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Medical Research Seminar: Super-resolution imaging methods
September 14 @ 12:00 pm - 1:00 pm
Dr Yuanqing Alex Ma
University of New South Wales
About the talk:
1. Clustering of TCR is sufficient to triggers T cell activation
The very fundamental question of how the TCR ligand binding event is transduced across the cell plasma membrane and causes the increase of phosphorylation of the intracellular motifs of the TCR complex remains unclear. One of the important molecular events occurring during T cell activation is the spatial clustering of the TCR, where critical downstream kinases and adaptor signalling proteins become associated with the clusters. Indicating TCR clustering plays an important role for T cell activation. However, the exact function of TCR clustering remains unclear. By using an optogenetic tool, I demonstrated that T cells can be activated by light induced clustering of the CD3ζ chain of the TCR complex, implying TCR clustering is the prerequisite for T cell activation.
2. Improve SOFI resolution by increase fluorophore blinking
Stochastic optical fluctuation super-resolution imaging SOFI is one of the variants of current super-resolution imaging methods. Compared to the single molecule localization based super-resolution imaging method, such as PALM, STORM, SOFI produces super-resolved images faster by taking fewer frames. Compared to the stimulated emission depletion STED super-resolution imaging approach, the optical setup of SOFI is simpler and less photodamaging to the sample. One of the obstacles that significantly limit the potential application of SOFI image is the slow and heterogeneity blinking of the dyes. Here, I demonstrated that by using electrochemistry, I can accelerate the dye blinking and improve SOFI image resolution.
3. Introduce axial contrast in STED imaging
Due to the diffraction of light, the laser focus of the confocal microscope is limited to a 200 nm diameter spot. Features smaller than 200 nm cannot be resolved. STED microscope overcomes the diffraction barrier by introducing the donut shaped depletion beam that turns the surrounding fluorophores off except the geometric centre of the laser focus. Several 3D STED techniques have been developed, which introduce STED depletion lobes above and below the focal plane by either using a 4-pi two objective approach or a top-hat phase plate in a separate excitation light path. Here, we present a straightforward approach to increase the axial contrast of the traditional STED imaging by using the Metal Induced Energy Transfer method. By imaging the fluorophores on the metal coated coverslip, we generated fluorescence lifetime contrast along the axial direction that enables 3D STED image without additional modification to the STED microscope.
About the speaker: Yuanqing Ma, often known as Alex by his colleagues here in Australia, was born and educated in life science in China. He did postgraduate research in Sydney Australia, where he worked on cloning and optimizing fluorescent proteins from corals and jellyfish in the great barrier reef in Australia and coast in Japan. For his PhD program in Professor Katharina Gaus’ group at UNSW, his work was to understand the critical molecular events that are responsible for triggering T cell activation.