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Diffusion Measurement Package
Olympus has introduced the new Diffusion Measurement module for Olympus ASW 2.1 software.
Designed specifically for use with the Olympus FluoView FV1000 confocal microscope system for live cell imaging, this new software module provides researchers with the flexibility to undertake three different types of diffusion study: point fluorescence correlation spectroscopy; raster scan image correlation spectroscopy (RICS) and fluorescence recovery after photobleaching (FRAP). As a result, the Diffusion Measurement Package is ideal for a broad range of cell biology and biophysics applications.
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Intracellular diffusion is frequently analysed to assess the movements, interactions and microenvironments of intracellular molecules to determine the patterns and rates of molecular movement. In order to meet this need, the Olympus Diffusion Measurement Package enables researchers to execute three different types of diffusion study:
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Point Fluorescence Correlation Spectroscopy
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Data is obtained from a single location using point scanning, enabling fluctuations in fluorescence intensity to be recorded. The number of moving particles within the confocal volume is therefore easily calculated. With its high temporal and spatial resolution, this technique provides data on the diffusion constant or number of molecules, as well as the capability to analyse cross correlations between different molecules.
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RICS
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With the ability to provide diffusion measurements in various cellular regions, the software is able to present the user with a spatial mapping of diffusion time constants. As a result, users can easily define small regions of interest and subsequently create a diffusion map from a series of 2D images. A wide range of intracellular structures, from molecules in solution to cell membrane proteins, can therefore be accurately measured. Resulting data can be expressed as either a diffusion constant or the number of available molecules.
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FRAP
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As a widely used fluorescence analysis technique, researchers commonly use FRAP to analyse the diffusion constant in a particular region of the cell, as molecules diffuse into the area after photobleaching.
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Analysis of the molecular interaction within cells :
Diffusion Measurement Package
This optional software module enables data acquisition and analysis to investigate the molecular interaction and concentrations by calculating the diffusion coefficients of molecules within the cell . Diverse analysis methods(RICS/ccRICS, point FCS/point FCCS and FRAP) cover a wide range of molecular sizes and speeds.
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Diverse analysis methods of intracellular molecular interactions
RICS
(Raster Imaging Correlation Spectroscopy)
Raster image correlation spectroscopy (RICS) is a method for analyzing the diffusion and binding dynamics of molecules in an entire, single image. RICS uses a spatial correlation algorithm to calculate diffusion coefficients and the number of molecules in specified regions.Cross correlation RICS (ccRICS) characterizes molecular interactions using fluorescent-labeled molecules in two colors.
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Spatial Correlation Algorithm
When the spatial correlation algorithm is applied between pixels, a higher correlation is obtained as the speed of movement of the molecule nears the scanning speed. When calculating the spatial correlation in the X-direction, because the scanning speed in the X-direction is fast, a higher correlation is obtained for fast-moving molecules than for slow-moving molecules. When the scanning speed in the Y direction is slow, a higher correlation is obtained for slow-moving molecules. RICS using LSM images scans in both X- and Y-directions, so it can be used to analyze the movements of a wide range of molecules, both fast and slow.
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Application
RICS can be used to designate and analyze regions of interest based on acquired images. EGFP is fused at protein kinase C (PKC) for visualization, using live cells to analyze the translocation with RICS.
The diffusion coefficient close to cell membranes was confirmed to be lower than in cytoplasm, after stimulation with phorbol myristate acetate (PMA).
This is thought to be from the mutual interaction between PKC and cell membrane molecules in cell membranes. In addition to localization of molecules, RICS analysis can simultaneously determine changes in diffusion coefficient, for detailed analysis of various intracellular signaling proteins.
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Point FCS
(Point scan Fluorescence Correlation Spectroscopy)
point scan fluorescence correlation spectroscopy (point FCS) analyzes intensity fluctuations caused by diffusion or binding/unbinding interactions of a protein complex.
point FCS uses an auto correlation function to carry out operations on fluorescence signals obtained by continuous scanning of a single pixel on the screen.
point scan fluorescence cross-correlation spectroscopy (point FCCS) analyzes the fluctuation of fluorescent-labeled molecules in two colors.
The coincidence of fluctuations occurring in two detection channels shows that the two proteins are part of the same complex. point FCS and point FCCS can now be performed with a standard detector, eliminating the need for a special high-sensitivity detector.
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FRAP
(Fluorescence Recovery after Photobleaching) analysis
The Axelrod analytical algorithm is installed as a FRAP(Fluorescence Recovery after Photobleaching) analysis method. The algorithm is used to calculate diffusion coefficients and the proportions of diffusing molecules.
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Fluorescence Recovery after Photobleaching
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For specific details on clinical applications for this product, please contact your local Olympus Australia/New Zealand representative.
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