Computational protocol: Label-Free Determination of Hemodynamic Parameters in the Microcirculaton with Third Harmonic Generation Microscopy

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Protocol publication

[…] Images were processed in ImageJ (http://imagej.nih.gov/ij/) or the derivate Fiji (http://fiji.sc/Fiji) and/or in Imaris 7 (Bitplane, Zürich, Switzerland). Point spread functions were calculated with the ImageJ macro “MIPs for PSFs all microscopes” kindly provided by Laurant Gelman, Basel (http://www.imaging-git.com/science/light-microscopy/routine-assessment-fluorescence-microscope-performance). Graphs and p-value calculations were made with Prism 6 (GraphPad Software, La Jolla, California). Figures for publication were assembled in Photoshop 9 (Adobe Systems, Mountain View, California). For presented micrographs, each color channel was mapped from the original 14 bit to 8 bit such that maximum detail was visible. No further processing was applied, except where explicitly noted in the respective figure legend. [...] RBC displacements between pairs of line-scans were determined using spatial cross-correlation analysis. They were computed with a MATLAB (The MathWorks, Inc) routine developed and generously made publicly available by Kim et al. . The program was adapted in minor points such as to write results in a table for import in spreadsheet software, where the conversion from pixels to millimeters and seconds was performed. For arterioles, every 5th time point was evaluated, for venules and capillaries every 10th. When the signal intensity decreased in the center of a vessel, the LS-PIV algorithm tended to calculate artificial velocities lower than in the vessel periphery. In some cases this could be avoided by careful visual inspection of x-t-diagrams and restriction of the evaluation to those areas of the scan line which showed visually detectable RBC streaks. […]

Pipeline specifications

Software tools ImageJ, Imaris, LS-PIV
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Xenopus laevis, Mus musculus