In common with other optical sectioning techniqueslike total internal reflection microscopy (TIRFM) or structured illumination, this approach uses the strategyof getting crisper images by restricting the fluores-cence
excitation
volume, rather than restricting thefluorescence
collection
volume, as is done in confocalmicroscopy. Although one could in principle combinerestricted fluorescence excitation and collection ap- proaches, we will argue in Section 3 that restricting thefluorescence collection volume is detrimental to col-lecting the maximal signal in nonlinear microscopy,especially for deep-tissue imaging. We note that yet another important means of enhancing image quality isto restrict the
“
fluorophore volume
”
(i.e., specific tar-geting of the fluorophore to the structure of interest).Thus, combining the
‘
new microscopies
’
and the newtools of molecular biology offers cell biologists, neu-roscientists and anatomists unprecedented power toimage cellular structures and processes.The aim of this article is to give an overview of nonlinear microscopy, sufficient to provide the reader with the background to understand the compendium of articles appearing in this volume on
in vivo
imaging.Although the field of multi-photon microscopy is inrapid expansion, there are several excellent reviewsthat give a fairly complete overview of different as- pects of nonlinear microscopy[2
7].We start by briefly discussing the basics of single- photon fluorescence excitation, then consider multi- photon excitation and the means of creating images byraster-scanning the multi-photon excitation volumeacross the specimen. We then discuss some alternativemodes of imaging thin optical sections and comparetheir respective advantages and disadvantages. Thereview terminates with a perspective on the current frontiers in nonlinear imaging.
2. Fluorescenceasacontrastgenerator inbiologicalimages
Fluorescence has been used extensively to generateand enhance contrast in biological samples that, due totheir low refractive-index variations, present little con-trast. A detailed discussion of the basis of fluorescencemicroscopy is beyond the scope of this article. Wereview just the essentials in as much as they are ne-cessary for understanding the concepts of nonlinear fluorescence excitation. For further reading, many freetutorialsonfluorescencearefoundonline(see,e.g.,[8],also the websites of major microscope manufacturers,e.g.,www.microscopyu.com,www.olympusmicro.
com, etc.). An exhaustive and very didactic treatment ofgeneralfluorescence isfoundintheclassical bookof Lakowicz[9]. A concise review of fluorescence tech-niques as applied to drug delivery research is found inan earlier volume of this journal[10].
2.1. One-photon excitation fluorescence (1PEF)
Fluorescence occurs when a photon is absorbed bya fluorophore, raising an electron to an excited energystate, from which it relaxes to the electronic groundstate and emits a lower energy photon. For absorptionto occur, the energy of the exciting photon has toapproximately match the energy difference betweenthe excited state and the ground state (Fig. 1A). At room temperature, absorption of a photon takes placefrom the vibrational ground state S
0
within
∼
10
−
18
s.Photon absorption excites the molecule to the singlet state S
2
from which it undergoes a radiation-less lossof energy by internal conversion to reach S
1
.From S
1
, there are several possible fates. By far most molecules will return to the electronic groundstate without emitting a photon. Their electronic ener-gy will be transferred to the surroundings and con-verted into heat when molecules collide. However, insome cases, after a delay of
∼
10
−
8
–
10
−
9
s, the elec-tron drops back to the ground state and a photon isemitted. This emitted fluorescent photon is of lower energy (i.e., longer wavelength) than the photon origi-nally absorbed, a phenomenon known as the
Stokes shift
. The
fluorescence lifetime
refers to duration of time between absorption of an excitation photon andthe emission of a fluorescent photon. The fluorescencelifetime is typically exponentially distributed and ishighly sensitive to the fluorophore environment and,therefore, may be used to report changes in the localfluorophore environment.From S
1
, another possible fate is to emit a phos- phorescent photon. In this case, the excited electronundergoes intersystem crossing (ISC) to attain thetriplet state, before it exits to the ground state. Thelifetime of the excited state in phosphorescence issignificantly longer than that in fluorescence
—
on theorder of
∼
10
−
4
s to minutes or even hours. The dropto the ground state is accompanied by emission of a
790
M. Oheim et al. / Advanced Drug Delivery Reviews 58 (2006) 788
–
808
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