Firstly, measurements in cells are often performed under over-expression of proteins of interest. This can lead to changed interaction patterns and physiological effects (compare, for instance, the different expression levels of proteins in cancerous and non-cancerous cells). Therefore, we need to develop techniques that can measure biomolecular interactions at physiological expression levels.
Secondly, measurements are often performed in 2D cell cultures. However, these conditions do not reflect the actual three dimensional organization of organisms and crucial cell-cell interactions are not well replicated. Thus, it is important to develop methods that can measure biomolecular functions and interactions in a three dimensional context of 3D cell cultures, tissues, or whole living organisms.
Thirdly, many spectroscopy methods which allow quantitation of biological interactions in vivo are restricted to single point measurements. Therefore, there is a need to multiplex existing techniques and develop imaging spectroscopy techniques that allow the quantitative analysis of every single point in an image with good spatial (diffraction limited) as well as good temporal resolution.
The most sensitive biophysical tools to date are fluorescence microscopy and spectroscopy which are routinely applied in cells and can have single molecule sensitivity. Therefore, recently several attempts have been made to a) bring fluorescence spectroscopy into living organisms, and b) provide imaging capabilities for spectroscopy to allow simultaneous measurements of biological processes in the complex 3D structure of living tissues with good temporal and spatial resolution.
We have developed four new fluorescence techniques called Single Wavelength Fluorescence Cross-correlation Spectroscopy (SW-FCCS), Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS), Line scan FCS, and Single Plane Illumination Microscopy Fluorescence Correlation Spectrscopy (SPIM-FCS) which address at least some of these issues. SW-FCCS can quantitatively measure biomolecular interactions with single molecule sensitivity in cells and organisms. (SW-FCCS Publications) ITIR-FCS on the other hand is a spectroscopic imaging technique which provides good spatial (diffraction limited) and temporal (~0.3 ms) time resolution.(Imaging FCS Publications) Line-scan FCS provides a tool to measure flow profiles with good spatio-temporal resolution in organisms (e.g. in the zebrafish vascular system). SPIM-FCS is capable of performing multiplexed measurements in cells and organisms (e.g. blood flow in zebrafish). By using state-of-the-art fluorescence techniques we hope to help elucidate biomolecular interactions under physiological relevant conditions and connect some molecular events to macroscopic effects, e.g. the development of organisms. (FCS in organism Publications).
For instance, here we study the dynamics of Wnt3, Lyn and Sec in zebrafish cerebellum. (A) Confocal image of zebrafish cerebellum expressing Wnt3EGFP at 34 hpf. Scale bar: 50 μm. (B) Zoom of A at 3× magnification with focus on the cerebellum boundary and flanking brain ventricle. Scale bar: 20 μm. Images were taken in dorsal view. BV, brain ventricle; ce, cerebellum. The images were modified using Imaris to increase the contrast. (C) Normalized fluorescence intensity from the cerebellum boundary cell to the brain ventricle along the white arrow in B of Wnt3EGFP (red), LynEGFP (green) and secEGFP (blue). Data are the average of three scans of three embryos for each type to the highest point. (D) Normalized ACF curves taken within a ventricle at 100 μm from the cerebellum boundary. Color-coding is the same as in C together with wild type (WT, dotted gray). The results show the free diffusion of Wnt3EGFP and secEGFP in the brain ventricle, whereas no fluorescence can be detected either for LynEGFP or for WT. (E) Diffusion coefficients extracted from fit for different types of EGFP-labeled proteins in both the cerebellum and the brain ventricle. secEGFP serves as an intercellular indicator of protein mobility in multicellular tissue and extracellular indicator in the brain ventricle. EGFP reporter Tg(-4.0wnt3:EGFP)F2 and LynEGFP transgenics Tg(-8.0cldnB:lynEGFP) serve as an indicator of intracellular protein mobility. Data are mean±s.d. Light gray bar, brain ventricle (BV); dark gray bar, cerebellum (ce).