22 February 2011 Fluorescence correlation spectroscopy as tool for high-content-screening in yeast (HCS-FCS)
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Abstract
To measure protein interactions, diffusion properties, and local concentrations in single cells, Fluorescence Correlation Spectroscopy (FCS) is a well-established and widely accepted method. However, measurements can take a long time and are laborious. Therefore investigations are typically limited to tens or a few hundred cells. We developed an automated system to overcome these limitations and make FCS available for High Content Screening (HCS). We acquired data in an auto-correlation screen of more than 4000 of the 6000 proteins of the yeast Saccharomyces cerevisiae, tagged with eGFP and expanded the HCS to use cross-correlation between eGFP and mCherry tagged proteins to screen for molecular interactions. We performed all high-content FCS screens (HCS-FCS) in a 96 well plate format. The system is based on an extended Carl Zeiss fluorescence correlation spectrometer ConfoCor 3 attached to a confocal microscope LSM 510. We developed image-processing software to control these hardware components. The confocal microscope obtained overview images and we developed an algorithm to search for and detect single cells. At each cell, we positioned a laser beam at a well-defined point and recorded the fluctuation signal. We used automatic scoring of the signal for quality control. All data was stored and organized in a database based on the open source Open Microscopy Environment (OME) platform. To analyze the data we used the image processing language IDL and the open source statistical software package R.
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Christopher Wood, Christopher Wood, Joseph Huff, Joseph Huff, Will Marshall, Will Marshall, Elden Qingfeng Yu, Elden Qingfeng Yu, Jay Unruh, Jay Unruh, Brian Slaughter, Brian Slaughter, Winfried Wiegraebe, Winfried Wiegraebe, } "Fluorescence correlation spectroscopy as tool for high-content-screening in yeast (HCS-FCS)", Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 79050H (22 February 2011); doi: 10.1117/12.873947; https://doi.org/10.1117/12.873947
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