This technology uses customizable, transparent polymer substrates with precise pores and indexing patterns to facilitate correlative light and electron microscopy, enabling accurate correlation of live cell fluorescence imaging with ultrastructural details of sectioned cells on permeable supports for advanced biological research.
Background: Correlative Light and Electron Microscopy (CLEM) is a powerful technique that combines the molecular specificity and live-cell imaging capabilities of fluorescence light microscopy (LM) with the ultrastructural information provided by electron microscopy (EM). This integration allows researchers to study dynamic cellular processes and their ultrastructural context, making CLEM an essential tool in cell biology, neuroscience, and pathology. A particular area of interest is the study of cells grown on permeable supports, such as polarized epithelial cells and 2D primary monolayer organoids, which require nutrient exchange mimic physiological conditions more accurately than impermeable surfaces. However, the complexity of CLEM, especially after resin-embedding sample processing and sectioning, has created a pressing need for improved substrates and workflows that can reliably support these advanced imaging studies. Current CLEM remains a technically demanding technique with significant limitations that hinder its effectiveness and accessibility. Traditional gridded glass coverslips are restricted to impermeable surfaces and are incompatible with advanced sample preparation techniques like high-pressure freezing for preserving fine cellular structures. Metal TEM grids, another reported solution for facilitating the location of regions of interest, must be painstakingly removed before ultramicrotomy and often lead to sample disruption. Additionally, existing permeable supports, such as Transwell membrane, track-etched membrane-based cell culture filters or inserts, suffer from non-uniform pore sizes, limited customization, and incompatibility with CLEM, which further complicates high-precision correlative imaging and limits the types of biological questions that can be addressed.
Technology Overview: This technology utilizes microfabricated polymer-based porous substrates to enhance Correlative Light and Electron Microscopy (CLEM) workflows. Fabricated via photolithography, these substrates feature customizable indexing patterns and tunable pore sizes. On the one hand, these polymeric indexing substrates can replace conventional impermeable coverslips and metal TEM finder grids, facilitating resin-embedded EM specimen preparation for CLEM studies. On the other hand, these optically transparent and chemically stable polymeric indexing substrates serve as supports for cell culture—including polarized cells that require culture on permeable supports—and can be directly embedded in epoxy resin after LM examination. The mechanical properties of these substrates are engineered to match those of the embedding resin, enabling smooth ultramicrotomy and serial sectioning without the need for substrate removal. Integrated indexing patterns facilitate precise localization, tracking, and computational alignment of regions of interest between light and electron microscopy images, streamlining the CLEM workflow from live-cell imaging to ultrastructural analysis. What differentiates this technology is its comprehensive solution to longstanding technical challenges in CLEM, particularly for imaging cells on permeable supports. Unlike traditional glass coverslips or metal finder grids, which are limited by impermeability, incompatibility with high-pressure freezing, and difficult removal processes, the substrates offer unmatched flexibility and performance. Their tunable mechanical properties ensure seamless integration with resin embedding and sectioning, eliminating artifacts and preparation failures. The ability to design pore sizes and indexing patterns enables precise correlation between imaging modalities, while the biocompatibility and optical clarity of this technology support advanced cell culture and imaging needs. This innovation not only improves the reliability and accessibility of CLEM but also opens new avenues for research in cell biology, pathology, and drug development by enabling high-resolution, correlative imaging of complex cellular systems.
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Advantages: • Enables on-the-fly correlation between light and electron microscopy images through integrated indexing patterns • Supports cell culture on permeable substrates, including polarized cells, enhancing biological relevance • Customizable pore sizes and substrate thicknesses for diverse applications • Biocompatible substrate that maintains cell viability and specialized functions, such as ensuring cell polarity and healthy cilia formation • Uses porous substrates with an indexing pattern, replacing cell culture filters or inserts and enabling CLEM • Optically transparent and chemically/thermally stable polymer suitable for advanced specimen preparation methods • Mechanical properties match embedding resin, allowing ultramicrotomy without substrate removal • Improves CLEM workflow and success rate and reduces specimen preparation time • Facilitates advanced correlative imaging, accelerating research in cell biology, pathology, and drug development
Applications: • Correlative Light and Electron Microscopy • Porous cell culture inserts • Customizable advanced substrates for cell growth • TEM finder grid replacement for resin-embedded applications
Intellectual Property Summary: Patent application filed
Stage of Development: • TRL 3 • https://en.wikipedia.org/wiki/Technology_readiness_level
Licensing Status: This technology is available for licensing.