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Development of polymer-based cell culture substrates for correlative light and electron microscopy (CLEM)

Award Number: R01GM152716
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/30/2024
PERIOD OF PERFORMANCE END DATE: 07/31/2027

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Development of polymer-based cell culture substrates for correlative light and electron microscopy (CLEM) - PROJECT SUMMARY/ABSTRACT Correlative light and electron microscopy (CLEM) combines dynamic information from florescence light microscopy (LM) with ultrastructural information from electron microscopy (EM) of the same biological specimen, addressing fundamental questions neither LM nor EM alone can answer. However, performing CLEM is technically challenging even for many experienced scientists. In particular, CLEM study is currently impossible or impractical for cells cultured on permeable substrates (e.g., Transwell inserts, thin 3D substrates) that are required by a broad range of biomedical research. This discrepancy is due to the lack of a convenient method or tools to target, track and locate the regions of interest in EM specimens of the cells previously examined by LM. Our long-term goal is to develop convenient tools and methods to simplify CLEM studies for cells, tissues and organoids. Our objective is to develop a working prototype for CLEM technology, including a new type of substrate and associated protocols that will enable CLEM studies allowing functional cells cultured on permeable/porous substrates to regain their functional state/morphology, polarity or desired orientations. Our strategy is to use microfabrication to develop and produce a designable polymer-based porous substrate with easy-to-recognize indexing patterns for cells to culture on, enabling fluorescence LM imaging and resin embedding, followed by EM of serial sections of the same object. The substrate will also be developed and validated for CLEM studies using focused ion beam (FIB) milling for FIB-scanning electron microscopic (SEM) tomography. We will develop the substrates and CLEM protocols to address two typical needs for functional cell culture, confluent cell culture and thin 3D cell culture. We will accomplish the research through three specific aims: (1) Develop a method for routine production of polymer substrates with a cutting property matching that of resin-embedded cell samples; (2) Develop accessible substrates/protocols for CLEM of functional cells on porous substrates; and (3) Develop accessible substrates/protocols for CLEM of cells cultured on desired thin 3D substrates. This innovative research will develop a novel CLEM tool by combining techniques in cellular EM specimen preparation with a unique method of microfabrication to produce a previously unavailable, polymer- based, indexing patterned substrate, and enable, the first time, CLEM studies for functional cells that must be cultured on porous substrates. The newly developed substrate will also facilitate convenient CLEM of EM specimens prepared by a high pressure freezing/freeze substitution method. The anticipated contribution will be significant because it will not only enable CLEM applications for a broad range of cell types that must be cultured on porous substrates for their polarity and functional status, but also facilitate EM specimen preparation by using a high pressure freezing/freeze substitution for these cells. Additionally, it opens the gate for further development of substrates and methods for 3D-CLEM that correlate information from 3D-LM live cell imaging with the ultrastructure of 3D cell cultures on 3D substrates.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $303,826 )
2025	2025	HEALTH RESEARCH, INC.	150 BROADWAY STE 280	MENANDS	NY	12204	ALBANY	USA	Biomedical Research and Research Training	001	2	7/25/2025	NON-COMPETING CONTINUATION	$303,826
2025	2024	HEALTH RESEARCH, INC.	150 BROADWAY STE 280	MENANDS	NY	12204	ALBANY	USA	Biomedical Research and Research Training	000	1	12/17/2024	NEW	$0
														Subtotal = $303,826

Issue Date FY: 2024 ( Subtotal = $303,582 )
2024	2024	HEALTH RESEARCH, INC.	150 BROADWAY STE 280	MENANDS	NY	12204	ALBANY	USA	Biomedical Research and Research Training	000	1	9/18/2024	NEW	$303,582
														Subtotal = $303,582

Grand Total All Awards = $607,408

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Development of polymer-based cell culture substrates for correlative light and electron microscopy (CLEM)

Award Number: R01GM152716

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/30/2024

PERIOD OF PERFORMANCE END DATE: 07/31/2027

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