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 [2018/09/14 14:34] – admin | howto:calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script [2019/02/07 12:13] – [Background Information] admin | ||
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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 " | + | 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 " |
| 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: | FCS measurements are usually point measurements acquired with very sensitive detectors ([[glossary: | ||
| FCS is a technique applicable over a wide range of concentrations, | FCS is a technique applicable over a wide range of concentrations, | ||
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| ===== Step-by-Step Tutorial ===== | ===== Step-by-Step Tutorial ===== | ||
howto/calibrate_the_confocal_volume_for_fcs_using_the_fcs_calibration_script.txt · Last modified: 2019/02/07 12:16 by admin