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Marine Biological Laboratory, Woods Hole, Massachusetts
AIST, Tokyo, Japan
We reported earlier on a centrifuge polarizing microscope (CPM) that was designed for observing the weak birefringence of organelles and fine structures in living cells as they became stratified and oriented under centrifugal fields of up to 10,500 times Earth’s gravitational field. In this earlier model (1), one chamber A contained the specimen under observation, while the contents of the opposed second chamber B, which acted solely to balance the rotor, could not be viewed. We have now improved the CPM so that either chamber can be viewed and selected at the flick of a lever, within the duration of a video frame. In the CPM, an electronic timing circuit synchronizes the firing of the light source laser precisely to the transit of the specimen under the microscope (freezing the image to less than 0.5-µm specimen motion at up to 11,700 rpm, regardless of the speed of the 16-cm diameter rotor). The timing circuit, in turn, is triggered by the signal from a photodiode that picks up the light originating from a stationary diode laser, and reflected by a small mirror (M1) mounted on the spinning rotor near its axis. The complexity of the electronic timing circuit led us to keep the electronic circuit undisturbed and instead to devise an optical system for switching between the display of the two chambers. To this end, we installed a second timing mirror (M2) on the rotor, exactly opposite the one for chamber A, but tilted up by a few degrees, rather than oriented parallel to the rotor axis as is M1. In front of the photodiode we also placed a mounted pair of small mirrors on a "beam switcher" that could either be flipped up out of the way so that the photodiode would capture the light reflected from M1, or flipped down into position so that light reflected from the tilted mirror M2 would be reflected by the mounted pair of beam-switcher mirrors and enter the photodiode. Thus, depending on the position of the beam switcher, the timing light would enter the photodiode, reflected either from mirror M1 or M2. The timing circuit would then trigger the light source laser at precisely (to within a few nanoseconds) the time point required to display a stable image of the specimen in chambers A or B. The response time of the electronic timing circuit and laser firing device turned out to be so short that no video frames were lost in flipping the beam switcher and capturing the images from either of the two chambers.
Figure 1, left panel, shows the recorded image of sea urchin eggs stratified in a density gradient in chamber A, while the right panel shows density-standard beads (Nycomed Amersham, Oslo, Norway) that reveal the gradient of the identically prepared seawater/Percoll mix in chamber B. As the figure shows, the density of the unfertilized Arbacia eggs is approximately 1.060. Immediately after fertilization, the negative birefringence disappears from the membranes stacked in the upper half of the clear zone of the stratified eggs (Figs. 1 and Ref. 2). Concurrently, the (de-jellied) egg becomes lighter over the next minute, presumably by influx of water, and starts to float upward in the gradient until its density is somewhat less than 1.040.
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We thank Hamamatsu Photonics K.K. and Olympus Optical Co. for generous support of this project.
Literature Cited
This article has been cited by other articles:
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  J. F. Hoffman and S. Inoue Directly observed reversible shape changes and hemoglobin stratification during centrifugation of human and Amphiuma red blood cells PNAS, February 21, 2006; 103(8): 2971 - 2976. [Abstract] [Full Text] [PDF]
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