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Biological Nonlinear Microscopy CHRIS XU, WARREN ZIPFEL, JASON B

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Biological Nonlinear Microscopy CHRIS XU, WARREN ZIPFEL, JASON B Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10763-10768, October 1996 Biophysics Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy CHRIS XU, WARREN ZIPFEL, JASON B. SHEAR*, REBECCA M. WILLIAMS, AND WATT W. WEBB Applied Physics, Cornell University, Ithaca, NY 14853 Contributed by Watt W Webb, July 16, 1996 ABSTRACT Intrinsic, three-dimensionally resolved, mi- copy, however, requires knowledge of the photophysics of croscopic imaging of dynamical structures and biochemical multiphoton excitation, especially the multiphoton excitation processes in living preparations has been realized by nonlin- properties of fluorophores and biological molecules. ear laser scanning fluorescence microscopy. The search for Nonlinear LSM gains several advantages from two- or useful two-photon and three-photon excitation spectra, moti- three-photon excitation of fluorescence. Because the absorp- vated by the emergence of nonlinear microscopy as a powerful tion increases quadratically or cubicly with the excitation biophysical instrument, has now discovered a virtual artist's intensity, the fluorescence (and potential photobleaching and palette of chemical indicators, fluorescent markers, and na- photodamage related to fluorescence excitation) are all con- tive biological fluorophores, including NADH, flavins, and fined to the vicinity of the focal point. This spatial localization green fluorescent proteins, that are applicable to living bio- not only provides intrinsic three-dimensional resolution in fluorescence microscopy but also provides unprecedented logical preparations. More than 25 two-photon excitation capabilities for three-dimensionally localized photochemistry spectra of ultraviolet and visible absorbing molecules reveal in subfemtoliter volumes, including photolytic release of caged useful cross sections, some conveniently blue-shifted, for effector molecules (9). Because two or three photons are near-infrared absorption. Measurements of three-photon flu- absorbed for each transition event, a red or near-infrared laser orophore excitation spectra now define alternative windows at is used to excite fluorophores that normally absorb in the UV relatively benign wavelengths to excite deeper ultraviolet or deep-UV region. Such a wavelength shift avoids many fluorophores. The inherent optical sectioning capability of limitations of UV lasers and UV optics. Because wide-field nonlinear excitation provides three-dimensional resolution detection is used in nonlinear microscopy, even multiply scat- for imaging and avoids out-of-focus background and photo- tered fluorescence photons contribute equally to the image damage. Here, the measured nonlinear excitation spectra and formation, in contrast with confocal microscopy, where the their photophysical characteristics that empower nonlinear unscattered photons form the confocal image (21). This effi- laser microscopy for biological imaging are described. cient detection of fluorescence photons, combined with the relatively deep penetration of IR excitation light in most Molecular two-photon excitation (TPE) was predicted by biological preparations (22), enables nonlinear microscopy to Goppert-Mayer in 1931 (1). Experimental observations of image deep (>200 ,um) into turbid biological specimens. In multiphoton processes awaited the invention of pulsed ruby general, the background interference from Rayleigh and Ra- lasers in 1960. Closely following the demonstration of second- man scattering is negligible because multiphoton excited flu- harmonic generation (SHG), the first demonstration of non- orescence occurs at a much shorter wavelength region than the linear optics, two-photon absorption was utilized by Kaiser and excitation light. Thus, ultrasensitive measurements are possi- Garrett to excite fluorescence emission in CaF2:Eu3+ (2). ble, including detection of single dye molecules (23, 24) and excited low quantities of fluorogen-labeled neurotransmitters (25). Three-photon fluorescence was observed and the Finally, in addition to background interference, photodamage three-photon absorption cross section for naphthalene crystals to living cells by deep UV excitation has previously limited the was estimated by Singh and Bradley in 1964 (3). Subsequently, use of imaging based on native fluorescence. Three-photon multiphoton excitation and fluorescence has been used in excited fluorescence provides the unique opportunity to excite molecular spectroscopy of various materials (4-8). intrinsic chromophores, such as amino acids, proteins, and A significant biological application of multiphoton excita- neurotransmitters, using relatively benign excitation wave- tion began with the invention of two-photon laser scanning lengths accessible with commercially available near-IR lasers microscopy (TPLSM) by Denk, Strickler, and Webb in 1990 (26). The combination of two- and three-photon excited (9). Originally devised for localized photochemical activation fluorescence microscopy extends the useful range of nonlinear of caged biomolecules, TPE of photochemical polymer laser microscopy. crosslinking also has provided a means for high-density three- A basic parameter of fluorescence is the fluorescence dimensional optical data storage at 1012 bits/cm3 (10). excitation cross section. Although one-photon absorption This article on multiphoton excitation is motivated by the spectra are well documented for a wide range of molecules, emergence of TPLSM as a powerful new microscopy for little is known of two- or three-photon absorption spectra of three-dimensionally resolved fluorescence imaging of biolog- biologically useful fluorophores (27). Furthermore, it is often ical samples (11, 12). The development of TPLSM has been difficult to predict multiphoton excitation spectra, especially propelled by rapid technological advances in laser scanning two-photon spectra, from the one-photon data because of microscopy (LSM) (13), fluorescence probe synthesis, mode- differences in selection rules and the effects of vibronic locked femtosecond lasers (14, 15), and computational three- coupling. We report measurements of two- and three-photon dimensional image reconstruction (16). Recently, three- fluorescence excitation cross sections of biological indicators photon excited fluorescence and its potential applications in imaging have also been reported for several fluorescent dyes Abbreviation: TPE, two-photon excitation; LSM, laser scanning mi- (17-20). Effective implementation of nonlinear laser micros- croscopy; TPLSM, two-photon LSM; GM, Goppert-Mayer; DiI, 1,1- dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole; n.a., The publication costs of this article were defrayed in part by page charge numerical aperture; FWHM, full width at half maximum. payment. This article must therefore be hereby marked "advertisement" in *Present address: Department of Chemistry, University of Texas, accordance with 18 U.S.C. §1734 solely to indicate this fact. Austin, TX 78712. 10763 Downloaded by guest on October 6, 2021 10764 Biophysics: Xu et al. Proc. Natl. Acad. Sci. USA 93 (1996) and native chromophores. Detailed methods for obtaining the multiphoton excitation cross sections and spectra were re- ported elsewhere (28, 29). The potential of three-photon excited fluorescence in LSM is also demonstrated. Two-Photon Excitation I-- u 0 c Fig. la summarizes results of measurements of two-photon 0 fluorescence excitation spectra [o-2(A)] of common fluoro- ux phores in the spectral range of 690-1050 nm. TPE spectra of Qw various Ca2+ indicators are reported in Fig. lb. TPE cross- section measurements of fluorescein, rhodamine B, and 1,1- dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlor- lW'- ate (DiI) are shown in Fig. 2, with their corresponding one-photon absorption spectra. For comparison, the tuning 10 - ranges of several mode-locked laser sources are also plotted in 1- , , Fig. 1. These spectra show that nonlinear fluorescence micros- I fluorescein 700 800 900 iobo II 10 - Wavelength (nm) 103l2- a FIG. 2. Comparison of one-photon (broken lines) and two-photon 10 (solid lines) fluorescence excitation spectra of rhodamine B, fluores- D; tyll cein (pH 13), and DiI. The y-axis values represent two-photon 100 1- absorption cross sections for rhodamine B and fluorescein, and 2 10 - DP. two-photon action cross sections for DiI. For the one-photon results, the x-axis values represent twice the one-photon transition wave- lengths. The one-photon results are plotted in arbitrary scale. 102 py CB C.) 0) 600 copy based on TPE can be expected to work well with most U, 600 700 800 900 1000 1100 existing fluorophores and many available laser sources. For U, nonratiometric Ca21 indicators, such as calcium crimson, 0 calcium orange, and calcium green-1, we found that the TPE 102 spectra of the Ca2+-free and Ca2+-bound forms are indistin- guishable. The ratios of fluorescence intensity for the Ca2+- 10 free to Ca2+-bound forms of these indicators are also compa- 0- rable to their reported one-photon values. 10 Green fluorescent proteins (GFPs) have attracted tremen- dous interest as biological reporters for gene expression (30). 1 0 Images of cultured cells using two-photon excited GFP fluo- rescence have been reported recently (31). Wild-type GFPs 10 2 have high UV absorption at 400 nm and relatively low visible 600 700 800 900 1000 1100 Wavelength (nm) absorption at 480 nm. To facilitate the use of GFPs with visible Ti
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