VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)
Lumkwana, D.; Lightley, J.; Vesga, A. G.; de Folter, J.; Domart, M.-C.; Maclachlan, C.; Kumar, S.; Evans, J. R.; Peddie, C.; Burrell, A.; Yoshimura, A.; Mangali, A.; Vahrjmeijer, N.; Bhaga, M.; Smit, C.; Titze, B.; Horrocks, M. H.; Cobra Straker, L.; Garcia, E.; Jones, M. L.; Mclean, A.; Roufosse, C.; Loos, B.; Gandhi, S.; Strange, A.; Henriques, R.; French, P. M.; Collinson, L.
Show abstract
Visual proteomics (VP) aims to allow researchers to visualise, measure and analyse proteins in the context of cell and tissue structure in health and disease. VP is becoming a reality through technological advances across several domains, including in situ structural biology and correlative light and electron microscopy (CLEM). However, widespread adoption remains limited due to the complexity and cost of the various VP approaches reported to date. Here we present the VP-CLEM-Kit, a disruptive cost-effective pipeline for super resolution volume CLEM (SR-vCLEM) that can be implemented with minimal advanced electron microscopy expertise and equipment, making it accessible to light microscopy facilities and research labs. SR-vCLEM is based on in-resin fluorescence (IRF), where fluorophores are preserved through processing into resin. The easyIRF protocol reported here reduces the requirement for complex costly sample preparation equipment and toxic chemicals compared to standard IRF protocols. easyIRF blocks are cut into ultrathin sections that are imaged using tomoSTORM, a new modular and cost-effective openFrame-based light microscope controlled by the open-source software package Micro-Manager that provides serial single molecule localisation microscopy in array tomography format. Sections are then post-stained and imaged using a tabletop scanning electron microscope controlled by open-source SBEMimage software to run in array tomography format. We demonstrate the potential of the VP-CLEM-Kit by imaging organelle reporters in human cell lines and stem cell derived neurons, fluorescently labelled protein in neurons, and immunolabelled cells in human kidney biopsy tissue from transplant patients. The VP-CLEM-Kit delivers a [~]5-fold improvement in resolution at a [~]7-fold lower cost, and thus provides new technical capability as well as a blueprint for more equitable access to advanced imaging workflows.
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