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Trouble-free 2-Photon Microscopy Two-Photon Microscopy with Fixed-Wavelength Femtosecond Lasers
| Content Provider | Semantic Scholar |
|---|---|
| Copyright Year | 2014 |
| Abstract | Two-photon (2P) absorption spectra of fluorescent dyes are broader than single-photon ones. Simultaneous 2P excitation of several dyes with a single wavelength (WL) is feasible and can be seen as an advantage. We used femtosecond (fs) pulsed fixed-WL infrared lasers with center WLs at 780 nm and 1040 nm, respectively, for Two-photon microscopy (2PM) in biologically relevant samples. The 1040 nm laser proved to be very efficient in exciting fluorescence from YFP and red fluorescent dyes, but also in generating second harmonic generation (SHG) from muscle tissue and collagen. We demonstrate that economical, small-footprint fixed-WL lasers can present adequate replacement for commonly used Ti:Sapphire (Ti:Sa) lasers, the size, price and handling of which causes considerable pain for microscope users. Both fixed-WL lasers were coupled simultaneously (fig. 1). 780 nm laser power attenuation to 10 mW was performed by changing pump power; 1040 nm laser power was attenuated by retarder plate and polarizer to 10 to 20 mW. The Nikon 25x objective lens aperture (Nikon CFi75 Apo Water LWD, N/A 1.1, w.d. 2 mm) was modestly over-illuminated. End-to-end Transmission of the system was 40% at 780 nm / 30% at 1040 nm. The 2PM was equipped with two nondescanned GaAsP PMTs with large sensitive surface. Dichroic mirrors were used to split signals into color channels. Laser safety was achieved by boxing lasers and beam path. Using typical samples taken from ongoing research we tested if fixed-WL lasers present a viable alternative to tunable laser systems. |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | https://www.spectra-physics.com/assets/client_files/files/documents/SP-AR-20131001_Trouble-free%202-Photon%20Microscopy.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
