Centrifuge polarizing microscope. I. Rationale, design and instrument performance.
We first describe early uses of the centrifuge for deciphering physical properties and molecular organization within living cells, as well as the development and use of centrifuge microscopes for such studies. The rationale for developing a centrifuge microscope that allows high-extinction polarized light microscopy to observe dynamic fine structures in living cells is next discussed. We then describe a centrifuge polarizing microscope (CPM) that we developed for observing fine structural changes in living cells which are being exposed to up to approximately 11 500 times earth's gravitational field (g). With the specimen housed in a rotor supported on an air spindle motor, and imaged through an external microscope illuminated by a precisely synchronized flash of less than 10 ns duration from a Nd:YAG laser, the image of the spinning object remains steady up to the maximum speed of 11 700 rev min-1, or up to approximately 11 500 x g. The image is captured, at up to 25 frames s-1, by an interference-fringe-free CCD camera that is synchronized to the centrifuge rotor. At all speeds (in 100 rev min-1 increments), the image is resolved to better than 1 microm, while birefringence of the specimen, housed in a specially designed specimen chamber that suffers low-stress birefringence and prevents leakage of the physiological solutions, is detected with a retardance sensitivity of better than 1 nm. Differential interference contrast and fluorescence images (532 nm excitation) of the spinning specimen can also be generated with the CPM. The second part of this study (Inoué et al., J. Microsc. 201 (2001) 357-367, describes several biological applications of the CPM that we have explored. Individual live cells, such as oocytes and blood cells, are supported on a sucrose or Percoll density gradient while other cells, such as cultured fibroblasts and Dictyostelium amoebae, are observed crawling on glass surfaces. Observations of these cells exposed to the high G fields (centripetal acceleration/g) in the CPM are yielding many new results that lead to intriguing questions regarding the organization and function of fine structures in living cells and related quasi-fluid systems.
Marine Biological Laboratory, Woods Hole, MA 02543, U.S.A.
S
S
Inoué
R A
RA
Knudson
M
M
Goda
K
K
Suzuki
C
C
Nagano
N
N
Okada
H
H
Takahashi
K
K
Ichie
M
M
Iida
K
K
Yamanaka
D000818:Animals; D001718:Birefringence; D002498:Centrifugation / Q000295:instrumentation; D004867:Equipment Design; D051379:Mice; D008859:Microscopy, Polarization / Q000295:instrumentation* / Q000379:methods