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29 Dec 2015 Scientists in NUS have developed new methods to image dynamics in live samples for a better understanding of molecular processes in biology.Biology and light microscopy have developed hand in hand over the last ~400 years with new inventions in microscopy leading to new discoveries in biology. However, most imaging was based on simple intensities and thus provided only spatial information of the presence or absence of particular molecules. Even modern super-resolution approaches mostly image intensities. However, as important as the spatial information is the temporal information, i.e. is the dynamics of the imaged molecules.A team led by Prof Thorsten WOHLAND from the Departments of Biological Sciences and Chemistry in NUS and Prof Jörg LANGOWSKI from the German Cancer Research Centre (DKFZ) has developed over the last years new approaches to imaging dynamics. They exploited fast and sensitive cameras, which can record 1000 images per second and can detect single molecules with new microscopy techniques. In particular they used so-called total internal reflection (TIR) and single plane illumination microscopy (SPIM, see Figure1a) to illuminate whole planes in live specimens to record molecular dynamics at each pixel in the images (see Figure 1b). In this way, they can determine the diffusion and transport of molecules as well as the interaction between molecules in live samples. This technology thus opens up new ways to investigate molecular mechanisms even in small organisms. Protocols with details how to perform these measurements including free software packages for the data analysis have just been published.
Figure 1a shows the principle of a light sheet setup. Shown is a single plane illumination microscopy (SPIM) arrangement. From left enters the laser sheet through the illumination objective. The fluorescent emission excited by the light sheet is collected by the detection objective and imaged onto a fast ultrasensitive camera. The light sheet allows illumination of any plane in a 3D sample. Figure 1b shows the results from a SPIM-FCS measurement of eGFP-labeled histones H2A in HeLa cells, with an intensity image, a map of the concentration and of the fraction of the slow diffusing component.
Reference
Krieger JW, AP Singh, N Bag, CS Garbe, TE Saunders, J Langowski, T Wohland. “Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms.” Nat Protoc 10 (2015) 1948.
