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, Quantitative evaluation of software packages for single-molecule localization microscopy Nat Meth advance online publication. [24] Herbert A o Available from: http://www.sussex.ac.uk/gdsc/intranet/microscopy/imagej/smlm_plugins Supplementary Information Supplementary Figure S1. Dual-color imaging under a single-wavelength excitation. A) The absorbance spectra of both JD165 and JD193 FONs partially overlap allowing for a single excitation laser beam between 400 and 500 nm (in this paper, a 488 nm illumination is used, shown as a lightblue line). B) Since the emission spectra of both FONs only slightly overlap, their fluorescence can be spectrally separated thanks to a 655 nm dichroic beam-splitter and optimized emission filters. C) Both imaging channels were designed to avoid any cross-talk between them when imaging either or both types of FONs For each image, the detection is split in two Left: NIR emission detection Right: Green emission detection, Top row: JD165 FONs in water. Middle row: JD193 FONs in water. Bottom row: Mixed JD165/JD193 FONs, 2015.
, Detection and tracking of single green FONs in water JD165 FONs were imaged freely diffusing in water. For each frame, detected point-spread functions were fitted with 2D- Gaussians (left, colored boxes) and fitted localizations tracked in trajectories (right, same color code)
, Detection and tracking of single NIR FONs in water JD193 FONs were imaged freely diffusing in water. For each frame, detected point-spread functions were fitted with 2D- Gaussians (left, colored boxes) and fitted localizations tracked in trajectories (right, same color code)
, Simultaneous dual-color imaging of green and NIR FONs under a monochromatic excitation JD165 and JD193 FONs were mixed (4:1, v/v) and imaged freely diffusing in water under 488 nm inclined illumination. The detection was split in two channels thanks to a 655 nm dichroic beam-splitter, and represented superposed (NIR channel in red, green channel in green) One frame of the movie is shown in Figure S1, C (bottom row) Scale bar, p.5
, Simultaneous detection and tracking of green and NIR FONs in a living COS7 cell. COS7 cells were incubated with JD165 and JD193 FONs for 2h, washed and imaged live under 488 nm inclined illumination. For each channel of the dual-view (top and bottom rows correspond to the NIR and green channels, resp.), emitting nanoparticles were detected, fitted with a 2D-Gaussian (colored boxes in the left column) and tracked between consecutive frames (ongoing traces in right column) One frame of the movie is shown in Figure 6, C&D. A contour of the imaged cell is given in Figure 6, p.5
, Superposed trajectories in a living COS7 cell. Tracks of JD165 and JD193 FONs observed in living COS7 cells in Movie S4 are represented superposed (red and green traces for the NIR and green channels, resp.). To superpose the tracks from both channels, a simple linear transformation was applied to the super-resolved coordinates. One frame of the movie is shown in Figure 6