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Biography

Prof.  Kun  Chen
University of Electronic Science and Technology of China

Title: Excitation fluorescence spectral microscopy for highly multiplexed and quantitative cellular imaging

Abstract:

We discuss our recent efforts to achieve highly multiplexed fast fluorescence imaging and quantitative biosensing through camera frame-synchronized scanning of the excitation wavelength in the wide field. For live-cell microscopy, we thus attain low (~1%) crosstalks and ~10 ms temporal resolutions for up to six fluorophores via linear unmixing. By covering the excitation/emission bandwidth across the full visible range, we extend this approach to the simultaneous imaging of up to ten targets, with unambiguous identification of different intracellular structures labeled by common fluorophores of substantial spectral overlap with minimal color crosstalks. To overcome spatial variations of reference spectra in unmixing caused by the heterogeneity of local structure and environment in live biological samples, we propose a novel spectral unmixing technique without measured reference spectra based on a regularized mutual information minimization scheme for spectral separation of the overlapped fluorophores, enabling high accuracy and faster acquisition in multiplexed fluorescence microscopy. The possibility to unmix and quantify different fluorescent species in the same sample through the excitation spectrum next enables us to devise fast, quantitative imaging schemes for different modes of both bi-state and FRET fluorescent biosensors in live cells. These capabilities are further integrated to multiplex absolute pH imaging with three additional target proteins in the mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging to quantitatively reveal intracellular activities and parameters of interactive organelles, beyond the qualitative visualization of their interactions.

Biography:

Kun Chen is a full professor in the College of Optoelectronic Science and Engineering at Univ. of Elect. Sci. & Tech. of China, (UESTC). His aims to develop and apply new optical imaging tools to better understand biology at the cellular and molecular level by integrating advanced laser techniques, single-molecule biophysics, Raman & fluorescence spectroscopy and nonlinear & super-resolution microscopy.

Kun did his postdoctoral research at University of California, Berkeley with Professor Ke Xu from 2017 to 2021. At Berkeley, he led the development of single-molecule displacement/diffusivity microscopy and excitation spectral fluorescence microscopy, and their applications in cell biology.

Kun received his B.S. in Opto-electronic science and technology from Nankai University in 2013. He obtained his Ph.D. in Optical Engineering from Tsinghua University in 2017, where he developed a highly advanced nonlinear multiphoton vibrational microscopy method to achieve fast label-free CARS imaging based on optical frequency combs and nonlinear optical spectroscopy with Professor Yan Li.

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