Biomolecules can be investigated at the nanoscale with quantitative single molecule localization microscopy (qSMLM). This technique, which achieves single molecule sensitivity, can probe how membrane receptors are organized under both normal and pathological conditions. While a number of receptors have been extensively studied in cultured cells, technical challenges have largely impeded their robust quantification in tissue samples. To rigorously interrogate tissue samples, methodological advancements are needed in three areas: analytical preparation of the sample, proper characterization of fluorescent reporters, and rapid/unbiased data analysis. Towards these ends, we have combined qSMLM with a touch preparation technique (touch prep-qSMLM). In this new method, touch prep is first used to obtain monolayers of patient cells. Then, highly selective, fluorescently labeled probes are used to detect the receptors of interest on the plasma membranes of cells. Finally, quantitative algorithms are used to analyze the imaging data. Using this touch prep-qSMLM methodology, we interrogated the density and nano-organization of human epidermal growth factor receptor 2 (HER2) in fresh breast cancer tissues. Touch prep-qSMLM agreed well with current clinical methods. Importantly, touch prep-qSMLM can be easily extended to other pathological conditions and ultimately used in precision medicine.
Keywords: Breast cancer; Human epidermal growth factor receptor 2; MATLAB data analysis; Quantitative single molecule localization microscopy; Touch preparation.
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