Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction

Total internal reflection fluorescence microscopy (TIRFM) is becoming an increasingly common methodology to narrow the illumination excitation thickness to study cellular process such as exocytosis, endocytosis, and membrane dynamics. It is also frequently used as a method to improve signal/noise in other techniques such as in vitro single-molecule imaging, stochastic optical reconstruction microscopy/photoactivated localization microscopy imaging, and fluorescence resonance energy transfer imaging. The unique illumination geometry of TIRFM also enables a distinct method to create an excitation field for selectively exciting fluorophores that are aligned either parallel or perpendicular to the optical axis. This selectivity has been used to study orientation of cell membranes and cellular proteins. Unfortunately, the coherent nature of laser light, the typical excitation source in TIRFM, often creates spatial interference fringes across the illuminated area. These fringes are particularly problematic when imaging large cellular areas or when accurate quantification is necessary. Methods have been developed to minimize these fringes by modulating the TIRFM field during a frame capture period; however, these approaches eliminate the possibility to simultaneously excite with a specific polarization. A new, to our knowledge, technique is presented, which compensates for spatial fringes while simultaneously permitting rapid image acquisition of both parallel and perpendicular excitation directions in ∼25 ms. In addition, a back reflection detection scheme was developed that enables quick and accurate alignment of the excitation laser. The detector also facilitates focus drift compensation, a common problem in TIRFM due to the narrow excitation depth, particularly when imaging over long time courses or when using a perfusion flow chamber. The capabilities of this instrument were demonstrated by imaging membrane orientation using DiO on live cells and on lipid bilayers that were supported on a glass slide (supported lipid bilayer). The use of the approach to biological problems was illustrated by examining the temporal and spatial dynamics of exocytic vesicles.