New approach to the study of cell cytotoxicity using high-resolution coherence phase-interference microscopy

摘要

An important role of immunological factors in the pathogenesis of many pathological conditions allows for their consideration as diagnostic and prognostic disease markers. A promising approach to the diagnosis of immunological abnormalities is based on the non-invasive cell diagnostic technology of quantitative phase imaging (QPI), with underlying principles of laser interferometry and holography. Our investigation is aimed to assess the QPI potential to analyze cell cytotoxicity and identify diagnostic criteria for cell immunity abnormalities in female patients with endometriosis. Measurements were performed in an original Cytoscan coherent-phase microscope (Russia). The optical scheme of the microscope is a modified Linnic microinterferometer optical scheme. He-Ne laser radiation (λ=633 nm, 1 mWatt) passes through a semi-transparent beam splitter, the object wave passes through the analyzed object twice, while the reference wave is reflected without any wave-front aberrations by a flat mirror fixed at a piezoconverter. We studied phase interference pictures of native lymphocytes to identify morphological and densitometry characteristics for CD8+cells. Two groups of cells were identified in the cytotoxic lymphocyte population, which differed in their values of the phase thickness (Hcyt) and the area of cytoplasm adjacent to the membrane and containing the perforin granules (Scyt). It was found, that in the healthy controls the proportion of perforin-positive cytotoxic CD8+lymphocytes was 37.1 ± 5.15%, in the patients with endometriosis 29.8 ± 6.34%. The reagent-free quantitative phase imaging technology (QPI), based on interference microscopy and holography, provides visualization, recording, monitoring, and multiparameter analysis of the data obtained in the study of living cells.