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Development of a sample preparation protocol for brain ultrastructural analysis, immunolabeling, and neuronal tracing by light microscopy

Award Number: R43MH128999
ORGANIZATION: NATIONAL INSTITUTE OF MENTAL HEALTH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/16/2022
PERIOD OF PERFORMANCE END DATE: 09/15/2023

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Development of a sample preparation protocol for brain ultrastructural analysis, immunolabeling, and neuronal tracing by light microscopy - Abstract The brain is the most complex organ in the human body. Understanding its neural connections and molecular anatomy requires imaging technology that is capable of mapping the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. The currently best available option is correlative light and electron microscopy (CLEM), which combines the resolving power and global contrast of EM with the high molecular specificity of fluorescence microscopy. While very powerful, CLEM is laborious, low throughput, expensive and often inadequate in spatial correlation resolution. To date, there is no imaging technology that can resolve specific proteins within the ultrastructure of a synapse. We recently developed an all-optical method called pan-Expansion Microscopy (pan-ExM) that has the potential to allow standard confocal microscopy users to do just that: by combining ~20-fold linear expansion of biological samples with novel bulk (pan) staining of total proteins, hallmark ultrastructural features such as post- and pre-synaptic densities and mitochondria cristae can now be resolved by their characteristic pan-staining pattern, analogous to heavy-metal stains in EM. Panluminate Inc. is a new company built around this core technology. Our proposed Phase I project is to validate pan-ExM protocols that will establish pan-ExM as a brain imaging technology. Our overall goal is to enable every neuroscientist to locate specific protein labels within their 3D contextual compartments as well as perform EM-like neuronal tracing in brain tissue. We specifically propose to (1) validate and optimize antibody-labeling compatible protocols for synapse identification, and (2) optimize and validate a novel lipid pan-stain we developed to delineate cellular boundaries to complement our proteome pan-stain. Ultimately, the developments proposed here will enable Panluminate to streamline and deploy pan-ExM sample preparation kits to every neuroscientist interested in simultaneous 3D ultrastructural analysis, antibody labeling, and neuronal tracing in brain tissue and to offer pan-ExM sample preparation and imaging as a service.


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: 2024 ( Subtotal = -$316 )
2024	2022	PANLUMINATE INC	123 YORK ST	NEW HAVEN	CT	06511	SOUTH CENTRAL CT	USA	Mental Health Research Grants	000	1	4/3/2024	NEW	-$316
														Subtotal = -$316

Issue Date FY: 2023 ( Subtotal = $0 )
2023	2022	PANLUMINATE INC.	123 YORK ST	NEW HAVEN	CT	06511	SOUTH CENTRAL CT	USA	Mental Health Research Grants	000	1	2/15/2023	NEW	$0
														Subtotal = $0

Issue Date FY: 2022 ( Subtotal = $248,545 )
2022	2022	PANLUMINATE INC.	123 YORK ST	NEW HAVEN	CT	06511	SOUTH CENTRAL CT	USA	Mental Health Research Grants	000	1	9/14/2022	NEW	$248,545
														Subtotal = $248,545

Grand Total All Awards = $248,229

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Development of a sample preparation protocol for brain ultrastructural analysis, immunolabeling, and neuronal tracing by light microscopy

Award Number: R43MH128999

ORGANIZATION: NATIONAL INSTITUTE OF MENTAL HEALTH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 09/16/2022

PERIOD OF PERFORMANCE END DATE: 09/15/2023

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