Fluorescence Correlation Spectroscopy

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Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.

Conventional FCS can provide absolute diffusion constant and concentration values only when the exact shape and size of the confocal volume is known. In practice, the shape is approximated by a 3D gaussian function. Size and elongation of the volume is obtained by calibration measure­ment of a reference compound with a known diffusion constant.
In order to get correct confo­cal volume para­meters, the experimental conditions during calibration (wavelengths, excita­tion power, cover slip thickness, solvent and immersion medium) must be the same as during the measurement. Precise diffusion coefficients of several reference compounds suitable for calibration purposes are summarized in this compilation.

ABSTRACT, The design and characterization of an imaging,sensor based on single,photon avalanche,diodes is presented. The sensor was fully integrated in a 0.35µm CMOS technology. The core of the imager,is an array of 4x112 pixels that independently,and simultaneously detect the arrival time of photons,with picosecond,accuracy. A novel event-driven readout scheme,allows parallel column-wise and non-sequential, on-demand row-wise operation. Both time-correlated and time-uncorrelated

Recently, thermal properties of the landscaped rooftops and walls have attracted the interest of researchers because of the potential to minimize energy consumption in urban areas and to aid summer-time thermal control. For this reason the creation of a plant-based shade for walls or above buildings is highly important. In this paper we evaluate using Lygodium japonicum, one of the many ferns and fern allies traditionally used in Japanese gardening, as a component of thermal-buffering green walls. Lygodium japonicum, the only climbing fern species in Japan, is fast-growing, adheres easily to walls and has a climbing nature. A simple thermal analysis of the sun-shading effect of Lygodium canopy suggested that local surface temperature above the ceramic tiles placed on the rooftop of a building can be buffered (lowered in daytime and maintained relatively warm at night) by the presence of leafy climbing ferns covering the tiles, possibly due to the reflection and absorbance of solar radiation. Furthermore, the presence of the plants may also slow the night-time release of heat from the building surface. Because plants installed on tall walls or on the tops of buildings are not easily accessed for manual care, we performed a real-time routine monitoring and control of plant growth status using various optical sensors that could be automated and monitored remotely for large-scale applications. For this purpose, the optical properties of a L. japonicum canopy under solar incident light have been determined. In order to evaluate the natural shading and growing properties of a green canopy, the incident solar radiation spectrum (J), leaf canopy-filtered light spectrum (transmittance, T) and leaf-reflectivity spectrum (R) were measured. By reading the reflectivity spectrum, concomitant chlorophyll fluorescence signals (F) from Lygodium leaves were also detected at 760 nm, which corresponds to the O2-A Fraunhofer line. Our data suggests that the daily change in photosynthetic status (P) can be traced by monitoring the change in relative F in relation to the estimated heat loss (H) and measured J, R, and T using a series of practical equations designed to roughly estimate the gross photosynthetic response within the plant canopy. Using our equations, the photosynthetic capacity in the plant canopy structure could be simply simulated and predictable by optical sensors.

Network data-transfer times in distributed simulation environments can be reduced by performing data analysis at the remote source, if the analytical technique does not require the entire set of data at once. This novel multi-tau autocorrelation algorithm allows time-domain data records to be processed in discrete, distributed segments and combined at a later point in time. The new approach agrees with autocorrelation results performed by concatenating the discrete segments before correlation, but it operates with significantly shortened processing times. The multi-tau algorithm also benefits from reduced memory requirements since it does not require access to the entire data record at once, and from improved scalability since the multi-tau algorithm has order O(N), while fast Fourier transform autocorrelation algorithms have order O(N log N). This distributed algorithm has particular utility in simulations of fluorescence correlation spectroscopy or photon correlation spectroscopy.

Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation,...

Genomes are tremendous co-evolutionary holistic systems for molecular storage, processing and fabrication of information. Their system-biological complexity remains, however, still largely mysterious, despite immense sequencing achievements and huge advances in the understanding of the general sequential, three-dimensional and regulatory organization. Here, we present the GLOBE 3D Genome Platform a completely novel grid based virtual “paper” tool and in fact the first system-biological genome browser integrating the holistic complexity of genomes in a single easy comprehensible platform: Based on a detailed study of biophysical and IT requirements, every architectural level from sequence to morphology of one or several genomes can be approached in a real and in a symbolic representation simultaneously and navigated by continuous scale-free zooming within a unique three-dimensional OpenGL and grid driven environment. In principle an unlimited number of multi-dimensional data sets can...

Fluorescence correlation spectroscopy (FCS) can measure dynamics of fluorescent molecules in cells. FCS measures the fluctuations in the number of fluorescent molecules in a small volume illuminated by a thin beam of excitation light. These fluctuations are processed statistically to yield an autocorrelation function from which rates of diffusion, convection, chemical reaction, and other processes can be extracted. The advantages of this approach include the ability to measure the mobility of a very small number of molecules, even down to the single molecule level, over a wide range of rates in very small regions of a cell. In addition to rates of diffusion and convection, FCS also provides unique information about the local concentration, states of aggregation and molecular interaction using fluctuation amplitude and cross-correlation methods. Recent advances in technology have rendered these once difficult measurements accessible to routine use in cell biology and biochemistry. Th...