Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments.
The life sciences require new highly sensitive imaging tools, which allow the quantitative measurement of molecular parameters within a physiological three-dimensional (3D) environment. Therefore, we combined single plane illumination microscopy (SPIM) with camera based fluorescence correlation spectroscopy (FCS). SPIM-FCS provides contiguous particle number and diffusion coefficient images with a high spatial resolution in homo- and heterogeneous 3D specimens and live zebrafish embryos. Our SPIM-FCS recorded up to 4096 spectra within 56 seconds at a laser power of 60 microW without damaging the embryo. This new FCS modality provides more measurements per time and more, less photo-toxic measurements per sample than confocal based methods. In essence, SPIM-FCS offers new opportunities to observe biomolecular interactions quantitatively and functions in a highly multiplexed manner within a physiologically relevant 3D environment.
Wohland
Thorsten
T
Department of Chemistry, National University of Singapore, Singapore. chmwt@nus.edu.sg
Shi
Xianke
X
Sankaran
Jagadish
J
Stelzer
Ernst H K
EH
D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation*; D021621:Imaging, Three-Dimensional / Q000295:instrumentation*; D008029:Lighting / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*