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howto:calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script

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howto:calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script [2019/02/07 13:13]
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howto:calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script [2019/02/07 13:16] (current)
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 **Note:** Calibration is a necessary task for FCS measurements. In a calibration procedure, the confocal volume VEff as well as the structural parameter κ (the ratio of z to xy extension) is determined using a reference dye. This is the prerequisite to determine correct diffusion coefficients and concentrations for the sample of interest.\\ **Note:** Calibration is a necessary task for FCS measurements. In a calibration procedure, the confocal volume VEff as well as the structural parameter κ (the ratio of z to xy extension) is determined using a reference dye. This is the prerequisite to determine correct diffusion coefficients and concentrations for the sample of interest.\\
 ATTO488 (ATTO-Tec, Germany) is a suited dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection around 500 – 540 nm.\\ ATTO488 (ATTO-Tec, Germany) is a suited dye to calibrate the confocal microscope at ~470 – 490 nm excitation and detection around 500 – 540 nm.\\
-The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "​Absolute Diffusion Coefficients:​ Compilation of Reference Data for FCS Calibration"​ available from PicoQuant ​[see [[https://​www.picoquant.com/​images/​uploads/​page/​files/​7353/​appnote_diffusioncoefficients.pdf]]]. This list is extensive, but does not claim to be complete.\\+The diffusion coefficient of ATTO488 carboxilic acid has been measured to be 400 µm²/s at 25°C. Published diffusion coefficients of various fluorophores are summarized in the Technical Note "​Absolute Diffusion Coefficients:​ Compilation of Reference Data for FCS Calibration"​ available from PicoQuant ​(see  [[https://​www.picoquant.com/​images/​uploads/​page/​files/​7353/​appnote_diffusioncoefficients.pdf]]). This list is extensive, but does not claim to be complete.\\
 Note that the diffusion coefficients of all dyes are temperature dependent.\\ Note that the diffusion coefficients of all dyes are temperature dependent.\\
 FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary:​spad|SPAD]] or [[glossary:​Hybrid PMT]] detectors).\\ FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary:​spad|SPAD]] or [[glossary:​Hybrid PMT]] detectors).\\
howto/calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script.txt · Last modified: 2019/02/07 13:16 by admin