Microscopy Facility
Total Internal Reflection Fluorescence (TIRF) Microscopy
Total internal reflection fluorescence microscopy permits high signal-to-noise imaging of fluorescently-labeled molecules at surfaces and interfaces. By adjusting the angle of the illuminating light beyond the critical angle at an interface between media of two different refractive indices (i.e. achieving total internal reflection), a shallow evanescent wave is generated into the medium of lower refractive index that can excite fluorescent molecules that lie within 100 nm of the interface. This results in high signal-to-noise images, due to the lack of signal beyond the 100 nm depth of penetration. This is ideal for studying signal molecule fluorescence, cell-substrate interactions and membrane dynamics.
The facility houses an Olympus TIRF microscope system (IX 81 inverted stand) equipped with a 100x/1.45 NA Plan Apochromat TIRF objective lens (in addition to 10x, 20x, 40x, and 60x lenses), DIC and phase optics, filter sets for detection of blue, green and red fluorescence, an Exfo X-cite illumination system and a Hamamatsu Orca ER CCD camera. The system has Argon (458, 476, 488, 514 nm) and 561 nm diode lasers, each with independent beam steering optics for two-channel TIRF and an LSM Technologies AOTF to regulate beam intensity.
