Wednesday, December 31, 2025 12/31/2025

Super-resolved multiphoton microscopy with dual output ultrafast laser

Award Number: K25EB032864
ORGANIZATION: NATIONAL INSTITUTE Of BIOMEDICAL IMAGING & BIOENGINEERING
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP
PERIOD OF PERFORMANCE START DATE: 03/01/2023
PERIOD OF PERFORMANCE END DATE: 11/30/2026

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Super-resolved multiphoton microscopy with dual output ultrafast laser - Project Summary/ Abstract Nervous system disease may yield devastating impact on cognition, emotion, or sensorimotor function. Gene delivery using adeno-associated virus (AAV) vectors has demonstrated immense potential for treatment of congenital and acquired diseases impacting the central and peripheral nervous systems. Advancing mechanistic understanding of vector uptake and trafficking within nervous system cells would inform viral vector capsid design. Heretofore, visualizing viral vector cellular transduction in vivo has been hampered by a lack of optimal means for resolving nanoscale particles in thick tissues. Imaging viral particles whose dimensions are below the ~250 nm diffraction limit resolution of light microscopy is typically achieved using electron microscopy, a resource-intensive technique incompatible with life. There is a critical need to develop intravital imaging techniques that enable high-speed and deep nanoscale imaging of living systems. Two- photon excitation (2PE) microscopy is a powerful technique for intravital imaging of the nervous system that employs ultrafast near-infrared laser light capable of penetrating deep into tissues. Though the technique enables intravital imaging of thick tissues, the achievable resolution and image quality of 2PE microscopy is inadequate for study of nanoscale processes. 2PE microscopy may be paired with stimulated emission depletion (STED) techniques to enable resolution of nanoscale fluorescent-tagged targets. To enhance imaging depth and signal-to-background, 2PE may be achieved via spatiotemporal overlap of two ultrafast lasers of different wavelengths in a process termed non-degenerate 2PE. Heretofore, wide dissemination of 2PE-STED and non-degenerate 2PE microscopy techniques have been hampered by the cost and complexity associated with synchronization and alignment of two ultrafast laser sources. Herein, we propose to develop efficient super-resolved multiphoton microscopy approaches to enhance spatiotemporal resolution and imaging depth within living tissues by employing a single dual-output commercial ultrafast laser. Once developed, we will employ these novel imaging platforms to study intracellular trafficking of single AAV particles within cells of the murine nervous system. In Aim 1, the dual-output ultrafast laser will be utilized to achieve 2PE-STED microscopy via pulsed depletion and employed to image AAV trafficking in cultured Schwann cells and primary sensory neurons of murine dorsal root ganglia. In Aim 2, the dual-output ultrafast laser will be utilized to achieve non-degenerate 2PE and paired with a continuous wave depletion beam for deep super-resolution imaging of AAV trafficking in corneal Schwann cells and sensory neurons in live anesthetized mice. A liquid lens will be employed to enhance volumetric imaging speed. If successful, this work carries potential to advance understanding of viral vector transduction of nervous system cells by identifying intracellular trafficking bottlenecks, while illustrating the potential of super-resolution 2PE microscopy techniques to advance knowledge discovery in biomedicine.


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: 2026 ( Subtotal = $0 )
2026	2025	THE GENERAL HOSPITAL CORPORATION	55 FRUIT ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	3	12/8/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2025 ( Subtotal = $146,621 )
2025	2025	THE GENERAL HOSPITAL CORPORATION	55 FRUIT ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	001	3	2/18/2025	NON-COMPETING CONTINUATION	$146,621
2025	2024	THE GENERAL HOSPITAL CORPORATION	55 FRUIT ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	2	10/24/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$0
														Subtotal = $146,621

Issue Date FY: 2024 ( Subtotal = $146,621 )
2024	2024	THE GENERAL HOSPITAL CORPORATION	55 FRUIT ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	004	2	9/16/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$28,935
2024	2024	THE GENERAL HOSPITAL CORPORATION	55 FRUIT ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	002	2	7/30/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$146,621
2024	2023	MASSACHUSETTS EYE AND EAR INFIRMARY	243 CHARLES ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	003	1	9/16/2024	NEW	-$28,935
2024	2023	MASSACHUSETTS EYE AND EAR INFIRMARY	243 CHARLES ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	001	1	7/29/2024	NEW	$0
2024	2023	MASSACHUSETTS EYE AND EAR INFIRMARY	243 CHARLES ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	1	2/13/2024	NEW	$0
														Subtotal = $146,621

Issue Date FY: 2023 ( Subtotal = $142,848 )
2023	2023	MASSACHUSETTS EYE AND EAR INFIRMARY & PHYSICIAN STAFF, INC.	243 CHARLES ST	BOSTON	MA	02114	SUFFOLK	USA	Discovery and Applied Research for Technological Innovations to Improve Human Health	000	1	3/1/2023	NEW	$142,848
														Subtotal = $142,848

Grand Total All Awards = $436,090

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Super-resolved multiphoton microscopy with dual output ultrafast laser

Award Number: K25EB032864

ORGANIZATION: NATIONAL INSTITUTE Of BIOMEDICAL IMAGING & BIOENGINEERING

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: TRAINING/TRAINEESHIP

PERIOD OF PERFORMANCE START DATE: 03/01/2023

PERIOD OF PERFORMANCE END DATE: 11/30/2026

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