Abstract
Transformation in chromatin organization is one of the most universal markers of carcinogenesis. Microscale chromatin alterations have been a staple of histopathological diagnosis of neoplasia, and nanoscale alterations have emerged as a promising marker for cancer prognostication and the detection of predysplastic changes. While numerous methods have been developed to detect these alterations, most methods for sample preparation remain largely validated via conventional microscopy and have not been examined with nanoscale sensitive imaging techniques. For these nanoscale sensitive techniques to become standard of care screening tools, new histological protocols must be developed that preserve nanoscale information. Partial Wave Spectroscopic (PWS) microscopy has recently emerged as a novel imaging technique sensitive to length scales ranging between 20 and 200 nanometers. As a label-free, high-throughput, and non-invasive imaging technique, PWS microscopy is an ideal tool to quantify structural information during sample preparation. Therefore, in this work we applied PWS microscopy to systematically evaluate the effects of cytological preparation on the nanoscales changes of chromatin using two live cell models: a drug-based model of Hela cells differentially treated with daunorubicin and a cell line comparison model of two cells lines with inherently distinct chromatin organizations. Notably, we show that existing cytological preparation can be modified in order to maintain clinically relevant nanoscopic differences, paving the way for the emerging field of nanopathology.
Highlights
Over the past few decades, despite a tremendous amount of research into discovering new molecular targets and improving precision therapies, cancer remains a leading cause of death worldwide
By quantifying the standard deviation (S) of the spectra acquired by Partial Wave Spectroscopic (PWS) microscopy, PWS obtains subdiffractional information from cellular structures, in particular the internal structures of nucleus
Length scale sensitivity of PWS depends on the illumination and light collection geometry of the microscope and is typically optimized to sense the chromatin length scales that correspond to the supranucleosomal chromatin structure from the size of chromatin chains to the size of topologically associated domains are the most significantly altered in early carcinogenesis (20-350nm; from ~1kb to 1– 10Mbp) [9, 17, 19]
Summary
Two models were developed for the analysis of cytological preparation on cellular ultrastructure. The first is a drug-based model of Hela cells differentially treated with 10μM daunorubicin for 15 minutes in order to induce alterations in higher-order chromatin structure potentially due to nucleosomal eviction [12]. The second is a cell line comparison model of two cell lines with inherently distinct chromatin organizations. The two cell lines compared are originally derived from A2780 and A2780.m248 lines (STR profiles included in SI). Nanoscopic changes in chromatin folding were detected and quantified by livecell PWS microscopy prior to nanocytological preparation.
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