very same detection optical path, involving dierent contrast mechanisms, to get
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Fig. 38. Colour-coded image of immunostained collagen bres in the RAFT. The image is an overlay of two images obtained from the same sample site by using an SBG39 (322654 nm) wide-pass emission lter and a 520/40 nm bandpass emission lter for 800 nm excitation, and P=60 mW. Collagen bers are shown in cyan, and the spotted staining pattern of the antibody is shown in yellow (bar, 3 mm). (Adapted from Zoumi et al. 2002.)
complementary information regarding biological system structure and functioning. 2PE uorescence is generally measured in epi-illuminating geometry but the forward propagating nature of SHG seemed to restrict SHG microscopy to the transmission detection mode. Zipfel and colleagues (2003a) achieved multi-colour nonlinear microscopy of living tissue using 2PE and 3PE intrinsic uorescence combined with SHG by supermolecular structures producing images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provided crucial physiological information. They demonstrated applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimers disease and cancer. In vivo SHG signals of intrinsic components were generated by scanning small regions of the specimen. Since collagen has been demonstrated to be a very eective upconverter of light by SHG there is a growing number of applications in biomedical imaging. Dierent types of collagen can be distinguished at low perturbation and without specic sample preparations (Roth & Freund, 1979 ; Cox et al. 2002; Zoumi et al. 2002) Figure 38 illustrates a colour-coded image, which displays SHG signals in cyan originated from type I collagen and antibody uorescence in yellow revealing that the bres in the raft (membrane microdomains enriched in cholesterol and glycosphingolipids) correspond to collagen (Zoumi et al. 2002). The fact that SHG is collected in the forward direction, due to its physical inherent properties, hampered potential experiments especially in thick or in optical congurations where it is not possible to place forward detectors. Recently reected SHG signals were collected from the Tromberg and Diaspro groups (Zoumi et al. 2002 ; Diaspro et al. 2002c) opening new application perspectives. Mohler and colleagues (2003) have recently shown how structural protein arrays
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consisting largely of collagen, myosin, and tubulin, and their associated proteins can be imaged in three dimensions with high contrast and resolution by laser-scanning SHG microscopy. In particular, structural protein arrays that are highly ordered and birefringent produce large SHG signals without the need for any exogenous labels. The authors, combining SHG with TPE GFP imaging were able to infer the molecular origin of the SHG contrast in Caenorhabditis elegans sarcomeres. From the Laboratoire de Neurophysiologie et Nouvelles Microscopies (CNRS, Paris, France), the combination of 2PE and SHG allowed a new outstanding possibility of fast-targeted control of the local inter-leaet distribution of markers in biological membranes. In fact, Pons et al. (2002) demonstrated an interesting mechanism of localized photoinduced ipop of stilbazolium markers in model lipid bilayer membranes. The ip-op mechanism and dynamics were determined by combined 2PE uorescence and SHG microscopy. Upon illumination of labelled membranes with a femtosecond laser beam, two-photon absorption induced photoisomerization, with a signicant increase in the cis marker population, whose ip-op rate was determined to be at least 1000 times faster than that for trans markers. Other interesting applications of high-order optical generation have appeared recently. Thirdharmonic generation can also be exploited to form images without the need of uorescent markers (Mueller et al. 1998 ; Squier et al. 1998). Moreover surface-enhanced second-harmonic generated (S-SHG) signals have been detected from granular gold structures exhibiting local plasmon resonance (Anceau et al. 2003). Here, a TPE microscopy technique was used to perform high spatial resolution S-SHG imaging. Polarization measurements of local S-SHG responses revealed eld anisotropy in the enhancement regions and, furthermore, proved the incoherent and strongly depolarized nature of the emission, which is attributed to ultra-fast uctuations of the enhancement location. 7. Conclusions Considering its impact on basic and applied research, including new diagnostic methods, the development of 2PE and multi-photon excitation imaging may be viewed as a great revolution for a new era in optical microscopy. Since 2PE broke into the scientic arena, it has grown in an unpredictably fast way, and has generated and still produces a tremendous number of related techniques. New experiments can be designed and performed and comparing one- and twophoton experiments can perform a critical reading of past results. It is true that a few impediments exist to the understanding and full exploitation of 2PE. One of the major limitations in this direction is the diculty of predicting or measuring two-photon absorption spectra of the uorescent molecules used. A further extremely practical disadvantage of 2PE resides in the cost of appropriate laser sources. The expectation is that their use will probably increase as cheaper and more reliable laser sources are developed. Furthermore, one should consider that in-plane optical resolution for 2PE is lower than in the conventional excitation case even if the overall signal-to-background ratio is larger (Jonkman & Stelzer, 2001). Photobleaching and photodamage can be greater than in 1PE at least in the focal plane (Drummond et al. 2002) indicating that the technique is better suited for thick samples than thin ones within this context. A precise control of the inuence of pulse duration on imaging eciency and potential photodamage would require an accurate control of pulse width in the focal plane that is not easy to accomplish on-line in an intensive utilization of the 2PE microscope (Koester et al. 1999). Finally, due to high peak powers, 3PE or plasma generation can aect imaging of biological samples (Konig et al. 1999b ; Koester et al. 1999), and in pigmented samples,
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single-photon absorbers such as haemoglobin, melanin or chlorophyll can cause damage through heating (Tauer, 2002). Despite these potential drawbacks 2PE and related techniques can be considered as a mainstay of the modern biological research milieu and a bright perspective in optical microscopy. In fact, 2PE brings an intrinsic 3D resolution, the absence of background uorescence, and the attractive possibility of exciting UV-excitable uorescent molecules at IR wavelengths increasing sample penetration. In 2PE, the uorescence emission occurs at a wavelength substantially shorter than the excitation wavelength, therefore reducing Rayleigh and Raman scattering contribution and increasing the signal-to-noise ratio. This fact makes single-molecule experiments, including spectroscopic ones, easier than in the conventional excitation case. On the nanotechnological side, 2PE architectures can be easily turned into active devices combining their 3D imaging ability with the possibility of performing destructive localized actions. Moreover, a further benet in establishing a 2PE optical system is the exibility in choosing the measurement modality favoured by the simplication of the optical design. As a matter of fact, a 2PE microscope oers a number and variety of measurement options without changing any optics or hardware. This means that one can really get multi-modal and multi-functional information within the same experiment from the very same sample. Finally, it should be remembered that photobleaching and natural uorescence are strictly conned in the focal volume of event, and multiple excitations of uorescent molecules can be more easily and eciently accomplished than by using conventional excitation. For these reasons, 2PE is rapidly becoming an essential imaging system for thick samples and live cells, even within their own organs or tissues.
8. Acknowledgements The authors are grateful to Alessandra Gliozzi, Giancarlo Baldini, Salvatore Cannistaro, Cristiana Ricci, Paolo Sapuppo and Enrico Gratton for believing in the 2PE project. A. D. is personally indebted to several scientists who provided helpful comments for the realization of the 2PE set-up, among them Enrico Gratton, David Piston, Tony Wilson, Colin Sheppard, Karsten Konig, Joseph Lakowicz, Weiming Yu, Steve Potter, Mary Dickinson, Martin Hoppe, Rolf Borlinghaus, Irmtraud Steinmez, Cesare Usai, Hans Gerritsen, Jerey Larson, Stan Schwartz, Peter So, Min Gu, Ammasi Perisamy, Fred Brakenho, Barry Masters, Michael Stanley, Osamu Nakamura, Stefan Hell, Christian Soeller, Marc Cannel. Special thanks are due to John Girkin for providing data on laser sources and important comments. All the users of the MPLSM (mplsmusers@yahoogroups.com) and Confocal (CONFOCAL@LISTSERV.BUFFALO.EDU) mailing list are acknowledged. The authors are indebted to their co-workers at LAMBS and LABS, namely (in random order) : Mario Faretta, Paolo Bianchini, Silke Krol, Fabio Cannone, Silvia Scaglione, Raaella Magrassi, Laura DAlfonso, Francesca Bertani, Mirko Corosu, Federico Federici, Sabrina Beretta, Alessandro Esposito, Cesare Usai, Paola Ramoino, Francesco Difato, Andrea Gerbi, Valentina Caorsi, Davide Mazza, Giuseppe Vicidomini, Mattia Pesce, Ilaria Testa, Marc Schneider, Munish Chanana, Francesca Cella, Federica Morotti, Emiliano Ronzitti. All the authors are indebted to their respective families. Special thanks are due to Brad Amos, Davide Neuhaus and Andrea Cranton. A. D. dedicates this work to the memory of Osamu Nakamura, and all the authors to Gianfranco Menestrina. The TPE project is performed under the auspices and grants of the National Institute for the Physics of Matter, Italy. Main sources of grants are the Fondazione San Paolo Torino, RTN Nanocapsule EU, IFOM and MIUR-FIRB grants.
2PE in biological microscopy 9. References

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Quarterly Reviews of Biophysics 38, 2 (2005), pp. 97166. f 2006 Cambridge University Press doi:10.

Two-photon fluorescence excitation and related techniques in biological microscopy

A. Diaspro, G. Chirico and M. Collini

8. Acknowledgements 9. References 155

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

S* 1020 nm 680 nm 340 nm 1020 nm 420 nm

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

z Important parameters Resolution: x, y, z

Position: x, y, z 2 Quantity: n2 n2 photons Probability of two-photon absorption

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

Output fluorescence rate (kHz)

A. Diaspro, G. Chirico and M. Collini

C1P 117 401

C2P 134 462

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

100000 C = 100 f M 10000 1000 Rate (Hz) 100 C = 1 pM

100000 10000 1000

100000 10000 1000 100

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

31 21 10 10 21 31 x (m) (c) 52 42 y (m) 31 21 10 10 21 42 52

0 002 005 007 010 012 015 017 020

6318 y80 y3 y20 y35* y2538* 3897 124 2106 y2 21055

A. Diaspro, G. Chirico and M. Collini

Coumarin xs Cascade Blue xs Lucifer Yellow xs Rhodamine xs

0001 (b) 1000

Ca-Orange wl Fluo3 xs Fura Ca xs

1 690 01 001 0001

Indo free xs Fura free mean xs

2PE in biological microscopy

30 20 10 0 750 10 05 00 1000 1050 1000

35 30 OPE (1015 cm2) 2PE (GM) 25 20 15

0 750 800 850 900 950

00 1000 1050 1100

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

Table 4. Collection of transmission data for Nikon CFI60 series of objectives

Table 5. Optical dispersion parameters for dierent glass types

2PE in biological microscopy 5.4 Laser sources

126 A. Diaspro, G. Chirico and M. Collini

Table 6. Laser sources for 2PE

Nd :YLF Nd :Glass Ytterbium Cr :LiSAF OPO

Not presently available

2PE in biological microscopy

A. Diaspro, G. Chirico and M. Collini

2PE in biological microscopy

Tsunami Variable N.D. filter 1P laser module

PCM2000 controller EZ 2000

A. Diaspro, G. Chirico and M. Collini