Journal: bioRxiv

Article Title: Self-inspired learning to denoise for live-cell super-resolution microscopy

doi: 10.1101/2024.01.23.576521

Figure Lengend Snippet: a , A representative example of live HeLa cells labeled with SiR-tubulin under STED (center pixel, STED depletion laser power as 50%, left) and its SN2N result (STED-SN2N, right). Data from ref. ( Methods ). b , Magnified views of the white boxed regions in a . First column: Confocal image (5 × 5 sum, top) and its SN2N result (bottom); the other columns: STED images (center pixel, top) under different depletion laser powers (0%, 8%, 16%, 30%, and 50% from left to right), its SN2N results (middle), and its ISM results (5 × 5 adaptive pixel reassignment, APR, STED-ISM, bottom). FRC-measured resolution values are marked at the left bottom. Notably, STED with a depletion laser power as 0% is equivalent to the confocal configuration. c , A sketch of different strategies for pixel assembly, including center pixel (‘Cent. pix.’), 5 × 5 sum, and APR. d , Fluorescence intensity profiles along the white arrow in b of confocal/STED (center pixel, left) and their SN2N results (right) under depletion laser powers. Different colors indicate different depletion laser powers. e , FRC analysis of the confocal/STED images (center pixel) before and after SN2N denoising under depletion laser powers. The black dashed line denotes the 1/7 FRC threshold. f , Average FWHM values of 5 × 5 sum confocal/STED, ISM/STED-ISM, center pixel confocal/STED, and center pixel SN2N results ( n = 6). g , STED snapshots of live COS-7 cells labeled with SiR-Tubulin (top), Lifeact-EGFP (middle), and Sec61β–EGFP (bottom) under a commercial STED microscope (Leica, Methods ). Left: the original STED images. Right: denoised images using SN2N. h , Raw STED frames (left) and SN2N (right) denoised counterparts of magnified views of the white-boxed regions in g . Three representative time points are provided from top to bottom. i , Three representative frames of PKMO labeled live COS-7 cells under another commercial STED microscopy (Abberior) at high depletion power (86%) and long duration time (100 μs per pixel). j , Examples STED raw images (left) and their SN2N results (right) at high depletion power (86%) and short duration time (10 μs per pixel). k , Photobleaching analysis of STED images used in i , j , and m , quantifying the normalized signal in each case. l , Fluorescence profiles along the white arrow in j imaged by STED and SN2N. j , Examples STED raw images (left) and their SN2N results (right) at high depletion power (86%) and short duration time (10 μs per pixel). m - p , Long-term STED imaging of PKMO labeled mitochondrial cristae in live COS-7 cells at high depletion power (41%) and short duration time (10 μs per pixel). m , Representative frames of STED (left) and RL-SN2N (right) results. n , Magnified views of the white boxed regions in m by raw STED, SN2N, and RL-SN2N. o , Representative montages of the mitochondrial fusion and fission events. The yellow and blue arrows highlight the regions of mitochondrial fusion and fission, respectively, while the white arrows indicate the moments before events. Centerline, medians; limits, 75% and 25%; whiskers, maximum and minimum; error bars, s.e.m. Experiments were repeated five times independently with similar results; scale bar, 2 µm ( a ), 1 µm ( b , g , h ), 500 nm ( i , m - o ).

Article Snippet: Long-term activities of mitochondrial cristae (PKMO labeled) were recorded by a commercial STED microscope (STEDYCON, Abberior Instruments, Germany) equipped with a wide-field objective (100×/1.45, CFI Plan Apochromat Lambda D, Nikon, Japan).

Techniques: Labeling, Fluorescence, Microscopy, Imaging