Thorsten Wohland

Research

Biophysical fluorescence

The research in my group is directed towards biophysics with an emphasis on biophysical fluorescence. This highly interdisciplinary domain requires the interaction of chemists, physicists, and biologists. Only a concerted effort of these three groups will allow us to tackle the problems in the life sciences. The close proximity of departments of biology, biochemistry, chemistry, and physics at the National University of Singapore on one side and the research institutes (RIs) on the other side, allows us to develop physical and chemical methods for the study of biological questions on one hand and to apply these methods to the frontier in biology on the other hand. Our interests lie accordingly in the different areas that interact freely to advance this research field.

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Schematic Sketches of [A] a selective plane illumination and [B] a confocal microscope.

Construction and development of new optical tools

Optical Spectroscopy is one of the most sensitive tools available in the life sciences. Proteins can be studied not only in ensembles but as well on the single molecule level. Besides using well established methods in our group (e.g. Fluorescence Correlation Spectroscopy, Fluorescence Resonance Energy Transfer), we also plan to develop new spectroscopy and microscopy tools and new mathematical procedures to study proteins on a single molecule level in vitro and in vivo.

Study of selected proteins and protein complexes on a single molecule level and in living cells

In collaboration with the Department of Biological Sciences, the Department of Microbiology and the RIs we will study the properties of selected proteins and peptides to elucidate their function on a molecular level.

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(A) Schematic overview of FP-fusion constructs. (B) Example images of cells expressing different constructs.

At the moment we concentrate on either antimicrobial peptides and their interaction with bacterial membranes, or on the study of transmembrane proteins (G-protein coupled receptors, growth factors) and their structure, function and interactions.

One of the most interesting questions in biology is the relationship between the structure and function of proteins. With the fluorescence tools developed in our group we hope to shed some light on this question by in vitro experiments. In a complementary approach we will study the proteins in living cells because proteins are in many cases very sensitive to their environment and only when studied under physiological conditions can we determine their exact function.

New record: More than one million fluorescence correlation spectroscopy (FCS) measurements performed simultaneously

Traditional confocal Fluorescence Correlation Spectroscopy (FCS) measurements, which determine the molecular dynamics and concentrations in a femtoliter sized confocal observation volume, are taken at one point at a time with measurement times of typically 10-30 seconds. This does not allow the simultaneous measurement of biomolecular properties at multiple locations in a live cell.

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Imaging Total Internal Reflection FCS (ITIR-FCS) provides thousands of observation volumes by using the pixels of a fast and sensitive camera in conjunction with the thin evanescent field using a total internal reflection (TIR) microscope. The xy-sectioning by the pixel along with the z-sectioning by the evanescent beam provides thousands of femtoliter sized observation volumes. Here, we show that by using a scientific CMOS (sCMOS) camera, we can record 1,152,000 (1920×600) autocorrelation functions at 25 fps. The sample being measured here is 0.2 µm sized fluorescent beads. Typically, a stack of around 1500 images are recorded.

Latest Publications

Marzinek JK, Bag N, Huber RG, Holdbrook DA, Wohland T, Verma CS, Bond PJ. A Funneled Conformational Landscape Governs Flavivirus Fusion Peptide Interaction with Lipid Membranes. J Chem Theory Comput. 2018 Jun 6. doi: 10.1021/acs.jctc.8b00438.

Ng J, Kamm RD, Wohland T, Kraut RS. Evidence from ITIR-FCS Diffusion Studies that the Amyloid-Beta (Aβ) Peptide Does Not Perturb Plasma Membrane Fluidity in Neuronal Cells. J Mol Biol. 2018 May 7. pii: S0022-2836(18)30340-1. doi: 10.1016/j.jmb.2018.04.030.

Sankaran J, Karampatzakis A, Rice SA, Wohland T. Quantitative imaging and spectroscopic technologies for microbiology. FEMS Microbiol Lett. 2018 May 1;365(9).

Harwardt MIE, Dietz MS, Heilemann M, Wohland T. SPT and Imaging FCS Provide Complementary Information on the Dynamics of Plasma Membrane Molecules. Biophys J. 2018 Apr 9. pii: S0006-3495(18)30351-5.

Hoischen C, Yavas S, Wohland T, Diekmann S. CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres. PLoS One. 2018 Mar 6;13(3):e0192572