Tracking the cells of tumor origin in breast organoids by light sheet microscopy - SPIM movie data
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ABSTRACT:
SUBMITTER: Ashna Alladin
PROVIDER: S-BSST317 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description: Not available
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Project description:Screening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregation in vitro assays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect development in vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.
| S-EPMC6062017 | biostudies-literature
Project description:Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 microm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.
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Project description:Cancer clone evolution takes place within tissue ecosystem habitats. But, how exactly tumors arise from a few malignant cells within an intact epithelium is a central, yet unanswered question. This is mainly due to the inaccessibility of this process to longitudinal imaging together with a lack of systems that model the progression of a fraction of transformed cells within a tissue. Here, we developed a new methodology based on primary mouse mammary epithelial acini, where oncogenes can be switched on in single cells within an otherwise normal epithelial cell layer. We combine this stochastic breast tumor induction model with inverted light-sheet imaging to study single-cell behavior for up to four days and analyze cell fates utilizing a newly developed image-data analysis workflow. The power of this integrated approach is illustrated by us finding that small local clusters of transformed cells form tumors while isolated transformed cells do not.
| S-EPMC7373425 | biostudies-literature
Project description:To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos.
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Project description:Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage.
| S-EPMC4674777 | biostudies-literature
Project description:MotivationLattice light sheet microscopy is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However analysis of such data sets still remains a major challenge.ResultsAutomated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis. Here we extend existing open-source kinetochore tracking software (KiT Armond et al. [2016]) to track (and pair) kinetochores throughout pro-metaphase to anaphase in lattice light sheet microscopy data. One of the key improvements is a regularization term in the objective function to enforce biological information about the number of kinetochores in a human mitotic cell, as well as improved diagnostic tools. This software provides quantitative insights into how kinetochores robustly ensure congression and segregation of chromosomes during mitosis.Availability and implementationKiT is free, open-source software implemented in MATLAB and can be downloaded as a package from https://github.com/cmcb-warwick/KiT. The source repository is available at https://bitbucket.org/jarmond/kit (tag v2.4.0) and under continuing development.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC9191203 | biostudies-literature
Project description:We developed a deconvolution software for light sheet microscopy that uses a theoretical point spread function, which we derived from a model of image formation in a light sheet microscope. We show that this approach provides excellent blur reduction and enhancement of fine image details for image stacks recorded with low magnification objectives of relatively high NA and high field numbers as e.g. 2x NA 0.14 FN 22, or 4x NA 0.28 FN 22. For these objectives, which are widely used in light sheet microscopy, sufficiently resolved point spread functions that are suitable for deconvolution are difficult to measure and the results obtained by common deconvolution software developed for confocal microscopy are usually poor. We demonstrate that the deconvolutions computed using our point spread function model are equivalent to those obtained using a measured point spread function for a 10x objective with NA 0.3 and for a 20x objective with NA 0.45.
| S-EPMC6879637 | biostudies-literature
Project description:Minimally-invasive optical imaging requires that light is delivered efficiently to limit the detrimental impact of photodamage on delicate biological systems. Light sheet microscopy represents the exemplar in tissue specific optical imaging of small and mesoscopic samples alike. However, further gains towards gentler imaging require a more selective imaging strategy to limit exposure to multiple yet discrete tissues without overexposing the sample, particularly where the information content is sparse or particularly optically sensitive tissues are present. The development of sample-adaptive imaging techniques is crucial in pursuit of the next generation of smart, autonomous microscopes. Herein, we report a microscope capable of performing 4D (x, y, z, t) light patterning to selectively illuminate multiple, rapidly reconfigurable regions of interest while maintaining the rapid imaging speed and high contrast associated with light sheet microscopy. We illustrate this utility in living zebrafish larvae and phantom samples.
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Project description:The combination of cellular-resolution whole brain light sheet microscopy (LSM) images with an annotated atlas enables quantitation of cellular features in specific brain regions. However, most existing methods register LSM data with existing canonical atlases, e.g., The Allen Brain Atlas (ABA), which have been generated from tissue that has been distorted by removal from the skull, fixation and physical handling. This limits the accuracy of the regional morphologic measurement. Here, we present a method to combine LSM data with magnetic resonance histology (MRH) of the same specimen to restore the morphology of the LSM images to the in-skull geometry. Our registration pipeline which maps 3D LSM big data (terabyte per dataset) to MRH of the same mouse brain provides registration with low displacement error in ∼10 h with limited manual input. The registration pipeline is optimized using multiple stages of transformation at multiple resolution scales. A three-step procedure including pointset initialization, automated ANTs registration with multiple optimized transformation stages, and finalized application of the transforms on high-resolution LSM data has been integrated into a simple, structured, and robust workflow. Excellent agreement has been seen between registered LSM data and reference MRH data both locally and globally. This workflow has been applied to a collection of datasets with varied combinations of MRH contrasts from diffusion tensor images and LSM with varied immunohistochemistry, providing a routine method for streamlined registration of LSM images to MRH. Lastly, the method maps a reduced set of the common coordinate framework (CCFv3) labels from the Allen Brain Atlas onto the geometrically corrected full resolution LSM data. The pipeline maintains the individual brain morphology and allows more accurate regional annotations and measurements of volumes and cell density.
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