Abstract
Through the formation of fluorescent self-interference (SELFI), quantitative intensity and phase imaging enables the 3D localization of single fluorescent molecules inside a fixed tissue with an accuracy well beyond the diffraction limit.. Here we demonstrate that this concept can be extended to 3D super-resolution microscopy and 3D single particle tracking in various living samples ranging from adherent cells to organotypic brain slices, using diverse fluorescent emitters (fluorescent proteins, organic dyes or quantum dots). This basically covers the most popular single molecule imaging techniques used for live sample studies. We also show that SELFI can be used in combination with different illumination schemes including highly inclined illumination and total internal reflection.
Highlights
Single molecule super-localization has revolutionized the field of quantitative biology, providing both images and molecular specificity with resolutions well-beyond the diffraction limit [1,2,3]
This work demonstrates that SELFI can be combined with the most common super-localization methods developed for live sample imaging [(u)PAINT, Photoactivated light microscopy (PALM), single particle tracking (SPT)]
Since SELFI obtains the 3D information without causing significant photon loss, it is possible to apply this concept to single emitters having limited brilliance such as fluorescent proteins as used in PALM, or nanoparticles deep inside thick living tissues
Summary
Single molecule super-localization has revolutionized the field of quantitative biology, providing both images and molecular specificity with resolutions well-beyond the diffraction limit [1,2,3]. To access thicker samples like 3D multicellular structures (organoids), we recently proposed a new 3D super-localization approach employing quantitative phase and intensity measurements. This technique, named SELFI, is based on fluorescent self-interferences [24] and can be considered as the transposition of lateral shearing interferometry [25, 26], a well-established technique for quantitative phase microscopy, to the field of 3D fluorescence microscopy. It was only demonstrated within fixed organoids and combined with direct Stochastic Optical Reconstruction Microscopy (dSTORM) [24] using AlexaFluor 647 which possesses optimal
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