Two-photon laser scanning

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... 

Tsai PS, NishimuraN, Yoder E, DolnickE, White GA, Kleinfeld D. Principles, design and construction of a two-photon laser-scanning microscope for in vitro and in vivo brain imaging. In Frostig RD (Ed.), In Vivo Optical Imaging of Brain Function. DCRC, Boca Raton, FL, 2002. 

RGDS-PEG PEG-DA Human dermal fibroblasts Two-photon laser scanning photolithography to crosslink peptide-PEG inside a precrosslinked hydrogel to create 3D cell adhesion patterns 2008 [175]... 

Lee SH, Moon JJ, West JL (2008) Three-dimensional micropatterning of bioactive hydrogels via two-photon laser scanning photolithography for guided 3D cell migration. Biomaterials 29(20) 2962-2968... 

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.

Martini, J., Schmied, K., Palmisano, R., Toensing, K., Anselmetti, D. and Merkle, T. (2007) Multifocal two-photon laser scanning microscopy combined with photo-acivatable GFP for in vivo monitoring of intracellular protein dynamics in real time. J. Struct. Biol, in press. 

Imaging tiphoton, or two-photon laser scanning microscopy) is an alter-... 

Of course, the Scan Sync In mode is not restrieted to eonfoeal and two-photon laser scanning microscopy or high-speed seanning. Due to the simple interfaeing with the scanner, it can be used for other seanning applieations as well [454]. 

Two-photon excitation by femtosecond NIR laser pulses can be used to obtain clear images of tissue layers as deep as 1 mm [132, 278, 279, 344, 462, 495, 534]. The efficiency of two-photon excitation depends on the square of the power density. It therefore works with noticeable efficiency only in the focus of the laser beam. With a microscope lens of high numerical aperture a lateral resolution around 300 nm and a longitudinal resolution of about 1 pm is obtained. Two-photon laser scanning microscopy has therefore become a standard technique of tissue microscopy. Two-photon laser scanning can be combined with... 

Dual-wavelength TCSPC detection in two-photon laser scanning microscopes is relatively simple [37]. Multispectral TCSPC detection in a two-photon laser scanning microscope requires a suitable relay optics between the objective lens and the polychromator [35, 60]. Details are described under TCSPC Laser Scanning Microscopy . 

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... 

Commercial confocal and two-photon laser scanning microscopes scan the laser beam by fast galvano-driven mirrors. Typical pixel dwell times are of the order of a few microseconds. Depending on the number of pixels, a complete frame is scanned within 25 ms to several seconds. 

Optically driven photon correlation experiments normally require confining the detection or the excitation to an extremely small sample volume. This is achieved either by confocal detection or two-photon excitation in a microscope. The optical principles are the same as in confocal and two-photon laser scanning microscopes (see Sect. 5.7, page 129). However, most correlation experiments do not require scanning and can be performed in relatively simple microscopes. 

Theoretically, the same procedure can also be used in a commercial confocal or two-photon laser scanning microscope if a sufficiently slow scan speed can be selected. To distinguish the individual pixels of the scan, the short scan control... 

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 ... 

Two-photon laser-scanning microscopy system. We use a Leica SP5 inverted 5 channel confocal microscope see Note 1 and Fig. la), a Ti Sapphire laser (Spectra Physics) with a 10-W pump tuned to 810 nm, a 20x multi-immersion objective with long working distance (for example, Leica 20x 0.7NA), an incubation cube chamber (Life Imaging Services, Basel, Switzerland) (rrrNote 1 and Fig. lb). 

Fig. 1. Two-photon laser-scanning microscopy system and operation for exposing the inguinal lymph node, (a) Two-photon laser-scanning microscopy system including a Leica SP5 inverted 5-channel confocal microscope. (b)The cube and box, cube chamber, maintains temperature at 37.0°C 0.5°C. (c) Anesthetized mouse is shaved and has small Incision on flank, (d) Immobilized iLN with cyanoacrylate glue on blade fitted to chamber slide. To protect tissue from drying add a few drops of PBS to exposed iLN. (e) Mouse in a warmed chamber slide. Blade can hold exposed ILN preventing respiratory movements, (f) Complete set up for imaging. Mouse is anesfhefized by fhe infraperifoneal injecfion of avertin with an infusion set.

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