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.

Selected Publications

(Full list of publications)

Bag N, Ng XW, Sankaran J, Wohland T. Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes. Methods and Applications in Fluorescence. 4 (3), 034003

Ng XW, Teh C, Korzh V, Wohland T. The secreted signaling protein wnt3 is associated with membrane domains in vivo: a SPIM-FCS study. Biophys J. 2016 Jul 26;111(2):418-29.

Macháň R, Foo YH, Wohland T. On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements. Biophys J. 2016 Jul 12;111(1):152-61.

Wang Y, Wang X, Wohland T, Sampath K. Extracellular interactions and ligand degradation shape the Nodal morphogen gradient. Elife. 2016 Apr 21;5.

Ge J, Zhang CW, Ng XW, Peng B, Pan S, Du S, Wang D, Li L, Lim KL, Wohland T, Yao SQ. Puromycin Analogues Capable of Multiplexed Imaging and Profiling of Protein Synthesis and Dynamics in Live Cells and Neurons. Angew Chem Int Ed Engl. 2016 Apr 11;55(16):4933-7.

Krieger, J.W; Singh, A.P; Bag, N; Garbe, C.S; Saunders, T.E; Langowski, J; Wohland, T. Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms. Nat Protoc. 2015 Dec;10(12):1948-74.

Bag, N; Huang, S; Wohland, T. Plasma Membrane Organization of Epidermal Growth Factor Receptor in Resting and Ligand-Bound States. Biophys J. 2015 Nov 3;109(9):1925-36.

Eshaghi, M; Sun, G; Grüter, A; Lim, C.L; Chee, Y.C; Jung, G; Jauch, R; Wohland, T; Chen, S. L. Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization. Angew Chem Int Ed Engl. 2015 Oct 8. doi: 10.1002/anie.201506686.

Teh C, Sun G, Shen H, Korzh V, Wohland T. Modulating expression level of secreted Wnt3 influences cerebellum development in zebrafish transgenics. Development. 2015 Nov 1;142(21):3721-33

Mistri, T.K.; Devasia, A.G.; Chu, L.T.; Ng, W.P.; Halbritter, F.; Colby, D.; Martynoga, B.; Tomlinson, S.R.; Chambers, I.; Robson, P.; Wohland, T. Selective influence of Sox2 on POU transcription factor binding in embryonic and neural stem cells, EMBO Rep 2015 Aug 11. pii: e201540467

Ng, X.W.; Bag, N.; Wohland, T. Characterization of Lipid and Cell Membrane Organization by the Fluorescence Correlation Spectroscopy Diffusion Law, CHIMIA International Journal for Chemistry 69 (3), 112-119.

Rashid, R.; Chee, S.M.L.; Raghunath, M.; Wohland, T. Macromolecular Crowding Gives Rise to Microviscosity, Anomalous Diffusion & Accelerated Actin Polymerization, Physical Biology (2015) Apr 30; 12(3): 034001

Sun, G.; Guo, S.M.; Teh, .; Korzh, V,; Bathe, M.; Wohland, T. Bayesian Model Selection Applied to the Analysis of FCS Data of Fluorescent Proteins in vitro and in vivo, Analytical chemistry (2015) Apr 21; 87(8): 4326-33