Digital Microscopy

Central to cytometry in bioimaging is digital microscopy, the digitization of images of biological states and processes.

These images may be acquired from optical (both emission and transmission: wide-field, scanning, confocal, evanescent wave, fluorescence, or phosphorescence) microscopes as well as, scanning and transmission electron microscopes. Image processing and analysis are coupled to digital microscopy to achieve quantitation, 2- and 3D restoration, as well as object and pattern recognition (cf image processing).  All of these microscopies add high-resolution spatial dimensions to cytometry which is missing from flow measurements. This spatial information may be used to distinguish objects or shape, to determine number (ref. 12) or surface properties, as in evanescent wave microscopy (ref. 15). 3-D reconstructions can be achieved from image processing of confocal or wide-field images. (cf. image processing)

The spatially and spectrally-resolved fluorescence digital microscope can be used for: gene and chromosome analysis (ref. 5, 9, 11; cf.  cytogenetics)  distinguishing intracellular metabolism or compartments, or pH and ion gradients  (ref. 1,4, 6, 16; cf cell metabolism)  The temporally-resolved (fluorescence) digital microscope can be used for kinetic processes (ref. 2) FRAP (fluorescence recovery after photobleaching) for diffusion or trafficking measurements FCS/M (fluorescence correlation) for diffusion measurements (ref. 13) The time/frequency-resolved fluorescence digital microscope can be used for: phosphorescence imaging (ref. 8, 14),   fluorescence lifetime imaging (ref. 7, 10), which can include proximity determinations through FRET (fluorescence resonance energy transfer)

References

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2. Bedner, E., Melamed, M. R. and Darzynkiewicz, Z. (1998). Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry. Cytometry 33, 1-9.
3. Bocker, W., Rolf, W., Bauch, T., Muller, W. U. and Streffer, C. (1999). Automated comet assay analysis. Cytometry 35, 134-144.
4. Bour-Dill, C., Gramain, M. P., Merlin, J. L., Marchal, S. and Guillemin, F. (2000). Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant MCF-7 cells using fluorescence microscopy image analysis. Cytometry 39, 16-25.
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6. Catlin, M. C., Kavanagh, T. J. and Costa, L. G. (2000). Muscarinic receptor-induced calcium responses in astroglia. Cytometry 41, 123-132.
7. Hanley, Q., Subramaniam, V., Arndt-Jovin, D. and Jovin, T. (2001). Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression. Cytometry in press  MS.  3494R.
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10. Murata, S., Herman, P., Lin, H. J. and Lakowicz, J. R. (2000). Fluorescence lifetime imaging of nuclear DNA: Effect of fluorescence resonance energy transfer. Cytometry 41, 178-185.
11. Poon, S. S. S., Martens, U. M., Ward, R. K. and Lansdorp, P. M. (1999). Telomere length measurements using digital fluorescence microscopy. Cytometry 36, 267-278.
12. Ramsden, J. J., Li, S. Y., Heinzle, E. and Prenosil, J. E. (1995). Optical method, for measurement of number and shape of attached cells in real time. Cytometry 19, 97-102.
13. Schwille, P., Korlach, J. and Webb, W. W. (1999). Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes. Cytometry 36, 176-182.
14. Tanke, H. J., Dehaas, R. R., Sagner, C., Ganser, M. and Vangijlswijk, R. P. M. (1998). Use of platinum coproporphyrin and delayed luminescence imaging to extend the number of targets fish karyotyping. Cytometry 33, 453-459.
15. Usson, Y., Guignandon, A., Laroche, N., Lafageproust, M. H. and Vico, L. (1997). Quantitation of cell-matrix adhesion using confocal image analysis of focal contact associated proteins and interference reflection microscopy. Cytometry 28, 298-304.
16. Vandeneijnde, S. M., Luijsterburg, A. J. M., Boshart, L., Dezeeuw, C. I., Vandierendonck, J. H., Reutelingsperger, C. P. M. and Vermeijkeers, C. (1997). In situ detection of apoptosis during embryogenesis with annexin v - from whole mount to ultrastructure. Cytometry 29, 313-320.