Fig. 1: Principles of ROCS microscopy. | Nature Communications

Fig. 1: Principles of ROCS microscopy.

From: 100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales

Fig. 1

a Setup scheme of ROCS microscope with azimuthal laser beam rotation at 100 Hz incident under high polar angles θ. Scattered laser light is imaged onto a camera integrating over sweep time. b ROCS images form upon the full 360° sweep of a laser beam. Coherent images from different azimuthal directions (see blue arrows) form speckle patterns, which generate the final ROCS image by incoherent superposition. c Wavelength spectra I(λ) for ROCS illumination and detection at the same wavelength (405 nm or 445 nm), without exciting fluorescence. Fluorescence excitation with laser (560 nm), fluorescence emission at orange wavelengths. d Four possible illumination and detection modes in ROCS microscopy, where TIR-ROCS in darkfield mode provides the highest contrast. e Parallel TIR-imaging of 200 nm PS beads in fluorescence mode (green at 505 nm, at NA = 1.46) and ROCS mode (blue at 405 nm, NA = 1.2). Spatial frequency spectra for TIR-F and TIR-ROCS are averaged in azimuthal direction (logarithmic intensity, STD in light gray), shown by green and blue line scans. TIR-F provides 240 nm spatial resolution, TIR-ROCS achieves 160 nm. f Kymograph I(s,ϕ) along direction s (dashed line in ROCS image). For totally n = 72 raw images from different azimuthal angles ϕ the speckle patterns show limited angular correlations. Below: Six line scans I(s,ϕn) at six angles with resulting sum intensity. g Comparison of image contrast for different imaging modes of a HeLa cell. Left to right: TIR-ROCS image (linear intensity) recorded within 10 ms at 445 nm with large dynamic range. The logarithmic intensity image shows lower intensity structures (filopodia) with strong signal contrast. The TIR-F image (200 ms integration time) with Lifeact-mCherry actin labeling reveals similar, but not the same structures (see ROI). An incoherent epi-image (inverted) of a blue LED (435 nm) reveals again different cellular details at low contrast and dynamic range (purple line). Source data are provided as a Source Data file.

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