Fast, multiplexed 3D imaging using an Airy beam light sheet microscope
Lead Research Organisation:
University of Glasgow
Department Name: College of Medical, Veterinary, Life Sci
Abstract
Generating 3D maps of tissue and organ structure is crucial to understanding disease. By examining the structure of diseased tissue and comparing it to healthy tissue we can begin to elucidate the underlying disease mechanisms and identify potential treatments or preventatives or determine why current treatments fail groups of patients.
Optical microscopy is an important tool in biomedical research which can be used to generate these 3D maps. We can attach fluorescent markers to cells and proteins of interest and use fluorescence microscopes to build up a 3D image of the spatial localisation of these markers within the tissue. In the past this was performed by slicing the tissue very thinly and computationally reconstructing the tissue from the imaged slices. Registration issues between slices can cause reconstruction artifacts and the slices are very delicate and can be damaged during the slicing and mounting process.
Another (the current gold-standard) method is to use a laser scanning confocal microscope to image thicker slices. The point-confocal microscope excites the sample at a single point using a tightly-focused laser, a pinhole in the detection path rejects out-of-focus light and we can "optically section" our samples without needing to physically slice the tissue. However, creating a 3D image requires scanning the point of excitation over the sample building up the image pixel-by-pixel. These samples are large (few mm) and to image entire organs and structures at cellular (or sub-cellular) resolution requires collecting very large datasets, typically requiring a few days per individual sample.
This application is to request a light sheet microscope (LSM). LSM uses a novel optical geometry whereby a sheet of light is used to illuminate a single plane within a sample, creating an optical section. This optical section is then imaged using a lens placed orthogonally to the illumination plane, directed onto a camera which can detect the emitted light from the whole plane simultaneously. The need for point-scanning is eliminated and the speed of acquisition is significantly increased. In particular, we are requesting a LSM with a specially shaped light sheet (known as an Airy beam light sheet) which is exceptionally long and thin so we can image even larger samples and further increase throughput. Our quoted Airy beam light sheet will also come with double the number of excitation lasers as is typically supplied with other commercial systems, allowing us to image twice as many markers during a single acquisition.
This increase in speed of acquisition and broader fluorescence detection will allow us to screen many more samples, meaning we can rapidly create 3D maps of a variety of tissue disease models, including for lung, liver and colorectal cancer, of the brain for chronic pain and understanding infection and inflammation throughout the body. It will also help to support the aims of the MRC National Mouse Genetics Network to use their sophisticated mouse models of disease to further increase the pre-clinical value by providing better 3D cell mapping for comparison of mouse and human tissues. It will also allow us to interrogate innovative in vitro 3D multicellular tissue culture systems.
Optical microscopy is an important tool in biomedical research which can be used to generate these 3D maps. We can attach fluorescent markers to cells and proteins of interest and use fluorescence microscopes to build up a 3D image of the spatial localisation of these markers within the tissue. In the past this was performed by slicing the tissue very thinly and computationally reconstructing the tissue from the imaged slices. Registration issues between slices can cause reconstruction artifacts and the slices are very delicate and can be damaged during the slicing and mounting process.
Another (the current gold-standard) method is to use a laser scanning confocal microscope to image thicker slices. The point-confocal microscope excites the sample at a single point using a tightly-focused laser, a pinhole in the detection path rejects out-of-focus light and we can "optically section" our samples without needing to physically slice the tissue. However, creating a 3D image requires scanning the point of excitation over the sample building up the image pixel-by-pixel. These samples are large (few mm) and to image entire organs and structures at cellular (or sub-cellular) resolution requires collecting very large datasets, typically requiring a few days per individual sample.
This application is to request a light sheet microscope (LSM). LSM uses a novel optical geometry whereby a sheet of light is used to illuminate a single plane within a sample, creating an optical section. This optical section is then imaged using a lens placed orthogonally to the illumination plane, directed onto a camera which can detect the emitted light from the whole plane simultaneously. The need for point-scanning is eliminated and the speed of acquisition is significantly increased. In particular, we are requesting a LSM with a specially shaped light sheet (known as an Airy beam light sheet) which is exceptionally long and thin so we can image even larger samples and further increase throughput. Our quoted Airy beam light sheet will also come with double the number of excitation lasers as is typically supplied with other commercial systems, allowing us to image twice as many markers during a single acquisition.
This increase in speed of acquisition and broader fluorescence detection will allow us to screen many more samples, meaning we can rapidly create 3D maps of a variety of tissue disease models, including for lung, liver and colorectal cancer, of the brain for chronic pain and understanding infection and inflammation throughout the body. It will also help to support the aims of the MRC National Mouse Genetics Network to use their sophisticated mouse models of disease to further increase the pre-clinical value by providing better 3D cell mapping for comparison of mouse and human tissues. It will also allow us to interrogate innovative in vitro 3D multicellular tissue culture systems.
Technical Summary
We are requesting funds to purchase an Airy-beam (AB) light sheet microscope (LSM). LSMs represent the forefront of rapid 3D imaging, functionality which is vital for rapid screening of cleared human and mouse tissue in 3D. This LSM will allow us to generate accurate tissue maps of mouse and human tissue, comparing disease against non-disease models.
The AB-LSM is particularly suited to our proposed experiments. Compared to imaging with a Gaussian or Bessel beam LSM, the AB-LSM uses a propagation-invariant beam to maintain a thinner sheet over a longer field of view. We will use this to image a larger volume at once, while maintaining the resolution required to allow us to distinguish single cells and sub-cellular structures within our tissues dramatically increasing the throughput of large tissue or organ imaging. We also request environmental control necessary for live cell imaging to capitalise on innovative 3D (co-)culture and ex vivo live tissue systems from the network of beneficiaries.
The requested Airy beam light sheet will also have eight excitation lasers. We have significant experience in imaging multiple fluorescent markers in tissue followed by spectral unmixing for generating highly multiplexed structural data, creating content-rich data for mining. By applying this technique in a light sheet (rather than point-scanning confocal) acquisition protocol we can significantly speed up our data acquisition pipeline.
The AB-LSM will be situated at the University of Glasgow's Garscube campus in the Wolfson Wohl Cancer Research Centre where it will be managed by the Beatson Advanced Imaging Resource team at the Cancer Research UK Beatson Institute. We will collaborate and integrate closely with the newly created UofG MVLS-CAF and other microscopy facilities within UofG to ensure maximum exposure and equitable access to this unique system. It will also help underpin the aims of the MRC National Mouse Genetics Network by allowing better 3D cell mapping.
The AB-LSM is particularly suited to our proposed experiments. Compared to imaging with a Gaussian or Bessel beam LSM, the AB-LSM uses a propagation-invariant beam to maintain a thinner sheet over a longer field of view. We will use this to image a larger volume at once, while maintaining the resolution required to allow us to distinguish single cells and sub-cellular structures within our tissues dramatically increasing the throughput of large tissue or organ imaging. We also request environmental control necessary for live cell imaging to capitalise on innovative 3D (co-)culture and ex vivo live tissue systems from the network of beneficiaries.
The requested Airy beam light sheet will also have eight excitation lasers. We have significant experience in imaging multiple fluorescent markers in tissue followed by spectral unmixing for generating highly multiplexed structural data, creating content-rich data for mining. By applying this technique in a light sheet (rather than point-scanning confocal) acquisition protocol we can significantly speed up our data acquisition pipeline.
The AB-LSM will be situated at the University of Glasgow's Garscube campus in the Wolfson Wohl Cancer Research Centre where it will be managed by the Beatson Advanced Imaging Resource team at the Cancer Research UK Beatson Institute. We will collaborate and integrate closely with the newly created UofG MVLS-CAF and other microscopy facilities within UofG to ensure maximum exposure and equitable access to this unique system. It will also help underpin the aims of the MRC National Mouse Genetics Network by allowing better 3D cell mapping.
