Fig. 2: Principle of fluorescence polarisation microscopy (FPM) with fluorescent proteins double tagged to membranes. | npj Imaging

Fig. 2: Principle of fluorescence polarisation microscopy (FPM) with fluorescent proteins double tagged to membranes.

From: High contrast fluorescence polarization microscopy through double tagged photoswitchable fluorescent proteins

Fig. 2

Illustration of the difference between single- (a) and double- (b) membrane-tagged rsFPs during constant polarization modulation of the excitation beam in an FPM setup such as shown in Fig. 1a. The rsFPs are preferentially excited by the light if the orientation of their transition dipole moment (shown as black arrows inside the barrel structure) is nearly parallel to the polarization vector (white arrow inside blue probability distribution of excitation). While st-FPs with one membrane tag are still flexible in the rotation around their anchor (a), the double-membrane tag restricts the free movement of the dt-FPs and leads to an orientation dependence on the shape of the tagged structure (b). This results in a much higher, better-defined modulation contrast of adjacent proteins in fluorescence polarization microscopy (FPM).

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