Fig. 5: Principle of frame-separated excitation polarization angle narrowing (FrExPAN) for the usage with negative-switching dt-rsFPs.

Shown is a the polarization orientation of the laser pulses and b the pulse duration, together with c a schematic spinehead-like structure with double membrane-tagged dt-rsFPs. At the end of the first frame, the dt-rsFPs get switched on by a short 2-ms pulse of 405 nm (d) directly followed by 2 ms of 488 nm with a polarization vector perpendicular to the 405 nm laser to achieve the excitation angle narrowing (e). The following readout step is moved into the next frame to enable fluorescence detection of dt-rsFPs with a transition dipole moment exactly parallel to the polarization vector of the switch-on and readout laser without detecting the intense fluorescence from the perpendicular polarized switch-off beam in the previous frame (f). Due to the camera readout time, there is a delay of 2 ms before the next pulse to read out the activated FPs. The readout pulse is 10 ms long and is positioned directly at the beginning of the second frame, with consideration of the 1 ms readout time of the camera between two frames to keep the time available for the diffusion of the proteins along the membrane as low as possible. In the future, the data can be analyzed by advanced algorithms, for example, localization of regions with multiple dt-rsFPs of narrowed and similar orientation angles (g). For details, see the text. h–k Experimental frame-separated FrExPAN from double membrane-anchored dt-p-rsGreenF-F HeLa cell and hippocampal neuron samples. The images show the average intensity of modulation averages observed with 50 frames (h) or 15 frames (i–k) per period. (The corresponding modulation signals are shown in Supplementary Movie 2). In addition, modulation signals of four selected ROIs are shown for each image as observed with (red, ExPAN) and without a second FrExPAN pulse (black, noExPAN). The inset in the lower right of each image (h-k) shows averaged ExPAN factors along with error bars from data with (red, ExPAN) and without second FrExPAN pulse (black, noExPAN) observed in at least 99 ROIs for FrExPAN and for noExPAN, respectively. h Experiments were conducted with a 5 ms switch-on with 405 nm, a 1 ms FrExPAN and 50 ms readout pulse and 50 frames per period. i–k were done with a 2 ms switch-on with 405 nm, a 2 ms FrExPAN and a 10 ms readout pulse and 15 frames per period. h Data from HeLa cell membranes (i), vesicles of HeLa cells (j), primary hippocampal neurons (k), and another HeLa cell membrane example. Scale bars 2 µm. For the plotting of the modulations signals a phase shift was applied to match the position of the maxima and the modulation signals were plotted twice for visual clarification of the narrowing effect. (for details see text).