Super-resolution microscopy (nanoscopy) enables the visualization of live cells at resolutions that exceed the diffraction limit of light, far beyond the capability of ordinary light microscopes. Observing living cells at resolutions <20 nm reveals details of organelle structure, function, and dynamics that deepen our fundamental understanding of cell biology. While there has been significant progress in the engineering, design, and development of improved nanoscopy hardware, the field is limited by the fuel?the dyes that support the ever more sophisticated requirements of both Stimulated Emission Depletion (STED) and Single Molecule Switching (SMS). These requirements, which include brightness, photostability, cell-permeability, and for SMS: the ability to ?blink,? represent real chemical challenges that currently limit the full potential of these methodologies. Recently, the Silicon Rhodamine (SiR) scaffold has been shown to exhibit many promising characteristics for live-cell super-resolution imaging (SiR-CO2H) such as cell-permeability, photostability, and the ability to blink spontaneously (HMSiR) but has in no way been fully optimized. Work in the Schepartz Lab addresses the aforementioned challenges from a chemical perspective through the design of probes and fluorophores that are tailored to overcome these challenges while meeting the needs of nanoscopy techniques. These efforts include the development of a lipid probe system for super-resolution imaging of the Golgi (published work) and Endoplasmic Reticulum (ER) (unpublished work). This work was performed in close collaboration with the Rothman, Toomre, and Bewersdorf labs in Yale Cell Biology. The proposed research seeks to develop new and improved fluorophores that will take full advantage of cutting- edge nanoscopy techniques available at Yale and elsewhere, providing new tools to advance our understanding of biology and medicine.

Public Health Relevance

Super-resolution microscopy (nanoscopy) enables the visualization of live cells at resolutions that exceed the diffraction limit of light, far beyond the capability of ordinary light microscopes. Observing living cells at resolutions <20 nm reveals details of organelle structure, function, and dynamics that deepen our fundamental understanding of cell biology. The proposed research seeks to develop new and improved fluorophores that will take full advantage of cutting-edge nanoscopy techniques. s

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31GM119259-01A1
Application #
9190951
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Barski, Oleg
Project Start
2016-09-01
Project End
2018-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Yale University
Department
Chemistry
Type
Graduate Schools
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
Takakura, Hideo; Zhang, Yongdeng; Erdmann, Roman S et al. (2017) Long time-lapse nanoscopy with spontaneously blinking membrane probes. Nat Biotechnol 35:773-780
Thompson, Alexander D; Bewersdorf, Joerg; Toomre, Derek et al. (2017) HIDE Probes: A New Toolkit for Visualizing Organelle Dynamics, Longer and at Super-Resolution. Biochemistry 56:5194-5201
Thompson, Alexander D; Omar, Mitchell H; Rivera-Molina, Felix et al. (2017) Long-Term Live-Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI-SiR. Angew Chem Int Ed Engl 56:10408-10412