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Kun Zhao
BME PhD Proposal Presentation
Date:2022-04-26
Time: Apr 26, 2022 09:00 PM Atlanta; Apr 27, 2022 09:00 AM Beijing.
Location / Meeting Link: Zoom Meeting https://emory.zoom.us/j/98717123427?pwd=cFdPd0hCV3NlYVgxalhnRHliY01mZz09 Meeting ID: 987 1712 3427 Passcode: 970280
Committee Members:
Dr. Peng Xi, Dr. Yonggang Ke, Dr. Changhui Li, Dr. Tianyu Xie, Dr. Juntao Gao.
Title: Extending spatiotemporal resolution of super-resolution microscopy
Abstract: Optical microscopes have limited resolution because of the natural phenomenon of light diffraction. Many super-resolution (SR) fluorescence microscopy techniques that overcome the limit have been developed, with widespread application for biological studies. An essential question for further development in super-resolution microscopy is that, can the boundaries of the spatial and temporal resolution be pushed further? This question concerns three major aspects: theory of optics, instruments and fluorophores. In this proposal, theory of optics and instruments are concerned. Here the following works are proposed: 1. Theoretical framework of two-photon MINFLUX. MINFLUX has the highest spatial resolution in current SR techniques, with ~ 1 nm localization precision and ~ 5 nm resolution. However, current MINFLUX doesn’t provide efficient multicolor capability, and contributes to only limited biological findings. Application of two-photon fluorescence to MINFLUX is proposed. Two-photon excitation increases gradient of intensity of donut minima through nonlinear effect. Localization precision of two-photon MINFLUX is doubled (to ~ 0.5 nm), and resolution is doubled as well. A single two-photon excitation wavelength may efficiently excite multiple fluorescence; registration-free multicolor 2p-MINFLUX is thus anticipated. 2p-MINFLUX takes a step closer to the ultimate resolution limit of size of a single molecule. 2. Further improvement of imaging speed of SIM. A parallel acquisition SIM is proposed to decrease the acquisition time of raw frames. Each raw frame has to be read-out completely before exposure of the next frame, which limits the imaging speed. By designing parallel acquisition scheme, multiple raw images are acquired with a single readout. Exploiting the rolling shutter of sCMOS camera, fluorescence exposure and camera readout are synchronized completely, saving the previously wasted time of camera readout. For field of view of 16.5 * 4.7 μm2, it is anticipated that 296 Hz SIM frame rate and 889 Hz rolling reconstruction rate could be achieved. 3. STED engineering prototyping with a single super-continuum laser. A STED microscope with 55 nm spatial resolution is built based on a single super-continuum laser, which is used for both excitation and depletion. It has the advantages of freely choices of excitation and depletion wavelengths, inherent synchronization of excitation and depletion beams and compactness of optics and hardware. In collaboration with Donglilai Optics Inc., the STED microscope is anticipated as a prototype for turn-key commercial STED microscope. Performances and limitations of the STED microscope are assessed with respect to parameters as wavelengths, power, and repetition rate.
