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Fig. 6. Capillary blood fl ow measurements with two-photon microscopy. Line scans are
used to acquire space-time images of capillary red blood cell (RBC) fl ow. The laser focus
is placed in the center of a straight part of a capillary as illustrated in the upper part of the
fi gure and scanned along the axis (30 μ m length). The fl uorescent dye (rhodamine-
dextran) labels the plasma ( red ) and blood cells appear as nonfl uorescent black interposi-
tions. The line is repetitively imaged for 1 s to acquire a space (horizontal axis)-time
(vertical axis) image. An individual RBC is scanned several times as it moves along the
vessel and creates a slanted stripe on the image. The velocity of individual RBCs is given
by the slope of this stripe and the blood cell fl ux is given by the number of RBCs per sec-
ond. RBC fl ux can be calculated by analyzing the variance of pixel intensity at a certain
column of the image (shown for the dotted line of the low-fl ow image). Detection of
regions with very low pixel intensity allows for automated RBC counting ( arrowheads ) .
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