603 Multiphoton microscopy in biological research Rebecca M Williams*, Warren R Zipfel and Watt W Webb. From its conception a decade ago, multiphoton microscopy has evolved from a photonic novell to an indispensable tool {or gleaning information from subcellular events within ‘organized tissue environments Its relatively deep optical penetration has recently been explolted for subcellulaly resolved investigations of disease models in ving transgenic mice. Ils enhanced spectral accessibilty enables aberration- free imaging of fluorescent molecules absorbing in deep-UV ‘energy regimes with simultaneous imaging of species having ‘extremely diverse emission spectra, Although excited fluorescence isthe primary signal for multiphoton microscopy, harmonic generation by multiphoton scattering processes are also valuable for imaging species with large anharmonic modes, such as collagen structures and membrane potential sensing dyes. Addresses ‘Appled and Engineering Physics, Corel University, thaca, NY 14853, USA ‘ema: 36@correll ed Current Opinion in Chemical Biology 2001, §:603-608 1367-5981/01/8 — see tron matter © 2001 Elsevier Science Lid. Al rights reserved ‘Abbreviations 1PE —one:photon excitation 2PE —_twophoton excitation SPE —_tvee-photon excitation GFP green fuocescent protein MPE —multphoton excitation MPM. multiphoton mecroscopy SHG Second harmonic generation Introduction Theoretically predicted in 1931 [1], multiphoton excitation (MPE) is based on the probability that multiple low-energy photons can arrive ‘simultaneously’ at a fluorophore and through interactions with it, induce an electronic transition normally excited by a single high-energy photon, For exam= ple, simultaneous absorption of ewo red photons ean excite Jar UV transition, The advantages of multiphoton over standard lincar microscopy stem primarily from the spatial isolation of this excitation event. MPE is highly restricted t0 the sub-micron-sized (with the best resolving, optics) volume at the microscope focus. Multiphoron microscopy (MPM) is accomplished by raster-scanning this, tightly focused beam within a specimen and chereby opti cally selecting an axially isolated plane of fluorescence information [2]. Ultimately, chis attribute enables deeper imaging into optically thick tissue, while restricting photo bleaching and photoxicity to the area being imaged. Primacily because of the development of robust ulteafast (pulsed) lasers (most commonly the ‘Ti Sapphire or Ne:YLF) and measurements yielding an MPE photophys- Jeal database for common bio-indicacors, MPM has now Figure 1 Lateral) and Asal (0) view ot plane of photoactivateable ‘iuorescain uncaged using a two-photon process. The cross-sectional \eew () shows that a rastor-scanned beam can ‘select an isolated ‘ce within the specimen, become a technically tractable tool for investigating com= plex biological problems. Here we review the main physical tenants of MPM, focusing on recent results in biological imaging, Sclected comprehensive reviews in the area [3-7] provide greater technical depth. Deeper imaging with less phototoxicity Because MPE fluorescence originates only at the beam focus, no out-of-focus background is produced. Confocal: like optical sectioning ean be achieved without confocal detcevion optics (Figure 1), Detectors for MPM are thus designed primarily for maximal fluorescence collection Even the fluorescent haze produced by particularly strong scattering biological specimens isnot background bur signal, because its ori to the appropriate image pixel by a clock timing the scanned bi he would have t© be mistouted by hundreds of meters in order to be temporally misplaced for typical microsecond pixel dwell times.) n within the focal plane is assigned ‘The penetration depth of confocal microscopy is limited o bi Because MPM can collect and utilize this scattered signal, logical tissue-scartering lengths of ~100m [8]604 Analytical techniques it offers clear images 2-3 times deeper than confocal microscopy [9]. This penetration advantage ean be further accentuated when chromophores, such as hemoglobin or pigments, are present at high enough concentrations 10 atvenuate a standard single-photon beam before it reaches the focal plane. Deleterious out-of-focus. absorptions, photobleaching and phototoxicity are all significantly alleviated with nonlinear excitation, I is these properties that have made MPM an essential tool for observing cellular events within the tissue envi- which they occur, It has been fully adopced by neuroscientists, who have utilized MPM to examine calei- tum [10%,11-13,14°,15-16] and sodium [17] dynamics and neuronal plasticity [18,19] in a multitude of intact brain preparations. ‘The extea penetration chat MPM affords is crucial because surface preparation artifacts such as dam- aged cells extend >70 sm into the brain slice interior (for example, in hippocampal slices, see [20)). Amazingly, MPM has been successfully extended 0 live mice for investigating such diverse processes as neuronal dynamics 2 ], Alzheimer’s plaque progression [24,25*), and tumor biology and growth [26"]. With everexpanding, resources being put towards transgenic animal develop- ment, the utility of MPM as a phenotyping tool will only hecome more important Enhanced spectral accessibility and flexibility ‘The optimal wavelength for MPE with # photons might be expected to be m times the single-photon absorption maximum. For example, one would expect two-photon excitation (2PE) of rhodamine at 1140 nm. For symmetric molecules, however, the quantum parity selection rule reverse berween one-photon excitation (IPE) and 2P processes. Thus, molecular states that are accessible with IPE. may not be accessible with 2PE and vite versa, especially for small aromatic molecules. (For tabulated examples sce [27].) Even for larger dye molecules, two- photon absorption spectra are often significantly different from their one-photon counterparts, though emission spec tra are in general identical [5,28,29]. Thus armed with the MPE spectra for standard biological fluorophores (ion indi- DNA and organelle probes, green Auorescent proteins [GFPs], ete.), one can often design experiments in which em spectrally shifted by hundreds of nanometers can be multiphoton excited simultaneously with a single wavelength. For example, the two-photon absorprion maximum for rhodamine is actually. 700 nm, close to that of DAPI (a DNA stain), which enables simultaneous imaging of both species. Vastly different color species can also be imaged using different order processes, for example three-photon excitation (SPE) of UV-emitting, nin with 2PE of a visible-cmitting dye [30*]. ‘Thus, llows researchers broad flexibility for various indica- tor combinations, which has been crucial for studies of secretion [30*.31], NADH metabolism [32], lipid phases [33] and cholesterol uptake [34], and for imaging multiple DNA hybridization targets in situ [35]. In one-photon confocal microscopy, UV excitation is fraught with materials problems such as les and low transmissivities, Unfortunately, U tend to be good performers; they are ly leys obtrusive. Although the development of visible-light indicators is a commercial priority, some probes, such as DAPI, Fura, glutathione ‘coumarins, still function better than theie visi competitors. In addition to synthesized dyes, the abil image intrinsic tissue fluorophores (for example, NADH, clastin and retinol) or introduced aromatic pharmaceuticals, (for example, tetracyclines, aminosalicylie acid and porphyrin-derived photosensitizers) requires UV In MPM, the red or infra-red light used to excite these ccnenies yields clean, aberration-free images. Perhaps the ‘most dramatic evidence of this increased probe ace ty is SPE experiments in which serotonin, normally excited in the deep-UV (<300 nm), can be imaged to identify and charseterize secretory geanules [36]. These cells are viable and release upon receptor-linked stimulation [37*]. So far, only indoleamines have been visualized in cells, but with the appropriate collection opties, the technique could be extended to the higher-energy-absorbing, eatecholemines, such as dopamine, norepinephrine and epinephrine. aberrations c Muorescent sibili- Working in energy regimes in which UV fluorophores are more accessible, however, comes with an associated cost Exciting intrinsic tissue chromophores, many of which possess extremely low quantum yields (for example 0.02 for NADH [38), requires unwanted energy input into the specimen. Clear enhancements in cellular viability with MPM over one-photon confocal microscopy have been shown only at MPE wavelengths greater chan 800 nm 14,39°40-41]. When performing MPM in the UV excita- tion regime, it is eruefal ro determine the levels at which the illumination begins to compromise the cellular func tion being monitored. With wavelengths fess than 750 nm, typical mode-locked pulses (100 fs at 80 MHZ), high NA optics, s beam dwell times and 10° frame intervals, safe operating conditions consist of specimen powers of a few mW [30°,32,37"]. At levels in which cell funerion begins to be perturbed, researchers generally agree that phototoxici- ty effects are caused by two-photon processes [41,42 which implies that for two-photon imaging the pulse width (D is not a critical parameter. With longer pulses simply increases the average power (P) such that P2/t is constant [5] to maintain the same imaging signal level. The resulting increase in the ratio of IPE to 2PE absorprion processes within the specimen is insignificant because the 2PE. processes ultimately limit cell viability. (The same conclusion is not true for 3PE imaging optimization. See [S7*].) We believe that this issue is somewhat confused by a recent publication suggesting a greater than two-photon dependence of phototoxicity [43], in which excitation doses (squared power « dwell time) were ~1000-fold high- cer than those previously identified as safe, Most ultrafast laser sources ean deliver a hug cells, and higher-order processes, by definition, will xcess of power to isolatedMultiphoton microscopy in ological research Wiliams, Zipfel and Webb 605 become more accentuated at higher powers. It should also be noted chat damage assessment procedures indicated by cell membrane leakiness occur at doses that are orders off ‘magnitude higher than chose indicated by cellular funetion (J Nichols, RM Williams, WW Webb, unpublished data). Nonlinear scattering microscopy An MPM apparatus can casily be rctiofitted to collect image information from # photon scattering processes, which generate harmonics of the laser frequency. In harmonie generation, multiple photons simultancously interact with non-centrosymmettic st tise there is no energy loss, these processes emit radiation at exactly Ifa of the exciting wavelength, Second harmonic generation (SHG) and third harmonic generation have been used in laserscanning microscopy 0 image membranes [44,45%46] and plant structure [47], respectively: jetures without being absorbed. Bet Because harmonie generation is a coherent process (seat tered photons maintain phase information), the scattered beam must satisfy phase-matching constraints and thus mits highly directed radiation. When using a fo beam (with a focal volume smaller than the specimen being imaged), these phas. strained by the Gouy phase anomaly and SHG forward atan angle that is approximately one half the max- imum angle of illumination, as determined by the NA of the microscope objective [45°46]. This general emission pattern is also dependent on the normal modes of the scatterer. For disordered, fluctuating nonlinear seatterers, the emitted “hyper Rayleigh’ radiation field, is more diffusely oriented [48] ised A tremendous advantage of SHG-MPM for investigating, disease models in tissue is its ability to yield high-resol tion images of unstained collagen structure (Figure 2).'The principle nonlinear signal from illuminated collagen is not fluorescence [49-51], bur almost entirely SHG. Because collagen is chiral, it contains anharmonic electronic normal ‘modes that vield a strong SHG radiation field, whose mag- nitude and direetionality are intimatel dependent upon the collagen structure at nanomokae sp: 2]. For in vivo tissue imaging, collecting forward propagating signal is cleaely not practical. Auspiciously and somewhat unexpectedly, a significant amount of collagen SHG propagates backwards. We believe that this is probably the result of a nonlinear enhancement in backward scattering, from wavelength-scale objects [53]. In tissue, this effect will significantly enhance MPE fluorescence collection in the epi mode as well [54]. seales Localized photochemistry and bioassays MPM was originally conceived as a tool for spatially resolved activation of “caged” bioactive molecules by photochemical release from an inactivating, chromophore ‘egeate micron-seale chemical stimuli [2,55], such as chose Within the synaptic cleft. MPE uneaging experiments have Figure 2 [SHG microscopy of colagen structure within a human skin explant ‘The scale bar s 20 =m been and remain somewhat stifled by the lack of caging, ‘groups that release faster than the microsecond diffusion times characteristic of micron-sized spatial scales [56%,57] However, the three-dimensionally defined nature of MPE has been exploited in a variety of other photochemical ‘measurements, manipulations and bioassays. MPE is ideally suited to the detection of minute quantities of biomolecules t ‘mens such as human blood SI, particularly in the presence of optically dense speci | Ic has enabled dilute molecule dynamical measurements based on fluorescence fluctuations to be performed in open volumes [61], with sev ceral labels [62,63] and in living. cells [64,65*,66]. Similarly the use of MPE in fluorescence photobleaching recovery ‘measurements [67*,68] has enabled diffusion measurements in tissue environments as diverse as plants [69] and brain slives [70]. Technical advances MPM techniques initially exploited the technological infrastructure generated around confocal microscopy. However, because background rejection is. unnecessary MPE microscopes are now being designed with detection pathways that are optically simpler and more compact than605 Analytical techniques their confocal counterparts [3,6,71]. Video rate MPMs have been constructed using resonant galvanometers [72] oF by illuminating many spots simultaneously using a microlens array [73,74]. Studies are also underway to develop probes optimized for MPM, with high nonlinear absorption eross- sections [45*,75,76] and excitation energies not limited by the usual one-photon laser illumination wavelengths. GFP. mutants, which provide the means for labeling, selected _zene products by transfection or by transgenic mouse tech- nologies, work well with MPE [21°,26*,29] Similarly, MPE photophysical measurements of a new red fluorescent [77] show a very large ewo-photon cross section, among other interesting properties. Conclusions MPM provides a specific new capability in_ biological imaging; it offers subcellularly resolved fluorescence and harmonic generation relatively deeply into living speci mens. We anticipate exciting new results from future integration of diverse imaging technologies that can pro Vide information from several vastly different perspective: For example, MRI or ultrasound, with deeper penetra but orders of magnitude less resolution, may be used 10 locate regions of interest or provide an important larg scale context or guidance to acquisition of the localized cellular information gleaned with MPM. Conversely, after recording dynamical information with MPM, a specimen can be fixed and sectioned for attaining the nanometer. scale information offered by electron microscopy. With increased optical penetration into seattering specimens, increased spectral accessibility and the potential for decreased phototoxicity, MPM has shown great promise for imaging cells and tissue explants to whole animals. With the developmental explosion of smart fluorescence indicators, many of which can be incorporated into the genome for specific gene product labeling, we expect MPM to become a standard cool for determining the molecular mechanisms of cell-based processes in basic biological research, tissue engineering and_ transgenic mouse models for disease and development. Acknowledgements The authrsthatk Ein Shects for sever erful reviews of his mmansrge This peon nos mde pie by Grant Numb Pt INDI fom she Natl Cer Resaeh Recursion instar of eh References and recommended reading Popes of pater rest. published wn fhe anual eran of reve rave been night os: of special interest of oustanding interest 1. Gopportayor M: Uber Elomontarakte mit wel (Guartensprungen. Ann Phys (Leiwig) 1931, 0273-208. (Te translation: About elementary events mit two quanta jumps 2. Denk W, Stickle JH, Webb WW: Two-photon laser ecanaing fluorescence microscapy Scionce 1080, 248 7376 3. Denk W, Pston DW. 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