Spinning-Spot Shadowless TIRF Microscopy

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.

Imaging Membrane Curvature inside a Model Immunological Synapse in RBL-2H3 Cells using TIRF Microscopy with Polarized Excitation

“…This can result in uneven illumination and interference fringes in the image, but this problem was not severe in our case. Spinning the laser beam around the periphery of the back aperture within a single camera exposure can help eliminate such interference patterns [29].…”

Myosin-II mediated traction forces evoke localized Piezo1 Ca2+ flickers

“…S3 were acquired at 200 Hz frame rate on a custom-built Spinning-Spot Shadowless TIRF microscope. Details of construction and comparison to traditional TIRF can be found in Ellefsen et al 2015 (Ellefsen, Dynes, and Parker 2015).…”

Theoretical background of light‐emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane‐associated proteins—Part II

“…Advantages over laser‐assisted TIRF include an inherently artifact‐free imaging, and the variable choice of excitation wavebands. In laser‐based systems, circular or multi‐spot variations of the laser illumination have been introduced to eliminate the image‐degrading impact of local scatters in the probe and fringing artifacts, while maintaining the control of the AOI and the brightness . light‐emitting diode (LED)‐TIRF shares this artifact‐free imaging with circular laser‐based illumination methods or with “white light TIRF”.…”