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Figure 2: Quanttatve imaging. (A) The frst image shows the plasma membrane of a neuroblastoma (SH-SY5Y) cell that expresses a fuorescent marker. The image, taken with an exposure tme of 50 ms, reports only on the brightness of the membrane. By imaging the sample very fast (50,000 images at 2 ms exposure), we can determine in each pixel the tme course of the fuorescence and deduce how partcles move and interact. (B) The difusion coefcient D , as determined ACF from the fuorescent tme traces at each pixel, showing regions of diferent mobility. (C) We can even determine whether the tracer can move freely (t < 0.2 s, where t is a measure for the freedom of movement) or whether the tracer encounters 0 0 obstacles (t > 0.2 s), even though the obstacles might be much too small to be seen by microscopy. Note that even in the 0 case of image (C), which has less pixels and is thus less resolved, it stll carries more detailed informaton as it reports on the mode of movement of the tracer at diferent parts of the cell. also how molecules interact (see their dynamics and follow molecular Figure 2) [3]. We have applied this The quantfcaton of imaging is just processes in a whole cell or even technology in the past to determine startng. There are many more ways organisms, to develop intelligent self- the interacton of drugs with proteins, to evaluate and analyse imaging driven data analysis programmes that measure how cancer-related receptor data. Ofen we do not even know help us determine how best to extract proteins interact in membranes, and which method is best and our images numbers. We need numbers to further how proteins move through tssues stll cover only a small part of a cell. our understanding of how the world and interact with proteins on cells to Therefore, our dream is to take 3D and we ourselves functon. Images control development of organisms. images of samples together with aren’t enough. Thorsten WOHLAND is a Professor with the Departments of Biological Sciences and Chemistry and is associate director for the Center for Bioimaging Sciences. He is a biophysicist by training, who studied Physics in Darmstadt and Heidelberg, Germany. He received his Ph.D. from the Swiss Federal Insttute of Technology in Lausanne (EPFL), Switzerland, did his postdoctoral research at Stanford University, USA, and joined NUS in 2002. His interests are in development and applicaton of fuorescence spectroscopy techniques. References [1] htp:// [2] Bag N; Wohland T, “Imaging fuorescence fuctuaton spectroscopy: New tools for quanttatve bioimaging” ANNUAL REVIEW OF PHYSICAL CHEMISTRY, VOL 65 Pages: 225-248 DOI: 10.1146/annurev-physchem-040513-103641 Published: 2014. [3] Krieger JW; AP Singh; N Bag; CS Garbe; TE Saunders; J Langowski; T Wohland, “Imaging fuorescence (cross-) correlaton spectroscopy in live cells and organisms” NATURE PROTOCOLS Volume: 10 Issue: 12 Pages: 1948-1974 DOI: 10.1038/nprot.2015.100 Published: 2015. ADVANCES IN SCIENCE | VOL. 22 | NUMBER 1 | JUNE 2017 5
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