0000000000123028
AUTHOR
Ludovico Silvestri
Swift light sheet volumetric charting of large human brain portions
Using a custom light sheet fluorescence microscope, we image large stained human brain portions, labelled for NeuN and GAD67 neuronal markers, discerning the inhibitory population via neural-network based image analysis and exposing the brain connectivity.
Power-effective scanning with AODs for 3D optogenic applications
Two-photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub-micrometric-resolution light targeting into the brain. However, besides structural and functional imaging, 2P optogenetic stimulations are less routinary, especially in 3D. This is because of the adopted scanning systems, often feebly effective, slow and mechanically constricted. Faster illumination can be achieved through acousto-optic deflectors (AODs) although their applicability to large volumes excitation has been limited by large efficiency drop along the optical axis. Here, we present a new AOD-based scheme for 2P 3D scanning that improves the power delivery…
Fast multi-directional DSLM for confocal detection without striping artifacts
In recent years light-sheet fluorescence microscopy (LSFM) has become a cornerstone technology for neuroscience, improving the quality and capabilities of 3D imaging. By selectively illuminating a single plane, it provides intrinsic optical sectioning and fast image recording, while minimizing out of focus fluorescence background, sample photo-damage and photo-bleaching. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. In this work we present an optical solution to this problem, via fast multi-directional illumination of the sample, based on an acousto-optical deflector (AOD). We demonstr…
A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy
The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence …
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 …
Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy
Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish bra…
Advanced fluorescence microscopy for in vivo imaging of neuronal activity
Brain function emerges from the coordinated activity, over time, of large neuronal populations placed in different brain regions. Understanding the relationships of these specific areas and disentangling the contributions of individual neurons to overall function remain central goals for neuroscience. In this scenario, fluorescence microscopy has been proved as the tool of choice for in vivo recording of brain activity. Optical advances combined with genetically encoded indicators allow a large flexibility in terms of spatiotemporal resolution and field of view while keeping invasiveness in living animals to a minimum. Here we describe the latest advancements in the field of linear and nonl…
Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector
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 t…
Two-photon high-speed light-sheet volumetric imaging of brain activity during sleep in zebrafish larvae
Although it is well known that zebrafish display the behavioural signature of sleep, the neuronal correlates of this state are not yet completely understood, due to the complexity of the measurements required. For example, when performed with visible excitation light, functional imaging can disrupt the day/night cycle due to the induced visual stimulation. To address this issue, we developed a custom-made two-photon light-sheet microscope optimized for high-speed volumetric imaging. By employing infra-red light (not visible to the larva) for excitation, we are able to record wholebrain neuronal activity with high temporal- and spatial-resolution without affecting the sleep state. In two-pho…
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…
3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy
AbstractThe combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH—H2O2—antigen Retrieval—TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging…
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.