MEMP PhD Thesis Defense: Ami Thakrar

Time-resolved High-pressure Freezing with Ligand Stimulation to Capture Nanoscale Cellular Dynamics

Ligand-triggered signaling processes govern much of cellular behavior and function across tissue types. Characterizing signaling from early-stage events (< 1 s) to measurable changes in downstream effectors (>1 min) is critical for deepening our mechanistic understanding of cellular physiology. However, existing imaging methods lack the combined spatiotemporal resolution to comprehensively resolve these events in situ. To overcome this limitation, we have developed a time-resolved cryo-vitrification method called HPF-X: High Pressure Freezing with buffer eXchange. Within our instrument, cells or tissue slices can be stimulated with agonists and subsequently high-pressure frozen at time-points as fast as ~800 ms after stimulation. Each sample can then be processed for high-resolution imaging (room-temperature electron microscopy, cryo-electron microscopy, or super-resolution optical microscopy). Taken together, the ensemble of images from samples frozen at different time-points enables us to map changes in cellular architecture with nanometer spatial resolution and millisecond temporal resolution. Although we anticipate HPF-X will be broadly useful in studying rapid dynamics of biological signal processing, as a first step, we have begun to use HPF-X to explore the sub-diffractive assembly of processing bodies in response to hyperosmotic shock. In this dissertation, we discuss the engineering and validation of HPF-X. We walk through the key elements of our instrument’s operation -- pressurization, cryo-vitrification, and buffer exchange -- demonstrating both technical and preliminary biological validation of our tool’s function.

Thesis Supervisor:
Maxim B. Prigozhin, PhD
Assistant Professor of Molecular and Cellular Biology and of Applied Physics, Harvard University

Thesis Committee Chair:
Mehmet Toner, PhD
Helen Andrus Benedict Professor of Biomedical Engineering, Harvard Medical School
Founding Co-Director, The Institute for Bioengineering and Biotechnology, a division of Surgery at Massachusetts General Hospital
Director, BioMEMS Center, Mass General

Thesis Readers:
Lindsay B. Case, PhD
Irwin and Helen Sizer Career Development Assistant Professor of Biology, MIT

Adam E. Cohen, PhD
Professor of Chemistry and Chemical Biology and Physics, Harvard University

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Ami Thakrar is inviting you to a scheduled Zoom meeting

Topic: Ami Thakrar MEMP PhD Thesis Defense
Time: Friday, May 2, 2025, 2:30 PM Eastern Time (US and Canada)

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