rsgreenf  (Addgene inc)

Journal: npj Imaging

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

doi: 10.1038/s44303-025-00094-y

Figure Lengend Snippet: Principle of single ( a ) and double ( f ) membrane-tagged RSFPs. FPM results from b living HeLa cell samples expressing the single-tagged Kohinoor-F, c HeLa cell samples expressing p-rsGreenF, HeLa cells expressing the single-tagged rsGreenF-F, e Dendritic branch of a hippocampal neuron expressing the single-tagged rsGreenF-F, g HeLa cells expressing the double-tagged p-Kohinoor-F, h HeLa cell expressing the double-tagged p-rsGreenF-F, i HeLa cell expressing the double-tagged p-rsGreenF-F, j Dendritic branch of a hippocampal neuron expressing the double-tagged p-rsGreenF-F. Shown is the average and phase colored FFT image of the different structures with the corresponding background-corrected raw data along with fluorescence polarization modulation data from selected ROIs(indicated in the images by red squares). Excitation wavelengths were 488 nm for Kohinoor and 405 and 488 nm for rsGreenF samples. Scale bars: 2 µm, a.u. arbitrary units. ROIs are highlighted with a red box and have a size of 495×495 nm for rsGreenF and 286 × 286 nm for Kohinoor results. The corresponding modulation signals are shown in Supplementary Movie . Very similar data have been observed with more biological replicates, and further examples can be found in Supplementary Movie - .

Article Snippet: The original expression plasmid for Kohinoor was provided by Takeharu Nagai and his research group, and rsGreenF was commercially purchased from Addgene (#78181) , .

Techniques: Membrane, Expressing, Fluorescence

Journal: npj Imaging

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

doi: 10.1038/s44303-025-00094-y

Figure Lengend Snippet: 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 ). 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).

Article Snippet: The original expression plasmid for Kohinoor was provided by Takeharu Nagai and his research group, and rsGreenF was commercially purchased from Addgene (#78181) , .

Techniques: Membrane, Plasmid Preparation, Fluorescence, Diffusion-based Assay, Clarification Assay

Journal: npj Imaging

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

doi: 10.1038/s44303-025-00094-y

Figure Lengend Snippet: a Setup for fluorescence polarization microscopy by rotation of the excitation polarization (FPM). b Diffraction-limited image of single molecules and c false color version of the diffraction-limited image, color-coded based on the different orientations. d Results of the deconvolution with the SPEED algorithm to locate the molecules at sub-diffraction-limited resolution (single molecule Data previously published in Hafi, N. et al., Nature methods 13 , 8–9 (2016) ). e Two selected phase ranges from this analysis, separating molecules in different orientation ranges. f–t Shows different examples of linear actin filaments on a coverslip labeled with phalloidin-Atto 590 (averaged raw data previously published in Hafi, N. et al., Nature methods 13 , 8–9 (2016) ) . f Averaged raw data with ( k , p ) orientation color-coded images of the diffraction-limited images. g , l , q Deconvolved images based on the entire signal and h , m , r deconvolved images based on only the modulating part (A in eq. , ) and i , j , n , o , s , t selected phase/orientation ranges using the ALPA algorithm (for details see methods section). u – ad FPM results of data from a double membrane-anchored dt-p-rsGreenF-F living HeLa cell without second FrExPAN pulse (NoExpan). u , z Averaged diffraction-limited raw data. v , aa FPM data deconvolved and orientation color-coded results after 500 iterations with Richardson–Lucy. w – y , ab – ad FPM data deconvolved and orientation color-coded images after 500 iterations with SPEED algorithm and with selected phase/orientation ranges (for details see text). ae Set-up for frame-separated fluorescence excitation polarization angle narrowing polarization microscopy (FrExPAN, for details see Fig. and corresponding text). af – an Results of the FrExPAN data obtained under identical conditions as in ( u – ad ) but with second ExPAN pulse. aj Averaged diffraction-limited raw data. af , ak Deconvolved images after 500 iterations with a Richardson–Lucy algorithm. ag – ai , al – an Deconvolved images after 500 iterations with the SPEED algorithm. Scale bars b – e 1 µm, f – t 0.25 µm, and u – an 2 µm. (The corresponding modulation signals are shown in Supplementary Movies , ) For details, see text.

Article Snippet: The original expression plasmid for Kohinoor was provided by Takeharu Nagai and his research group, and rsGreenF was commercially purchased from Addgene (#78181) , .

Techniques: Fluorescence, Microscopy, Labeling, Membrane