Total Internal Reflection Fluorescence (TIRF) Microscopy

Total Internal Reflection Fluorescence (TIRF) Microscopy image 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 illumination. This is ideal for studying signal molecule fluorescence, cell-substrate interactions and membrane dynamics.


The facility houses 2 TIRF systems. One is an Olympus cellTIRF 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 & phase optics, UVFITCTRITC, & quad filter sets, a low-profile stage incubator (to facilitate live cell imaging), Zero Drift Compensation autofocus, an Exfo X-cite illumination system and an Andor iXon DU-897E emCCD camera. The system has 405 nm diode, Argon (458, 476, 488, 514 nm) and 561 nm diode lasers, each with independent beam steering optics for three-channel TIRF and an AOTF to regulate beam intensity.

TIRF is also available on the Nikon N-SIM structured illumination microscope. TIRF may be run independent of SIM. The microscope (Eclipse Ti inverted stand) is equipped with a CFI Apochromat TIRF 100x oil objective (NA 1.49), 'Perfect Focus' autofocus system, an Andor iXon DU-897E emCCD camera and, for TIRF, a dedicated LU-N4 laser input (405, 488, 561 & 640 nm).