"Optomechanically Enhanced Fluorescence Imaging to Monitor Cellular Functions at Spatiotemporal Resolution" Semineri
Boğaziçi-BME/Lifesci & Inovita & ISEK Ortak Semineri
Optomechanically Enhanced Fluorescence Imaging to Monitor Cellular Functions at Spatiotemporal Resolution
Asst. Prof. Halil Bayraktar (Department of Chemistry, Koç University, Istanbul)
14 Mart 2017 (Salı) 13.00 – 15.00
Biyomedikal Mühendisliği Enstitüsü, AZ-19, Boğaziçi Universitesi Kandilli Yerleşkesi, İstanbul
Seminer Hakkında: This talk will introduce our recent work on developing optomechanically-enhanced fluorescence imaging methods to study cellular functions at spatiotemporal resolution. Local strain tracking microscope for mechanical perturbation of cells will be discussed. Cell-stretching techniques are a key method to regulate deformation magnitude, cyclic strain levels, and frequencies, therefore elucidating the biological processes involved in activation of mechanosensitive pathways, cell patterning and morphological changes at physiologically relevant mechanical loads. Although several approaches have been demonstrated to deform cells and usually compute ration cross-head displacement to initial length of sample as a percentage stretch, however in-plane strain components may have a non-uniform spatial distribution due to heterogeneous extracellular matrix of connective tissue around cells. This limitation has prompted us to develop an alternative approach. Here, we present uniaxial cell-stretching device integrated into inverted fluorescence microscope that provides a high spatial resolution to determine the local strain changes around fluorescent and non-fluorescent cells. Transparent and biocompatible PDMS elastomer modified with small fluorescent beads is used to deliver strain at the physiologically relevant magnitude and cycles.
Secondly, I will discuss our recent findings on developing photochromic fluorescence resonance energy transfer method to monitor opsin and heme protein dynamics. Super-resolution imaging microscopy provides an in-depth insight to investigate cellular pathways. Sub-diffraction localization provides unprecedented resolution for understanding the molecular interactions at bulk and single molecule level. Although biophysical imaging methods utilize different physical aspects to capture signals, optical efficiency is linked to the fluorescence labeling strategies. Hence, fluorescent probes are crucial for data acquisition. Advance reporters for imaging various functions are needed for stem cells, developmental biology and neuroscience. Different colors of fluorescent proteins are currently used to monitor the expression levels of proteins as well as intracellular dynamics of biomolecules by fluorescence resonance energy transfer (FRET) measurements. FRET provides a resolution of 1-10 nm, therefore it is used to measure the change in distance between donor and acceptor fluorophore, using the rate of energy transfer as a proxy sensor. I demonstrate the theory of photochromic FRET (pcFRET) and recent work for developing new biological applications. Briefly, spectral overlap affects the energy transfer efficiency if protein undergoes a large absorption change that can be detected by pcFRET. The dynamics of microbial membrane proteins such as sensory rhodopsin and redox proteins such as Cytochrome c were monitored. Principles of method provide a sensitive tool developed for monitoring rhodopsin dynamics and lately used for voltage imaging in neurons. My group uses pcFRET to identify changes in membranes and prepare novel constructs to enhance signal to noise ratio.
Konuşmacı Hakkında: Halil Bayraktar is currently an assistant professor in the chemistry department at Koc University. He received his BS. from Bogazici Unversity in 2003 and a Ph.D. from University of Massachusetts, Amherst, in 2008. He was a postdoctoral fellow until 2011 at Harvard University, Cambridge. He has developed photochromic fluorescence energy transfer imaging method to determine opsin protein dynamics. He currently works on developing optomechanical tools to study cell mechanics ant its influence in gene expression. His research group also develops genetically encoded fluorescent probes for imaging membrane and redox signals. His major research interests include cell mechanics, biophotonics, super-resolution microscopy methods, fluorescent probes and high content screening/tracking of cells.