Fig. 4From: Multispectral intravital microscopy for simultaneous bright-field and fluorescence imaging of the microvasculatureComposite picture of a 9 s image sequence of 150 images (red and green channel). (A): left image: The composite image shows the path (white arrow A) of a moving microbead observed in the fluorescent > 600 nm optical channel. A group of non-moving, adherent microbeads is also visible (white arrow B); right image: Velocity of microbead A in left image of panel (A). The distance of the microbead was estimated in each pair of successive images. Velocity was computed by determining the traveled distance in the interframe interval. (B), (C) and (D): Intravital microscopy image sequence of a microbead (streak) moving in the bloodstream’s centerline. With an image exposure time of 40 ms, this free-flowing fluorescent microbead’s average velocity was 468 μm/s (range: 270–710 μm/s). Arrows in image sequences (B and D), displaying the location of the fluorescent bead only visible in image sequence (C) (yellow arrow). Bright-field illumination wavelength range: 400–550 nm (B). Filters: band-pass filter: 565/24 nm (C) and long-pass filter: 600 nm (D). Microbeads: excitation 488 nm / emission 645 nm and excitation 540 nm/ emission 560 nm. Exposure: 40 ms; frame rate: 16.7 fps. Please note that since all images are captured on monochrome cameras in the MSIVM system, no color images can be generated directly. We used image processing to add color for visualization purposes only. White dotted lines indicate the approximate position of the vascular wallBack to article page