Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS. Here we provide guidelines for imaging FCS/FCCS measurements on commercial and custom-built microscopes (including sample preparation, setup calibration, data acquisition and evaluation), as well as anticipated results for a variety of in vitro and in vivo samples. For a skilled user of an available SPIM or TIRF setup, sample preparation, microscope alignment, data acquisition and data fitting, as described in this protocol, will take ∼1 d, depending on the sample and the mode of imaging.

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Abstract:Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, transport, flow velocities and concentrations in an imaging mode. SPIM-FCS records correlation functions over a whole plane in a sample, which requires array detectors for recording the fluorescence signal. Several types of image sensors are suitable for FCS. They differ in properties such as effective area per pixel, quantum efficiency, noise level and read-out speed. Here we compare the performance of several low light array detectors based on three different technologies: (1) Single-photon avalanche diode (SPAD) arrays, (2) passive-pixel electron multiplying charge coupled device (EMCCD) and (3) active-pixel scientific-grade complementary metal oxide semiconductor cameras (sCMOS). We discuss the influence of the detector characteristics on the effective FCS observation volume, and demonstrate that light sheet based SPIM-FCS provides absolute diffusion coefficients. This is verified by parallel measurements with confocal FCS, single particle tracking (SPT), and the determination of concentration gradients in space and time. While EMCCD cameras have a temporal resolution in the millisecond range, sCMOS cameras and SPAD arrays can extend the time resolution of SPIM-FCS down to 10 μs or lower. References and links1. K. M. Berland, P. T. So, and E. Gratton, "Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment," Biophy. J. 68, 694-701 (1995). 2. P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, "Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one-and two-photon excitation," Biophy. J. 77, 2251-2265 (1999). 3. R. Brock, G. Vàmosi, G. Vereb, and T. M. Jovin, "Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy," Proc. Natl. Acad. Sci. U.S.A. 96, 10123-10128 (1999 0-1 (2010). 53. Z. Petrásek, P. Schwille, and Z. Petrášek, "Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy," Biophy.

In curved environments, geometric constraints can result in cellular competition for space. Quantitative information about cell geometry was extracted to explore how columnar-like cells fit within the curved anterior of the Drosophila embryo. Cell deformations and cell rearrangements enable packing in this highly curved region.

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Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, flow velocities and concentrations in an imaging mode. Here we extend this technique to two-color fluorescence cross-correlation spectroscopy (SPIM-FCCS), which allows to measure molecular interactions in an imaging mode. We present a theoretical framework for SPIM-FCCS fitting models, which is subsequently used to evaluate several test measurements of in-vitro (labeled microspheres, several DNAs and small unilamellar vesicles) and in-vivo samples (dimeric and monomeric dual-color fluorescent proteins, as well as membrane bound proteins). Our method yields the same quantitative results as the well-established confocal FCCS, but in addition provides unmatched statistics and true imaging capabilities.

Integrating data from three independent data sources--USPTO patenting data, Shanghai Jiao Tong University's Academic Ranking of World Universities (ARWU) and the Times Higher Education Supplement's World University Ranking (WUR), we examine the possible link between patenting output and the quantity and quality of scientific publications among 281 leading universities world-wide. We found that patenting by these universities, as measured by patents granted by the USPTO, has grown consistently faster than overall US patenting over 1977-2000, although it has grown more slowly over the last 5 years (2000)(2001)(2002)(2003)(2004)(2005). Moreover, since the mid-1990s, patenting growth has been faster among universities outside North America than among those within North America. We also found that the patenting output of the universities over 2003-2005 is significantly correlated with the quantity and quality of their scientific publications. However, significant regional variations are found: for universities in North America, both the quantity and quality of scientific publications matter, but for European and Australian/NZ universities, only the quantity of publications matter, while for other universities outside North America and Europe/Australia/NZ, only quality of publications matter. We found similar findings when using EPO patenting data instead of USPTO data. Additionally, for USPTO data only, the degree of internationalization of faculty members is found to reduce patenting performance among North American universities, but to increase that of universities outside North America. Plausible explanations for these empirical observations and implications for future research are discussed.

Rhabdomyosarcoma (RMS) is a rare malignant soft tissue neoplasm comprised of cells derived from the primitive mesen¬chyme. About 35% of RMS arises in the head and neck, are are classified as parameningeal and non-parameningeal forms. These are the most common soft tissue sarcoma of the children, adolescents and young adults. Their etiopathogenesis and its molecular relevance have been emphasized. The first line of treatment is radical excision and this is usually supplemented by radiotherapy. It is believed that adjunct combination chemotherapy may greatly improve the prognosis. Inadequately treated tumours grow in an infiltrative manner and recur in a high percentage of cases. Bone does not constitute an effective barrier to the growth of the tumour and bone invasion is a frequent finding in head and neck rhabdomyosarcomas. Key words:Rhabdomyosarcomas, botryoid, spindle, alveolar, sarcomas, undifferentiated.