JUL 16, 2020 8:00 AM PDT

SRRF 'n' TIRF - simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

Sponsored by: Andor
C.E. Credits: P.A.C.E. CE Florida CE
Speakers
  • Professor of Biological Sciences and Chemistry., National University of Singapore (NUS)
    Biography
      Thorsten Wohland studied Physics at the Technical University of Darmstadt and the University of Heidelberg in Germany from 1989-1995. He completed his diploma thesis in physics at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, where he worked on the influence of light polarization on the forces in optical tweezers under the supervision of Ernst H.K. Stelzer. In 1997 he joined the research group of Prof. Horst Vogel at the Swiss Federal Institute of Technology in Lausanne (ETHL/EPFL), Switzerland. In the year 2000 he obtained his doctoral degree in the field of biophysics for the study of theoretical and practical aspects of fluorescence correlation spectroscopy (FCS) and its application to integral membrane proteins. Following another two years in the group of Richard N. Zare at Stanford in the USA working on single molecule detection and protein immobilization he joined the National University of Singapore (NUS) where he is now Professor in the departments of Biological Sciences and Chemistry. At NUS he developed several new fluorescence correlation spectroscopy methods including imaging fluorescence correlation spectroscopy. His current research aims at the integration of fluorescence microscopy and spectroscopy to yield quantitative microscopy methods that can extract information with high spatial and temporal resolution and the application of these methods to biological problems.
    • Product Specialist for Microscopy Cameras at Andor Technology.
      Biography
        My background in microscopy is built from my work and studies while at Queen's University Belfast. I always had a keen interest in bacteria and viruses as pathogens, as well as how we may exploit them for our benefit, so naturally specialised in microbiology. After my BSc (Hons) in 1999, I went on to do my PhD in microbiology within the School of Biology and Biochemistry at the Medical Biology Centre at Queens. My PhD was focused on polyphosphate metabolism of microorganisms- trying to get a better understanding of this molecule, how to quantify it, and the key roles this played within environmental and pathogenic bacteria. Roles include as a protective function in response to stress and in helping pathogens establish infections. An interesting application of this was in biological phosphate removal from wastewater for which we had a pilot scale study. This then led on to a Research Fellow position in the same research group of John Quinn and John McGrath in 2002. While in this position I worked further on connecting the biochemistry and genetics of polyphosphate metabolism. This included studies to characterise the bacterial transport systems involved and development of novel research tools, such as fluorescent assays to determine intracellular pH, and how to apply our luminescence and fluorescence imaging based methods to 96 well plate formats for faster screening and analysis. After Academia, I went on to work in the Medical Diagnostics and Pharmaceutical industries. I joined Andor Technology in 2012, initially working as a Technical Author for our camera and systems products. My current role is a Product Specialist for microscopy cameras. This role brings me in touch with the latest developments in camera technology, as well as a broad range of microscopy applications and techniques.

      Abstract
      DATE:  July 16, 2020
      TIME:   8am PT, 11am ET, 4pm BST, 5pm CEST
       
       
      Super-resolution microscopy and single molecule fluorescence spectroscopy often require mutually exclusive experimental strategies optimizing either time or spatial resolution. While the measurement of biomolecular dynamics on the single molecule level requires fast measurements on the millisecond scale, super-resolved images require acquisition times on the second scale to achieve the required signal-to-noise ratio. These complementary requirements render the combination of spatiotemporal super-resolution microscopy challenging. Past solutions either restricted time resolution, limited the field of view and number of recorded points, or they required specialized instrumentation or fluorescent labels, restricting access to the techniques. To achieve high spatiotemporal resolution, we implemented a GPU-supported, camera-based measurement strategy that resolves high spatial structures (~60 nm), temporal dynamics (≤ 2 ms), and molecular brightness analysis from the exact same data almost in real time. 
       
      In this webinar, we investigated the connection between the epidermal growth factor receptor (EGFR) dynamics, its oligomerization state and the cytoskeleton. For this purpose, we acquired images of mApple labeled EGFR and LifeAct, an actin binding protein labelled with EGFP, on whole cells with high sensitivity and high-speed using EMCCD or sCMOS cameras and GPU-based processing with spatial or temporal binning to optimize extraction of various parameters. The resulting single datasets are evaluated by a combination of spectroscopy and super-resolution techniques that include: imaging fluorescence correlation spectroscopy (imaging FCS) to measure dynamics; Number and Brightness analysis (N&B) to determine oligomerization or aggregation states; and super resolved radial fluctuation microscopy (SRRF) to obtain super-resolution images. The simultaneous acquisition of these multiple fluorescence parameters allows a direct cross-correlation analysis, which would not be possible in sequential measurements, that allows us to determine how EGFR diffusion is dependent on its oligomerization state, and whether the cytoskeleton has any influence on the receptor diffusion mode and dynamics.  This approach is easily extendable to other fluorescence parameters, does not require specialized instrumentation, and thus is immediately applicable to a wide range of situations. 
       
      Learning Objectives:
       
      This webinar will demonstrate how participants can extract more information from their fluorescence images by combining computational super-resolution microscopy with fluorescence fluctuation techniques using only existing, freely available ImageJ plugins and widely existing setups of commercially available TIRF/SPIM microscope and cameras (EMCCDs or sCMOS). For that purpose, the webinar will discuss imaging fluorescence correlation spectroscopy and number and brightness analysis and how it benefits from computational super-resolution techniques.
       
      Questions Answered:
      1. Is it possible to extract from a single measurement structure with a resolution below 100 nm and with dynamics on the millisecond scale?
      2. Given the huge amounts of data and the size of single data sets (~GB) is data treatment in real time possible?
      3. What is the advantage of combining multiple fluorescence techniques in a single measurement?
      4. Can one optimise the signal-to-noise ration of multiple fluorescence techniques simultaneously in a single measurement?
      5. What is the maximum information content of an image? 
       
       
      Webinars will be available for unlimited on-demand viewing after live event.
       
      LabRoots is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. ® Program. By attending this webinar, you can earn 1 Continuing Education credit once you have viewed the webinar in its entirety.

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