Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector

Abstract

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acoustooptic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast.

Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy

“…Furthermore, this characterization represents a useful guide to choose the properly-oriented linearly-polarized light when maximization of signal levels is needed. This is particularly important in high-speed 2P LSM, because in this situation (differently from 1P LSM) the acquisition frequency is usually limited by the signal-to-noise ratio and therefore increasing the signal levels is necessary to achieve a higher temporal resolution, by implementing for example strategies that double the frame rate [49].…”

Exploring the human cerebral cortex using confocal microscopy

“…It should be noted, however, that for many of these cellular markers, the penetration depth when using confocal is limited, and the whole sample is exposed to the excitation light, causing fluorophore bleaching during the acquisition. For these reasons, for 3D mesoscopic reconstruction, other optical techniques, like two-photon microscopy (Costantini and Mazzamuto et al, 2021) or light-sheet fluorescence microscopy (Gavryusev et al, 2019), are the best candidates for imaging such samples. Nevertheless, we believe that the characterizations performed in this study can be exploited in future studies of the fine structural anatomy of the human brain, both in normal and pathological conditions.…”

3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy

“…An acousto-optical-tunable-filter (AAOptoelectronic AOTFnC-400.650-TN) modulates the transmitted power and wavelength and a galvo mirror (Cambridge Technology 6220H) sweeps it across the detection focal plane, generating a digitally scanned light sheet. The induced fluorescence is collected by the objective onto a Hamamatsu ORCA Flash4.0v3 sCMOS camera, operating in the confocal detection mode (4,52). The sample, enclosed in a sandwich holder, is accommodated in a large plexiglass chamber filled with TDE and into which the objectives are immersed in order to allow for refractive index matching.…”

“…However, this strategy lacks three-dimensionality and can decontextualize the section from the surrounding environment, introducing artifacts. Several volumetric imaging technologies like confocal, multiphoton, and light sheet-fluorescence microscopy (LSFM) have been employed for visualizing deeper inside the tissue (2)(3)(4)(5)(6). To perform 3D reconstructions, these optical techniques require an optimized clearing protocol, which preserves the proteins of interest and removes the components that affect the imaging (generally lipids and chromophores) by producing a refractive index mismatching that is correlated with an increase in light scattering and absorption by chromophores and pigments (3,7,8).…”