Two-photon laser scanning microscopy

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... 

Denk, W, Strickler, J., and Webb, W. 1990. Two-photon laser scanning microscopy. Science 248 73-76. 

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... 

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.

Multiphoton or two-photon laser scanning microscopy is an alternative to confocal and time-resolved microscopy for bioimaging applications. The principle has been discussed in Lanthanides Luminescence Applications and concerns a two-photon excitation from the simultaneous absorption of two photons in a single quantized event. A bioprobe that normally absorbs ultraviolet light (Xex = 350 nm) can also be excited by two photons of NIR light, at 700 nm (the wavelength is twice that required for one-photon excitation). These two photons must interact simultaneously, which means in a very small lapse time. The instrumentation requires pulse lasers to provide sufficient power, as the photon density must... 

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. 

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