Periodic Reporting for period 1 - MesoBrainMicr (Novel high speed and high resolution microscopy setup for cytoarchitectonic studies of mesoscale sized human brain tissues, healthy and affected by Focal Cortical Dysplasia)
Reporting period: 2018-10-01 to 2020-09-30
The human brain is a complex information processing system with a hierarchy of different yet tightly integrated levels of organization. From such intricate interplay emerge our personality, emotions, memory and decision making capabilities, but understanding its functioning is a huge challenge. An open problem is how to investigate with both high resolution and high speed the brain structure over mesoscale (millimeters to centimeters) sized regions, which would provide unique insight on its neuroanatomy and help to develop novel medical diagnostic approaches.
In this project we aim to develop a one photon fluorescence microscope capable of resolving sub-cellular morphology over centimeter-sized tissue samples at state of the art speed and with micrometric resolution. This innovative instrument, called dual-view inverted dual-slit confocal light sheet fluorescence microscope (di2CLSFM), will enable new studies of the brain anatomy across different functional areas.
One traditional limitation of cytoarchitecture studies is their limited extension. Cell morphology and spatial distribution are analysed within single cortical columns or small brain volumes, potentially hiding any long-range correlations in the fine details of neuronal organization or missing unexpected neuroanatomical features in other brain areas. We will use the di2CLSFM to obtain a detailed characterization of the human brain cytoarchitecture, both on the level of local circuits and of long-range connections, in healthy and dysplastic mesoscopic tissues, in particular affected by Focal Cortical Dysplasia (FCD). This pathology is a common malformation of cortical development found in epilepsy surgeries and its causes are poorly understood. This approach will generate digital anatomical reconstructions of large brain volumes which will greatly advance the medical and neurobiological understanding of the healthy brain tissue structure and of the effects of neuro-degenerative pathologies.
In this project we aim to develop a one photon fluorescence microscope capable of resolving sub-cellular morphology over centimeter-sized tissue samples at state of the art speed and with micrometric resolution. This innovative instrument, called dual-view inverted dual-slit confocal light sheet fluorescence microscope (di2CLSFM), will enable new studies of the brain anatomy across different functional areas.
One traditional limitation of cytoarchitecture studies is their limited extension. Cell morphology and spatial distribution are analysed within single cortical columns or small brain volumes, potentially hiding any long-range correlations in the fine details of neuronal organization or missing unexpected neuroanatomical features in other brain areas. We will use the di2CLSFM to obtain a detailed characterization of the human brain cytoarchitecture, both on the level of local circuits and of long-range connections, in healthy and dysplastic mesoscopic tissues, in particular affected by Focal Cortical Dysplasia (FCD). This pathology is a common malformation of cortical development found in epilepsy surgeries and its causes are poorly understood. This approach will generate digital anatomical reconstructions of large brain volumes which will greatly advance the medical and neurobiological understanding of the healthy brain tissue structure and of the effects of neuro-degenerative pathologies.
Light sheet fluorescence microscopy (LSFM) is a well-established technique for volumetric imaging of large samples with high speed and good resolution and is compatible with tissue optical clearing techniques that enhance light penetration in highly scattering media and improve image quality by reducing optical aberrations. We built the di2CLSFM according to the optical scheme shown in panel A in the attached figure (photo in panel B), it works reliably and acquires new data daily. It has two identical optical pathways that alternately serve as fluorescence excitation and detection arms to capture two orthogonal views of a sample, obtaining in post-processing by computational fusion and deconvolution an improved almost isotropic micrometer resolution. We developed the image processing pipeline and will release it as open source. We optimized a tissue preparation protocol based on a refined SWITCH method to image samples with lateral size up to tens of centimeters and height up to 0.5 mm, greatly reducing the traditional need to cut smaller blocks. Up to four target neuronal populations can be rendered fluorescent through immuno-staining and then detected in different emission bands, one plane per image, when illuminated by the digitally scanned light sheet (DSLM), realized via a galvo mirror from a single beam coming from the matching laser. We developed a custom quartz holder to store and sequentially image multiple samples, by scanning them through the light sheet by moving the motorized precision three-axis translation stages in a serpentine pattern.
We leverage confocal line detection, for improved contrast and reduced background, by exploiting the rolling shutter of a fast sCMOS camera and have demonstrated a dual beam illumination scheme that doubles the imaging speed without affecting the image quality. We have also demonstrated two illumination schemes that suppress illumination artefacts and we have studied the effects of excitation light polarization on fluorescence emission in LSFM.
The acquired images are inspected with an advanced artificial intelligence-based (AI) image analysis algorithm to automatically recognize the complex biological content, discriminating the shapes of neurons and blood vessels from the background fluorescence. In the attached figure, panel C presents the algorithm’s image analysis steps, while panel D displays an example of a classified image and panel E of a whole tissue. Three large human brain regions with volume of several cubic centimetres were imaged with the di2CLSFM: a whole left hippocampus, part of the Broca’s area and of the motor cortex (examples of representative slices are shown in panels F, G and H). Their digital anatomical reconstruction is in progress and it will greatly advance the medical and neurobiological understanding of the healthy brain tissue structure. The study of tissues affected by FCD, unfortunately, was not realized due to the impact of the COVID-19 pandemic and related lock-down, making also impossible to do a comparative cytoarchitectonic investigation between healthy and dysplastic brain tissues. The obtained data was contributed to open repositories as a foundation for brain models.
These results have been presented to the scientific community at five conferences and through four publications and three conference proceedings, and more publications are in preparation. Moreover, we disseminated them among the general public through social media and at the Scienz’Estate 2019 and European Researchers’ Night BRIGHT 2020 outreach events.
We leverage confocal line detection, for improved contrast and reduced background, by exploiting the rolling shutter of a fast sCMOS camera and have demonstrated a dual beam illumination scheme that doubles the imaging speed without affecting the image quality. We have also demonstrated two illumination schemes that suppress illumination artefacts and we have studied the effects of excitation light polarization on fluorescence emission in LSFM.
The acquired images are inspected with an advanced artificial intelligence-based (AI) image analysis algorithm to automatically recognize the complex biological content, discriminating the shapes of neurons and blood vessels from the background fluorescence. In the attached figure, panel C presents the algorithm’s image analysis steps, while panel D displays an example of a classified image and panel E of a whole tissue. Three large human brain regions with volume of several cubic centimetres were imaged with the di2CLSFM: a whole left hippocampus, part of the Broca’s area and of the motor cortex (examples of representative slices are shown in panels F, G and H). Their digital anatomical reconstruction is in progress and it will greatly advance the medical and neurobiological understanding of the healthy brain tissue structure. The study of tissues affected by FCD, unfortunately, was not realized due to the impact of the COVID-19 pandemic and related lock-down, making also impossible to do a comparative cytoarchitectonic investigation between healthy and dysplastic brain tissues. The obtained data was contributed to open repositories as a foundation for brain models.
These results have been presented to the scientific community at five conferences and through four publications and three conference proceedings, and more publications are in preparation. Moreover, we disseminated them among the general public through social media and at the Scienz’Estate 2019 and European Researchers’ Night BRIGHT 2020 outreach events.
The di2CLSFM setup, image analysis pipeline and sample preparation protocol have been published in three conference proceedings (Pesce et al., Proc. SPIE 11226 2020; Pesce et al., Proc. SPIE 11360 2020; Gavryusev et al., Biophotonics Congress: Biomedical Optics 2020). It advances the best features of the digital confocal and of the dual-view inverted LSFM designs, for instance, the high speed and image quality of the first and the diffraction-limited isotropic resolution and compatibility with extended tissues deposited on a glass coverslip of the second, enabling novel mesoscopic cytoarchitecture investigations.
In Gavryusev et al. (BOE 2019), a dual-slit confocal detection scheme that allows doubling the acquisition speed without impact on the signal has been demostrated by realizing a DSLM with two parallel gaussian beams, controlled by an acousto-optical-deflector (AOD) and synchronized with the two rolling shutters of a sCMOS camera.
The images acquired with one-photon excitation presented significant illumination homogeneity defects in the form of striping artefacts, mostly due to absorption and scattering from occlusions within the sample. In Sancataldo et al. (Front. Neuroanat. 2019), we demonstrated the suppression of these striping artefacts in one-photon LSFM using AODs to generate multiple static light sheets or a dynamically pivoted one. Moreover, in Ricci et al. (BOE 2020), we extended this method to preserve confocal detection, for improved contrast. These results pave the way to a more quantitative structural and functional imaging in ex-vivo and in-vivo samples.
In de Vito et al. (BOE 2020), we investigated the effects of excitation light polarization on fluorescence emission in two-photon LSFM, identifying the optimal conditions for one- and two-photon illumination depending on the sample properties, which allows to improve the signal without negative consequences.
In Gavryusev et al. (BOE 2019), a dual-slit confocal detection scheme that allows doubling the acquisition speed without impact on the signal has been demostrated by realizing a DSLM with two parallel gaussian beams, controlled by an acousto-optical-deflector (AOD) and synchronized with the two rolling shutters of a sCMOS camera.
The images acquired with one-photon excitation presented significant illumination homogeneity defects in the form of striping artefacts, mostly due to absorption and scattering from occlusions within the sample. In Sancataldo et al. (Front. Neuroanat. 2019), we demonstrated the suppression of these striping artefacts in one-photon LSFM using AODs to generate multiple static light sheets or a dynamically pivoted one. Moreover, in Ricci et al. (BOE 2020), we extended this method to preserve confocal detection, for improved contrast. These results pave the way to a more quantitative structural and functional imaging in ex-vivo and in-vivo samples.
In de Vito et al. (BOE 2020), we investigated the effects of excitation light polarization on fluorescence emission in two-photon LSFM, identifying the optimal conditions for one- and two-photon illumination depending on the sample properties, which allows to improve the signal without negative consequences.