Abstract
Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing near-membrane cellular structures and processes, including imaging of local Ca2+ transients with single-channel resolution. TIRF is most commonly implemented in epi-fluorescence mode, whereby laser excitation light is introduced at a spot near the periphery of the back focal plane of a high numerical aperture objective lens. However, this approach results in an irregular illumination field, owing to interference fringes and scattering and shadowing by cellular structures. We describe a simple system to circumvent these limitations, utilizing a pair of galvanometer-driven mirrors to rapidly spin the laser spot in a circle at the back focal plane of the objective lens, so that irregularities average out during each camera exposure to produce an effectively uniform field. Computer control of the mirrors enables precise scanning at 200 Hz (5ms camera exposure times) or faster, and the scan radius can be altered on a frame-by-frame basis to achieve near-simultaneous imaging in TIRF, widefield and ‘skimming plane’ imaging modes. We demonstrate the utility of the system for dynamic recording of local inositol trisphosphate-mediated Ca2+ signals and for imaging the redistribution of STIM and Orai proteins during store-operated Ca2+ entry. We further anticipate that it will be readily applicable for numerous other near-membrane studies, especially those involving fast dynamic processes.
Highlights
The advent of total internal reflection fluorescence (TIRF) microscopy has greatly facilitated the imaging of cellular structures and processes in or very close to the plasma membrane [1]
TIRF microscopy has become the technique of choice for cellular imaging of near-membrane structures and dynamic processes [1,16]
Most TIRF microscopes, including commercial systems from the major manufacturers, employ ‘through-the-objective’ excitation, whereby laser light is introduced at a single spot near the edge of the back focal plane of a high numerical aperture TIRF objective
Summary
The advent of total internal reflection fluorescence (TIRF) microscopy has greatly facilitated the imaging of cellular structures and processes in or very close to the plasma membrane [1]. TIRF works by exciting fluorescence restricted to a very thin sheet (of the order of 100 nm) in an aqueous medium immediately above the cover glass. TIRF microscopy provides an optical sectioning effect analogous to confocal microscopy, but by a completely different mechanism and with a much thinner section. In contrast to the raster spot scanning of confocal microscopy, the entire section is illuminated at the same time, enabling fast camera-based imaging of dynamic processes. TIRF works by directing excitation light through a glass substrate toward an aqueous specimen at a sufficiently shallow angle so that total internal reflection occurs due to the refractive index decrease at the glass–water interface.
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