Increase the fluorescence signal

Selective Plane Illumination Microscopy (SPIM) or light sheet microscopy is currently the fastest imaging technique suitable for live imaging of voluminous biological samples with low phototoxicity. The samples and capillary, typically used as a sample holder, induce aberrations that limit the optical quality of the imaging system. Adaptive Optics proved to be very effective correcting this sort of aberrations. It drastically improves the resolution and contrast of the image and allows to reach deeper layers of the biological sample.

Light sheet excitation is also becoming an increasingly popular technique in single molecule localization microscopy (SMLM), where it excites fluorescence in only a thin section of the sample, instead of in the whole volume. For example, thanks to a specially designed sample holder, single objective SPIM (soSPIM) technique uses the same objective lens to send the light-sheet excitation to the sample and to collect the emitted fluorescence signal. Due to an increased signal-to-noise ratio, this method allows the user to reach deeper layers of the sample, where correcting aberrations becomes essential in order to maintain the imaging quality.

Figure caption: (A) Schematic representation of the soSPIM imaging system combined with the MicAO adaptive optics system from Imagine Optic for system and sample induced aberration correction, enabling in-depth 3D single molecule imaging. (B) Representation of the acquired volume 12 µm above the coverslip surface with an objective depth of focus of 1µm. (C) Raw single molecule DNA-PAINT data illustrating the 60nm RMS astigmatism induced with the deformable mirror of the MicAO system in order to allow for 3D single molecule localization. (D) Depth-coded projection of the localization acquired within the volume shown in (B) of the protein Lamin-B1, demonstrating the 3-D shape of the nuclear envelope in non-adherent cells.

* soSPIM was jointly developed by CNRS, University of Bordeaux and the National University of Singapore by Rémi Galland, Gianluca Grenci, Vincent Studer, Virgile Viasnoff and Jean-Baptiste Sibarita. https://doi.org/10.1038/nmeth.3402

** This collaborative work between Imagine Optic and the Quantitative Imaging of the Cell group at the Interdisciplinary Institute for Neuroscience was done thanks to the following funding: Cifre, ANR, soSPIM.

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