This tutorial shows step-by-step, how an autocorrelation curve of a fluorophore dissolved in water is calculated and fitted using the FCS script.
Response: The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
ATTO4888_485nm_pulsed.ptuby a single mouse click.
Response: The FCS-script is applied to the file
ATTO488_485nm_pulsed.ptu. Thereby, a new Window opens:
Note: The window contains three different regions:
Note: A possibility to remove the afterpulsing when working with cw excitation is splitting the light onto two detectors and calculate a cross correlation. Some detectors (mainly Hybrid PMTs) also have so low afterpulsing that the problem does not occur.
Note: If you want to check what is calculated with the FLCS background correction, click on “more” to open the graphs of the TCSPC curve and the calculated filters.
Response: The FCS curve is re-calculated. As a result, the steep decrease at the beginning of the curve is now missing and an artifact free curve is obtained.
Response: A result file is generated and linked to the raw data file.
Note: In order to provide correct results, the confocal volume VEff and the structural
parameter k have to be calculated first. Check the tutorial Calibration of the
confocal volume for FCS using the FCS Calibration Script for a description
about how to calibrate the confocal volume.
If no calibration values are stored, the effective volume VEff is set to 1 fl and the structural parameter k is set to 5. While a structural parameter value of 5 is usually reasonable, the confocal volume depends on the wavelength and can differ significantly.
Check your system regularly for correct alignment using a reference dye as ATTO488.
In this tutorial, we use the default values to keep the description easy. If your system is already calibrated, the results (especially the concentration C and the diffusion constant D) will depend on your calibration.
Note: It is also possible to open a stored FCS curve directly with the FCS fitting script.
Note: This window consists of different sections:
Left: fitting panel to select a fitting model and see the fitting parameter. If you need information about how the fitting models are defined and what is the meaning of the parameters, click on the “Help” button next to the fitting model. This opens the corresponding help window.
Center/right: here the FCS curve is plotted. Below the graph the residuals are plotted. As we have not performed a fit yet, this area is still empty.
Note: There are several models available. Click on the “Help” button to see the
corresponding fitting equations:
Pure Diffusion: Use this model to analyze diffusion of a dye in a solvent (i.e. water). It is assumed that the dye does not show any fluctuation between various states with different photophysics at a timescale short or equal to the diffusion time through the confocal volume. Examples for these dyes are ATTO655 or ATTO425. Also dyes with a moderate triplet contribution can be fitted with this model if the excitation intensity is so low that the triplet population can be neglected. See: 1); 2).
Triplet: Use this model if the dye you use shows some contributions caused by transitions into the triplet state or similar photophysics, while otherwise freely diffusing in a solvent. See: 3).
Triplet Extended (3D): Use this model if your dye is observed in conditions where either the average count rate is not completely stable and/or the dye is diffusing in all three space directions through a crowded environment as a polymer or a cell. For cell measurements, this is usually the model of choice. See also 4).
Triplet Extended (2D): Use this model if diffusion of a dye within an artificial or cell membrane is observed. This model is made for dyes moving within a plane, e.g. modified lipids in a membrane. If the diffusion is not anomalous, set the anomaly parameter to 1 and keep it fixed. See e.g. 5) or 6).
Conformational: Use this model if your dye is undergoing conformational changes when diffusing through the confocal volume, which are characterized by stretched exponential kinetics. See e.g. 7).
Protonation: Use this model if analyzing a diffusion of a sample with an internal + external acid/base equilibrium which has an effect on the photophysical properties of the dye. This model was used to characterize GFP. See e.g. 8).
Note: It can be clearly seen that at short correlation time ranges, the curve does not fit the data well. This is an indicator that the sample undergoes some photophysical fluctuations in the μs – time range, usually triplet fluctuations. Therefore, we have to change the fitting model.
Note: This time, the curve fits the data significantly better.
A diffusion time of a few tens of μs is typical for a dye in water, as well as triplet time in the range of a few microseconds.
The fitted diffusion coefficient D is significantly higher than the real value of 400μm²/s. This is due to the incorrect calibration. If the diffusion coefficient is known, the size of the confocal volume can be decreased and the curve refitted, until the fitted diffusion coefficient is realistic.
Please look at the corresponding tutorial to learn how to calibrate the confocal volume.
Response: A result file is generated and linked to the raw data. We can now close the fitting window.