Time shifting interferences for in depth tissue imaging in single molecule localization microscopy

In Single Molecule Localization Microscopy, the positions of the fluorophores are obtained from a fitted Point Spread Function. This spatially based localization precision will then strongly depends on the PSF shape, which can be degraded by defocusing and aberrations. We proposed a new localization method called ModLoc where the uniform excitation is replaced by a time-varying structured illumination over the entire field of view. The illuminated fluorophores will then have a modulated emission where the phase shift encodes their position. The demodulation requires extracting four intensities values for each single molecule event, and as emitters can exhibit fast ON-time dedicated strategies must be implemented. As a sequential acquisition is too slow and would discard events, we have developed dedicated detection by introducing an active optical element in front of the camera. The kHz modulation induced by the pattern shift is sampled on four different subarrays acquired simultaneously by the camera, this permits to achieve fast demodulation of all emitters without losing events. This technique can be implemented in different directions. When it is applied in the z direction, it provides a unique uniform axial precision not only within the objective capture range but also up to several microns in depth, allowing a cell to be imaged with uniform precision. We will discuss the various implementations of ModLoc and present results on complex samples such as organoids, or fibers in mouse muscle. The recent work on complex samples up to 40 microns deep will be described and the results will be shown. We will also present how multiple targets labeled with dyes presenting close spectrum (CF647/CF680) can be retrieved thanks to a specific implementation of spectral demixing. In depth multitarget imaging will be presented in cells and tissues.