Another method of measuring scattering from subcellular particles is CLASS (confocal light absorption and scattering spectroscopic) microscopy. This third distribution was reported to be responsible for 80% of the scattering. This information is used in fitting the angular dependent light scattering of a cell to a trimodal distribution in which three distributions are identified, one with a mean of 0.6 μm being the lysosomes, a second distribution with a mean of 0.2 μm that is assigned to secretory granules and a third distribution with a mean of 1.3 μm that is assigned to mitochondria. The addition of a strong absorber that localizes in lysosomes was shown to reduce angular light scattering between the angles of 15° – 60°. Light scatter from lysosomes has been modulated in EMT6 cells. The locations of light scatter changes in bovine aortic endothelial cells treated with staurosporine (an inducer of apoptosis) have been demonstrated to be correlated with the location of MitoTracker Green fluorescence from mitochondria. Changes in light scattering have been correlated with both alterations in lysosomes and mitochondria. To investigate what structures scatter light, some investigators have taken the approach of modifying one type of organelle and then measuring whether the light scattering properties changed.
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For example, in human skin fibroblasts, mitochondria are 2.4% and lysosomes are more than 2.7% of the cell volume. In other cell types, mitochondria are much less of the cell volume. Hepatocytes are unusual in that mitochondria make up 28% of the cell volume. However, these results can not be extrapolated to other cell types. demonstrated that mitochondria are a major contributor to light scattering from liver cells. A fundamental question is: What physical features of the cell are scattering light? Many indirect and a few direct measurements have been performed. In developing light scattering techniques, it is helpful to understand the origin of light scattering. Clinical trials on several different tissue or organ sites have been reported: breast, lymph nodes, cervical tissue, oral tissue, and esophageal tissue. Recently, this fact has motivated the development of in vivo light scatter techniques for nonintrusive diagnosis of precancerous conditions.
Work in the 1970’s demonstrated that light scattering properties are related to cell morphology. Light scattering measurements provide information about cells without the need for stains or other perturbation that might affect the cell or hinder subsequent measurements. Rather lysosomes, nuclei and unknown particles all have significant contributions to 90° scattering and the contributions of some of these particles can be modulated by changing the polarization of the incident light. In conclusion, mitochondria were not found to be either the most efficient scatterer or to have the largest contribution to scattering in either cell line, in contrast to previous reports.
This dependence on side scatter polarization indicates that lysosomes contain scattering structures that are much smaller than the wavelength of light used in the measurements (785 nm). The contribution of lysosomes to side scatter was much stronger when the incident light was polarized perpendicular to the scattering plane than when the polarization of the side scatter laser was parallel to the scattering plane. The contributions of lysosomes, mitochondria, the nucleus, and particles unstained by either Hoechst, LysoSensor or MitoTracker ranges from ∼20% to ∼30% in fibroblast cells. The nucleus was the largest contributor to side scatter in the cervical carcinoma cells. The percent of 90° scattering from the nucleus, mitochondria, and lysosomes as well as the group of other internal cellular objects was estimated.
The relative scattering efficiencies of lysosomes and mitochondria were the same for both cell lines, while the scattering efficiencies of the nuclei differed. Lysosomes or nuclei are the most efficient type of scatterer depending on the cell type and incident light polarization. The origins of side scattering from a fibroblast and cervical cell line were determined by comparing side-scatter images with images stained for lysosomes, nuclei, and mitochondria on a cell by cell basis.
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