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A first step towards truly quantitative TIRF microsopy
Total internal reflection and other near-field optical techniques excel in selectively imaging dynamic processes at or near the basal plasma membrane, e.g., single-receptor or single-vesicle dynamics. However, it has been difficult to quantify TIRF intensities and relate them to axial fluorophore distance. Now, in Klimovsk et al. researchers from SPPIN’s team1 and the Salomon’s Lab at Bar-Ilan University describe a simple optical technique for simultaneously characterising the refractive index, flurophore distance and field-homogeneity of evanescent-wave excited thin fluorophore films. Test samples were assembled by combining “nanosandwiches” of thin (<200 nm) spacer layer made from a bioimimetic polymer and thin dye layers. Using such test slides with different thickness, they could quantify the flurophore distance with <10 nm resolution. The technique is based on the detection in the far-field and the analysis of the fluorphore radiation pattern in the back-focal plane of a high-NA objective.
Characterizing nanometric thin films with far-field light, Hodaya Klimovsky, Omer Shavit, Carine Julien, Ilya Olevsko, Mohamed Hamode, Yossi Abulafia, Hervé Suaudeau, Vincent Armand, Martin Oheim, Adi Salomon, Advanced Optical Materials, 2022.08.15.503956; doi: https://doi.org/10.1101/2022.08.15.503956