Real-time imaging during centrifugation
People who lose blood or whose bodies
aren’t producing enough can receive transfusions. However, they sometimes
need only certain blood components, such as red blood cells, plasma and platelets.
In these cases, centrifugation is used to separate the components from the whole
But does centrifugation alter the blood? Studies
comparing human red blood cells before and after centrifugation have shown that
the procedure doesn’t affect the shape or volume of the cells. We still do
not know, however, whether and how these change during centrifugation. Researchers
at Yale University in New Haven, Conn., and at the Marine Biological Laboratory
in Woods Hole, Mass., have described changes in human and Amphiuma salamander
red blood cells during centrifugation using a specially designed microscope.
The study was made possible, said Yale
researcher Joseph F. Hoffman, by a centrifuge polarizing microscope. Developed
by Shinya Inoué of the Marine Biological Laboratory in collaboration with Olympus
America Inc. of Melville, N.Y., and Hamamatsu Photonics KK of Hamamatsu City, Japan,
the instrument enables video observation at the microscopic level, and thus allowed
them to record stratification of the various components in living cells during centrifugation.
A timing circuit triggers a 6-ns pulse from a 532-nm Nd:YAG laser as individual
cells pass below a 40x, 0.55-numerical-aperture objective. A photodiode acquires
sequential images with a resolution of 0.5 μm.
Hoffman and Inoué observed the red blood
cells suspended in solutions and spinning at rates of 200 to 11,700 rpm. They analyzed
the data by playing back the video sequences at various speeds and listening to
audio commentary that they had recorded in real time.
The experiments demonstrated that centrifugation
changes a red cell’s shape and the distribution of its various components,
but that this is completely reversible — indicating that the changes cause
In human cells, the researchers observed
two major changes in the cells’ appearance at higher centrifugation speeds.
First is a change of shape caused by sedimentation of hemoglobin inside the cell.
This leads to a bulging of the lower part of the cell, producing a sort of baglike
shape. Here, however, the upper part of the cell maintains its original biconcave
(dimpled) form, suggesting that the original shape is not dependent on the presence
of hemoglobin. In any event, the cells immediately revert to the original shape
when they touch cells in the classical rouleau formation, in which cells are stacked
on top of one another like a roll of coins.
Experiments with the specially designed microscope revealed significant
shape changes during centrifugation, particularly at high speeds. Sedimentation
of hemoglobin molecules led to a bulging in the lower parts of the cells. Because
the upper parts remained relatively thin, the cells took on baglike aspects.
Reprinted with permission of PNAS.
Changes in the salamander red cells were different, although they also proved reversible.
The most interesting question remains,
however: What is the basis of the cells’ normal, biconcave shape? This question
has been asked since 1827, Hoffman said, when use of a new microscope, corrected
for chromatic aberration, definitively established the biconcave shape of red blood
cells. Studying shape transformations of abnormal cells with the centrifuge polarizing
microscope might help provide an answer, he added.
PNAS, Feb. 21, 2006, pp. 2971-2976.
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