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.
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…
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…
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.