https://doi.org/10.1051/epjconf/202328709034
Colour-coded nanoscale calibration and optical quantification of axial fluorophore position
1 Chemistry department, Bar-Ilan University, 529000, Ramat-Gan, Israel
2 Institute of Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, 529000, Ramat-Gan, Israel
3 Université de Paris, CNRS, SPPIN – Saints-Pères Paris Institute for the Neurosciences, Paris, France
* Corresponding author: author@e-mail.or
Published online: 18 October 2023
Total internal reflection fluorescence (TIRF) has come of age, but a reliable and easy-to-use tool for calibrating evanescent-wave penetration depths is missing. We provide a test-sample for TIRF and other axial super-resolution microscopies for emitter axial calibration. Our originality is that nanometer(nm) distances along the microscope’s optical axis are color-encoded in the form of a multi-layered multi-colored transparent sandwich. Emitter layers are excited by the same laser but they emit in different colors. Layers are deposited in a controlled manner onto a glass substrate and protected with a non-fluorescent polymer. Decoding the penetration depth of the exciting evanescent field, by spectrally unmixing of multi-colored samples is presented as well. Our slide can serve as a test sample for quantifying TIRF, but also as an axial ruler for nm-axial distance measurements in single-molecule localization microscopies, supercritical-angle fluorescence, and related super-resolution.
© The Authors, published by EDP Sciences, 2023
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
