Abstract
A light-sheet-based 2D light scattering cytometer is developed for label-free characterization of senescent cells. The light-sheet provides an illumination beam with controlled thickness for single cell excitation, and 2D light scattering patterns are obtained by using a defocused imaging method. The principle of this cytometer is validated by distinguishing microspheres with submicron resolution. Automatic classification of senescent and normal cells is achieved at single cell level by using the support vector machine (SVM) algorithm, where a sensitivity of 89.1% and a specificity of 96.4% are obtained. Our results suggest that the light-sheet-based 2D light scattering label-free cytometry has the capability to perform size differentiation of beads with submicron resolution and to classify different groups of cells without fluorescent labeling, showing the potential for clinical diagnosis of senescence-related diseases.
Highlights
Cell senescence plays an important role in understanding of age-related diseases and tumorsuppressor mechanism [1,2,3]
The light sheet has a large extent in the x and y directions, which may cause background scattering that can be detected by the microscope objective with an numerical aperture (NA) of 0.4
Experiments on standard polystyrene microspheres of 3.87 and 4.19 μm in diameter were performed, and 2D light scattering patterns were obtained by using a 20× microscope objective with an NA of 0.4, which has a resolution of about 810 nm for size differentiation in conventional microscopy
Summary
Cell senescence plays an important role in understanding of age-related diseases and tumorsuppressor mechanism [1,2,3]. Conventional methods for the identification of senescent cells mainly depend on biomarkers [4, 5]. The most extensively used method for senescent cell detection is to measure the SA-β-gal positive activity by histochemical staining [6, 7]. The presence of senescence-associated heterochromatin foci (SAHF) can be used for the identification of senescent cells by detection of DNA dyes [8]. The morphometric analysis depending on the changes in nuclear geometry and DNA distribution has been proposed as a new indicator of cell senescence, which can be measured in a laser scanning cytometry with the aid of fluorescence labeling [10]
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