Quantitative videomicrofluorometry: empirical methods for an automatic determination of the threshold level for fluorescent objects and application to living lymphoblastoid cell lines

Jean Vigo; Pierre M. Viallet; Jean-Marie Salmon

doi:10.1117/12.297966

29 December 1997 Quantitative videomicrofluorometry: empirical methods for an automatic determination of the threshold level for fluorescent objects and application to living lymphoblastoid cell lines

Jean Vigo, Pierre M. Viallet, Jean-Marie Salmon

Author Affiliations +

Proceedings Volume 3197, Optical Biopsies and Microscopic Techniques II; (1997) https://doi.org/10.1117/12.297966
Event: BiOS Europe '97, 1997, San Remo, Italy

Abstract

Analytical methods on individual cells are essential for studying both the functions of normal cells and the possibility to monitor pathological cells. The development of some polyvalent techniques like flow cytometry and fluorescence imaging spectroscopy has led to more and more accurate methods for analyzing heterogeneous cell populations. Unfortunately, most of the quantitative morphometric or biological parameters extracted from fluorescence measurements rely on the accuracy of the determination of the surface of the biological objects (whole cell, nucleus, . . .). That determination is a difficult task for each molecule of the fluorescent marker act as a secondary light source emitting in a solid angle of 360 degrees. As a result the spot of any fluorescent object appears larger than the shadow that it displays on an image obtained with absorption techniques. Furthermore the limits of the fluorescent spot are more difficult to determine automatically due to a lack of sharp change in the intensity profile of the spot. Of course this problem is more important when images are recorded with a low magnifying objective, which is generally the case when people want to harvest data on a large number of cells. In that case it is necessary to have an accurate but also automatic protocol allowing to standardize the way that the surface of fluorescent objects are measured. The problem has been previously addressed by suggesting different mathematical protocols allowing an easier determination of the convenient thresholding level. In this paper are suggested two empirical solutions, one usable for the nuclei and the other one for the whole cell delineation. Of course these protocols are usable only when the fluorescent images have been corrected from all the potential distortions introduced by the equipment.

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Jean Vigo, Pierre M. Viallet, and Jean-Marie Salmon "Quantitative videomicrofluorometry: empirical methods for an automatic determination of the threshold level for fluorescent objects and application to living lymphoblastoid cell lines", Proc. SPIE 3197, Optical Biopsies and Microscopic Techniques II, (29 December 1997); https://doi.org/10.1117/12.297966

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