0000000000548645
AUTHOR
Giuseppe De Vito
Two-photon light-sheet microscopy for high-speed whole-brain functional imaging of zebrafish neuronal physiology and pathology
We present the development of a custom-made two-photon light-sheet microscope optimized for high-speed (5 Hz) volumetric imaging of zebrafish larval brain for the analysis of neuronal physiological and pathological activity. High-speed volumetric two-photon light-sheet microscopy is challenging to achieve, due to constrains on the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarization, and we implemented remote scanning of the focal plane to record without disturbing the sample. Two-photon illumination is advantageous for zebrafish larva studies since infra-red excitation does not induce a …
Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts
The development of light-sheet fluorescence microscopy (LSFM) has greatly expanded the experimental capabilities in many biological and biomedical research fields, enabling for example live studies of murine and zebrafish neural activity or of cell growth and division. The key feature of the method is the selective illumination of a sample single plane, providing an intrinsic optical sectioning and allowing direct 2D image recording. On the other hand, this excitation scheme is more affected by absorption or scattering artifacts in comparison to point scanning methods, leading to un-even illumination. We present here an easily implementable method, based on acousto-optical deflectors (AOD),…
Removing striping artifacts in light-sheet fluorescence microscopy: a review
Abstract In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented…
Visualization_1
Volumetric recording of a single CRIW event shown as a selected subset of coronal sections. To produce the time lapse, original 16-bit depth images were converted into 8-bit and JPEG compressed. Scale bar: 100 ��m.
Visualization_2
3D rendering of the lag map shown in Fig. 4a. The lag value is color-coded as specified by the color bar. Scale bar: 100 ��m.
Visualization_1
Volumetric recording of a single CRIW event shown as a selected subset of coronal sections. To produce the time lapse, original 16-bit depth images were converted into 8-bit and JPEG compressed. Scale bar: 100 ��m.
Visualization_2
3D rendering of the lag map shown in Fig. 4a. The lag value is color-coded as specified by the color bar. Scale bar: 100 ��m.