There seems to be no end to the stream of optical technologies hitting the market. Cambridge Research & Instrumentation (CRI) Inc., of Woburn, Mass., has developed CellView and SpindleView imaging systems to apply the company's LC-PolScope™ technology1,2 to the subcellular organization of living cells without stains or fluorescent labels.
Many subcellular structures are oriented polymers that are spatially organized, or anisotropic. This anisotropy causes the speed of light to vary in different spatial directions within these biomaterials, giving rise to two distinct refractive indices. Using these refraction indices, the relative orientation and even the number of filaments of a polymer may be estimated (based on thickness effects).
CRI Inc.'s LC-PolScope systems convert a light microscope into a quantitative retardance imager. Paired electro-optical liquid crystal retarders replace cumbersome mechanical polarizers and generate orientation-independent polarization images (360° view), eliminating moving parts and giving images in perfect register. Only a few seconds are needed to obtain four differentially polarized images to generate a retardance image. The process uses patented algorithms and produces a quantitative gray-scale or color-coded value for retardance and azimuth orientation of the slow axis (i.e., where light travels more slowly). The images, which have dark backgrounds similar to fluorescence-based ones, can be observed with the eyepiece or displayed on a computer monitor. Spatial resolution with high-NA lenses could be as good as 0.2 µm.
Using the contrast-enhancement intrinsic to the PolScope, CellView can image subcellular structures such as actin and tubulin filaments3 or nerve growth cones.4 An important application for the alternative SpindleView system is visualizing the meiotic spindle and zona pellucida of eggs being fertilized in vitro by intracytoplasmic sperm injection (ICSI). The technique gives substantially better intracellular detail than other nonlabeling methods and helps to preserve egg viability. According to Edward D. Salmon, professor of biology at the University of North Carolina, this technology can be applied to single-polymer detection and used to further understand cell division mechanisms such as intrachromosomal structure.
Quantitative, noninvasive techniques always seem in short supply for the increasing demands of live-cell experimentation. CRI's CellView and SpindleView systems have opened a new chemical-free window into the depths of the cell.
--Jorge Cortese (Jorge_Cortese@mindspring.com)
1. R. Oldenbourg, G. Mei, "New polarized light microscope with precision universal compensator," Journal of Microscopy, 180(2):140-7, 1995.
2. R. Oldenbourg, "A new view on polarization microscopy," Nature, 381:811-2, 1996.
3. R. Oldenbourg et al., "Birefringence of single and bundled microtubules," Biophysical Journal, 74:645-54, 1998.
4. K. Katoh et al., "Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones," Molecular Biology of the Cell, 10:197-202, 1999.
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