สถานวิจัยการวิเคราะห์สารปริมาณน้อยและไบโอเซนเซอร์ (Trace Analysis and Biosensor Research Center: TAB-RC) (TAB Research Forum ครั้งที่ 22) ขอเชิญฟังการบรรยายและแลกเปลี่ยนความคิดเห็น โดย Assoc. Prof. Dr. Thorsten Wohland Departments of Biological Sciences and Chemistry and Centre for Bioimaging Sciences (CBIS) Faculty of Science, National University of Singapore

Morning session (10:00 - 11:30 a.m.) “Basics of Fluorescence Correlation and Cross-Correlation Spectroscopy”

Afternoon session (1:30 – 3:00 p.m.) “Imaging Fluorescence Correlation Spectroscopy as a Tool to Investigate Membrane Structure and Dynamics in Live Cells and in Vivo”

ณ ห้องสัมมนาคณะวิทยาศาสตร์ มหาวิทยาลัยสงขลานครินทร์ วันจันทร์ ที่ 6 มิถุนายน 2559 เวลา 09.30 น. – 15.30 น.

ผู้ใดสนใจ กรุณาแจ้งชื่อที่ คุณกนกเลิศลักษณ์ สุจริตพนิต โทร 074-288453 (ภายใน 8453) e-mail: [email protected] สถานวิจัยการวิเคราะห์สารปริมาณน้อยและไบโอเซนเซอร์

Morning session Basics of Fluorescence Correlation and Cross-Correlation Spectroscopy Thorsten Wohland Departments of Biological Sciences and Chemistry and Centre for Bioimaging Sciences, National University of Singapore, Singapore E-mail: [email protected]. Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS) belong nowadays to the basic repertoire of fluorescence techniques in many biophysical laboratories. However, these techniques are perceived as technically difficult, requiring complex evaluations. This complexity originates from at least three different sources. First, the basic observables in FCS are signal fluctuations, not fluorescence signals; but the physical meaning of fluctuations and their interpretation is often not apparent to users. Second, the analysis of the data via correlation functions is not commonly used outside the physical sciences and requires some adaptation of the user. And third, the actual properties of correlation functions and their calculation, which is often done on semi-logarithmic time scales, make an understanding even more difficult and their dependence on experimental conditions is often not straightforward and data fitting, and thus interpretation, is complex and sometimes time-consuming. However, with a little bit of effort FCS can be understood quite intuitively and lead to rewarding results not achievable with any other technique. Here we will first explain the meaning of fluorescence fluctuations and correlation functions and then discuss their experimental measurement and calculations before delving into some actual applications to show how a wide range of parameters, e.g. molecular mobilities and interactions, can be determined from FCS measurements. We will then extend the discussion to FCCS, which is particularly useful in determining biomolecular interactions and can be used to obtain quantitative data about protein dimerization and biomolecular affinities. In the last part we will discuss about some of the limitations of FCS and and how they can be overcome with different FCS implementations.

Afternoon session IMAGING FLUORESCENCE CORRELATION SPECTROSCOPY AS A TOOL TO INVESTIGATE MEMBRANE STRUCTURE AND DYNAMICS IN LIVE CELLS AND IN VIVO Thorsten Wohland Departments of Biological Sciences and Chemistry and Centre for Bioimaging Sciences, National University of Singapore, Singapore E-mail: [email protected]. Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) is a camera based FCS modality that records the fluorescence fluctuations in each voxel of a sample plane [1]. By correlating the fluorescence time traces it allows to extract information about molecular concentrations, mobilities and interactions. Imaging FCS has been implemented either in total internal reflection (TIR) microscopes for the observation of membranes or in light sheet microscopes for the observation of 3D samples [2]. Both illumination methods allow the selective excitation of fluorescent probes in a single layer in a sample. By using fast and sensitive array detectors (EMCCD or sCMOS cameras; avalanche photodiode arrays) one can collect data with single molecule sensitivity with time resolutions down to the microsecond range, fast enough to resolve molecular motions. Here we show how these methods can be used to measure molecular mobilities in 2D cell cultures and in vivo in live zebrafish embryos and how they can be combined with the so-called FCS diffusion law [3, 4] to extract information about structures in cell membranes even below the diffraction limit. First we discuss measurements on the organization of the epidermal growth factor receptor (EGFR) in live cells before and after stimulation. The diffusion law analysis in this case demonstrates the dependence of the EGFR on the membrane composition, in particular cholesterol, and the cytoskeleton. In the 3D case of live zebrafish measurements, we focused on the membrane organization of the secreted signaling protein Wnt3 [5]. Wnt3 undergoes two lipid modifications by palmitoylation at C77 and S209. These posttranslational modifications have been linked with protein association with cholesterol-dependent lipid domains. Using Imaging FCS we are able to show that Wnt3 localizes to cholesterol dependent domains in live zebrafish embryos and that this localization is disturbed when either the lipid modification of Wnt3 is inhibited by the porcupine inhibitor C59, or when the cholesterol concentration is reduced by methyl--cyclodextrin.

References [1] Bag, N., and T. Wohland. 2012. Imaging Fluorescence Fluctuation Spectroscopy: New Tools for Quantitative Bioimaging. Annu. Rev. Phys. Chem. 65: 225–248. [2] Krieger, J.W., A.P. Singh, N. Bag, C.S. Garbe, T.E. Saunders, J.O.R. Langowski, and T. Wohland. 2015. Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms. Nat. Prot. 10: 1948–1974. [3] Wawrezinieck, L., H. Rigneault, D. Marguet, and P.-F. Lenne. 2005. Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. Biophys. J. 89: 4029–4042. [4] Ng, X.W., N. Bag, and T. Wohland. 2015. Characterization of Lipid and Cell Membrane Organization by the Fluorescence Correlation Spectroscopy Diffusion Law. CHIMIA 69: 112–119. [5] Teh, C., G. Sun, H. Shen, V. Korzh, and T. Wohland. 2015. Modulating the expression level of secreted Wnt3 influences cerebellum development in zebrafish transgenics. Development 142: 3721–3733.

กาหนดการบรรยาย 9:30 – 10:00 น. ลงทะเบียนภาคเช้า 10:00 – 11:30 น. บรรยายในหัวข้อ “Basics of Fluorescence Correlation and CrossCorrelation Spectroscopy” ณ ห้องสัมมนา คณะวิทยาศาสตร์ 13:00 – 13:30 น. ลงทะเบียนภาคบ่าย 13:30 – 15:00 น. บรรยายในหัวข้อ “Imaging Fluorescence Correlation Spectroscopy as a Tool to Investigate Membrane Structure and Dynamics in Live Cells and In Vivo” ณ ห้องสัมมนา คณะวิทยาศาสตร์ 15:00 – 15:30 น. ร่วมรับประทานอาหารว่างและสนทนากับวิทยากร