Two-Photon Microscope (Thorlabs)

Advantages

Multiphoton microscopy (also called non-linear laser scanning microscopy) utilizes simultaneous absorption of two or more photons for excitation of a fluorescent label. Fluorescent emission, following two-photon excitation, is exactly the same as emission generated in normal one-photon excitation. Out-of-focus light is eliminated by excitation only at the focal plane; no pinhole is necessary. In contrast to confocal microscopy, several different fluorescent labels can be excited with the same wavelength (e.g. Alexa488, Alexa 568, and Cy5 are excited by ~ 800 nm; GCamp6f, DsRed, and Evans Blue are excited by ~920 nm in living tissue). This microscopy is best suited for deep imaging in living and fixed tissue, as well as in vivo animal imaging (several mm deep from the surface of a cleared or transparent sample).

Description

Our two two-photon setups are similar. Both microscopes are upright. The acquisition is fast, with ~15 fps rate at 1024x1024 pixels. The microscopes are compatible with any near infrared Nikon objectives D25 and D32 diameters. The camera is a part of the setup with the images integrated into the software. Transmitted light and epifluorescent images/movies can be taken. There is a tiling option. No environmental chamber is available. The imaging can be synchronized with other applications for light stimulation, behavior etc.

Data analysis is typically conducted on a different computer using Fiji or other specialized programs.

Both two-photon setups are complex and require extended hands-on training prior to independent use. After completion of the training, researchers are given access to the instrument. Assistance with imaging and experimental design is available during normal business hours.

Sagdullaev Lab

ThorLabs Bergamo II two-photon microscope has a tunable femtosecond TI sapphire laser with a working range 720-1020 nm. The microscope has 16x (water, WD 3.0, NA 0.8, D32 diameter) objective. Two PMTs are available for simultaneous imaging at 500/40 nm and 607/70 nm separated by a dichroic mirror at 530 nm. For living non-sterile tissue, a perfusion with temperature control and oxygenation is available. The head of the microscope can be rotated. Equipment manager: E. Ivanova

Hollis Lab

ThorLabs Bergamo II two-photon microscope has two Spectra-physics Insight 3 lasers: a tunable laser with a working range 680-1300 nm and 1040-1045 nm laser. The microscope has 16x (water, WD 3.0, NA 0.8, D32 diameter) and 60x (water, WD 2.2, NA 1.0, D25 diameter) objectives. Two PMTs equipped with the following filter sets are available for simultaneous imaging: 525/50 nm and 607/70 nm separated by a dichroic mirror at 562 nm. Equipment manager: N. Serradj