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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A012
Light microscopy is very appealing as an automated tool in biology and
medicine. Electronic imaging and computerized analysis, together with a
wide range of sensitive and specific labeling methods for live and fixed
cells and tissues, can generate multiparametric molecular and morphological
information at cellular and subcellular resolutions to be used in basic
research, and for diagnosis. There are a number of aspects that require
attention. First, one must rely on a small sample size. As compared to
cell sorters or biochemical essays, microscopic imaging can scan and
analyze thousands of cells, as compared to millions. The inherently wide
distribution of cellular parameters imposes severe constraint on the
statistical quality of the results. In addition, distribution of parameters
quantified from images often behave as fractals, meaning that the number
of small values increase fast, and merge into noise. This makes average
values practically meaningless, and requires application of complex
multivariable statistical methods. The instrumental aspects of quantitative
light microscopy are not less important, and new technology offers solutions
for current and future applications. Microscopes were designed for optimizing
a compromise between intensity and homogeneity of the illuminated field. A
factor of two fall in intensity at the edges of the field are common, but are
not acceptable for quantitative transmission or fluorescence imaging.
Spatially and temporally stable illumination enable calibration, but arc
lamps hot-spot jumps introduce large changes. Fiber scramblers and intensity
integration for each exposure solve this problem. Sensitive and wide
dynamic range CCDs with computerized image processing allow acquisition of
multicolor and time dependent pictures of fields. But image interpretation
software is not an off-the-shelf product. We have developed segmentation
algorithms to identify regions in DIC and fluorescence microscopy, followed
by morphological and intensity-integration and background-subtraction for
multiple colors, in addition to colocalization, energy transfer and ratio
imaging that produce quantitative visual data. Aspect of intensity-integration
in confocal and wide field microscopy will be discussed with application
to cell cycle analysis on single cell basis. Cell suspensions and blood
cells are relatively easy specimens for automated analysis. Confluent tissue
cultures, and sections are much less so. They require three dimensional
imaging, and 3D algorithms for image interpretation are rare, and time
consuming. They are essential though diagnosis of biopsies.
QUANTITATIVE MICROSCOPY: PROBLEMS AND SOLUTIONS
Kam Z
Molecular Biology of the Cell, Weizmann Institute of Science, Rehovot,
Israel