Abstract
Fluorescence anisotropy imaging microscopy (FAIM) measures the depolarization properties of fluorophores to deduce molecular changes in their environment. For successful FAIM, several design principles have to be considered and a thorough system-specific calibration protocol is paramount. One important calibration parameter is the G factor, which describes the system-induced errors for different polarization states of light. The determination and calibration of the G factor is discussed in detail in this article. We present a novel measurement strategy, which is particularly suitable for FAIM with high numerical aperture objectives operating in TIRF illumination mode. The method makes use of evanescent fields that excite the sample with a polarization direction perpendicular to the image plane. Furthermore, we have developed an ImageJ/Fiji plugin, AniCalc, for FAIM data processing. We demonstrate the capabilities of our TIRF-FAIM system by measuring -actin polymerization in human embryonic kidney cells and in retinal neurons.
Highlights
Fluorescence anisotropy imaging microscopy (FAIM) is used to spatially resolve and quantify a range of physical and chemical properties, including rotational diffusion (RD) [1], polymerization reactions [2], and conformational changes of a molecule [3]
We propose here that it is possible to combine the collinear geometry of an anisotropy TIRF microscope with the precise G factor calibration method that is conventionally used for an L-format spectrofluorometer (see figure 1(d))
As sketched in figures 2(c) and (d), the FAIM set-up used for this work contained a half wave plate (HWP) (WPH05M-488, Thorlabs) placed after a vertically polarized 488 nm laser line (Coherent Sapphire) and a mirror mounted on a translation stage (BB2-E02 on NRT100/M, Thorlabs) to accurately and reproducibly switch between episcopic and TIRF illumination modes
Summary
Fluorescence anisotropy imaging microscopy (FAIM) is used to spatially resolve and quantify a range of physical and chemical properties, including rotational diffusion (RD) [1], polymerization reactions [2], and conformational changes of a molecule [3]. For the accurate measurement of the extent of depolarization of fluorescent molecules, one needs to know the systematic errors introduced by the optical set-up and the detectors in use, which are combined in a single parameter commonly referred to as the G factor. This name stems from polarization-sensitive spectrofluorometers, which use gratings, ‘G factor’, for spectral selection. Often two distinct cameras are used for their detection or the respective signals are used on separate parts of the same camera chip using an image splitter
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