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Total internal reflection fluorescence anisotropy imaging microscopy: setup, calibration, and data processing for protein polymerization measurements in living cells

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Wong, HHW 
holt, CE 
kaminski, CF 


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 β-actin polymerization in human embryonic kidney cells and in retinal neurons.



Actins, Bacterial Proteins, Calibration, Fluorescence Polarization, HEK293 Cells, Humans, Luminescent Proteins, Microscopy, Fluorescence, Optical Imaging, Polymerization, Retinal Neurons

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Methods and Applications in Fluorescence

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IOP Publishing
Engineering and Physical Sciences Research Council (EP/H018301/1)
Wellcome Trust (085314/Z/08/Z)
Engineering and Physical Sciences Research Council (EP/L015889/1)
Wellcome Trust (203249/Z/16/Z)
European Research Council (322817)
Medical Research Council (MR/K02292X/1)
Medical Research Council (MR/K015850/1)
Wellcome Trust (089703/Z/09/Z)
Biotechnology and Biological Sciences Research Council (BB/H023917/1)
Medical Research Council (G0902243)
This work was funded by grants from the Medical Research Council UK (MR/K015850/1 and MR/K02292X/1), the EPSRC (EP/L015889/1 and EP/H018301/1), theWellcome Trust (3-3249/Z/16/Z and 089703/Z/09/Z), and In nitus, China, Ltd (CFK); the Cambridge Trust, Croucher Foundation, Sir Edward Youde Memorial Fund (HHWW); a Wellcome Trust Programme Grant (085314/Z/08/Z) and an ERC Advanced Grant (322817) (CEH).