Two-photon laser scanning fluorescence

W. W. (1990) Two-photon laser scanning fluorescence microscopy. Science, 248, 73-76. 

As mentioned above, spectral imaging microscopy is a form of multidimensional fluorescent microscopy where a fluorescent emission spectrum is acquired at each coordinate location in the sample. This mode of imaging has been implemented for wide field, confocal, and two-photon laser scanning microscopy, and several excellent... 

Figure 5. (A) Scheme of two-photon laser scanning microscope (1) Ti Sa laser, 100 fs, 80 MHz, 750-980 nm, 1.6W 800 nm (TSUNAMI, Spectra Physics), (2) pre-chirp, (3) beam multiplexer, (4) scanning mirrors, (5) microscope (Olympus IX 71, XLUMPLFL20XW, WD = 2 mm, NA = 0.95), (6) fluorescent foci in sample, (7) filter wheel/spectrograph (SpectraPro 2300i, Acton Research Corporation)/spectrometer (home built), (8) back illuminated EMCCD camera (IXON BV887ECS-UVB, Andor Technology), (9) dichroic mirror (2P-Beamsplitter 680 DCSPXR, Chroma). (B) Experimental setup of two-photon laser scanning microscope.

An application of TCSPC FLIM to CFP-YFP FRET is shown in Fig. 5.87 and Fig. 5.88. The microscope was a Zeiss LSM 510 NLO two-photon laser scanning microscope in the Axiovert 200 version. An excitation wavelength of 860 nm was used. The nondescanned fluorescence signal from the sample was fed out of the... 

Two-photon laser scanning microscopes and microscopes for two-photon single molecule experiments use femtosecond lasers. Two-photon fluorescence excitation with a picosecond diode laser cannot be expected to work with the same efficiency. The average intensity is only a few mW. Moreover, the pulse duration is of the order of 100 ps, so that the peak power is many orders of magnitude lower than for a fs Ti Sapphire laser. The relative excitation efficiency of different lasers can be estimated as follows ... 

Recently polymer photonic crystal materials have been applied as optical data storage media. An optically active, core-shell latex colloidal crystal with dye-labeled polymer cores was atmealed into a material with a periodic array of fluorescent particles in an optically inert matrix. A two-photon laser scanning microscope was used to write information to, and read from, the material leading to increased optical storage density over conventional materials [133]. 

Potter S.M., Wang C.-W, Garrity P.A., and Fraser S.E. 1996. Intravital imaging of green fluorescent protein using two-photon laser-scanning microscopy. Gene 173 25-31. 

Strickler J FI and Webb W W 1990 Two-photon excitation in laser scanning fluorescence microscopy Proc. SPIE 13948107-18... 

Two-photon excitation provides intrinsic 3-D resolution in laser scanning fluorescence microscopy. The 3-D sectioning effect is comparable to that of confocal microscopy, but it offers two advantages with respect to the latter because the illumination is concentrated in both time and space, there is no out-of-focus photo-bleaching, and the excitation beam is not attenuated by out-of-focus absorption, which results in increased penetration depth of the excitation light. 

Synthesis and nonlinear optical characterization of a two-photon absorbing organic dye, trans-4-(dimethylamino)-4 -[N-ethyl-N-(2-hydroxyethyl)amino]stilbene (DMAHAS), were reported [49]. Linear absorption, single-photon-induced fluorescence, and two-photon-induced fluorescence of DMAHAS were experimentally studied. This dye showed a moderate two-photon absorption cross section of a2 = 0.91 X 10-46 cm s/photon at 532 nm as shown by an open aperture Z-scan technique. DMAHAS also showed strong two-photon-induced blue fluorescence of 432 nm when pumped with 800 nm laser irradiation. 

Bhawalkar J D, Swiatkiewicz J, Pan S J, Samarabandu J K, Liou W S, He G S, Berezney R, Cheng P C and Prasad P N 1996 Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore Scanning 18 562-6... 

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