I would like to know your opinion about a ratio imaging experiment with two excitation wavelengths and one emission. Is it acceptable to change the acquisition conditions (time, gain) between different cells or different experiments but keeping always the same modified acquisition time and gain for both images?
Usually you have to keep acquisition conditions (time, gain) between the cells you want to compare (control vs KO, experiment 1 for example). You can change your parameters between different experiments (exp 1 and 2) but you have to conserve your parameters inside one experiment for a couple "control/KO". For example, if you use a confocal, with laser power which fluctuates from one day to another, it is important to acquire control and KO with the same parameters on the same day and to avoid to take the control one day and the KO the following day. If you don't have enough time to take all your pictures on the same day, make shorter "control/KO" series with same parameters. The second day you can continue your acquistion of WT/KO with other parameters. At the end you can report your KO to your control, and see if your effect in percentage is reproducible from one experiment to another. Hope it can helps. Lydia.
You can setup different acquisition conditions for first and second image but once you will do it you have to keep them constant throughout your experiments. Alternatively, you can change acquisition parameters for both images with no need to keep them constant for all experiments.
Usually you have to keep acquisition conditions (time, gain) between the cells you want to compare (control vs KO, experiment 1 for example). You can change your parameters between different experiments (exp 1 and 2) but you have to conserve your parameters inside one experiment for a couple "control/KO". For example, if you use a confocal, with laser power which fluctuates from one day to another, it is important to acquire control and KO with the same parameters on the same day and to avoid to take the control one day and the KO the following day. If you don't have enough time to take all your pictures on the same day, make shorter "control/KO" series with same parameters. The second day you can continue your acquistion of WT/KO with other parameters. At the end you can report your KO to your control, and see if your effect in percentage is reproducible from one experiment to another. Hope it can helps. Lydia.
Since you are doing ratiometric imaging, changing contions should not cause any problem as long as you dont't change the the ratio. But since dyes and detecting system might not behave linear it's always risky to change settings, so i try to avoid that. Since cell loading is not allways the same it might become necessary to change the emission intensity. I would suggest not to change the excitation intensity or duration to reach this, but rather alter gain since the light might cause an effect on the cells. Since we cannot comleptely avoid it, we can at least keep that error constant. You have two excitation wavelenght and one emission and can alter the time; are you using BCECF on a TILL system?
All imaging techniques follow the same principle of contrast variation, so if wants a good image then should thing how to improve the contrast.
To check the systems linearity you can held or double the laser power & then make sure that everything halves or doubles respectively. Then you may be in a linear regime and ratios would be safe. I usually use an ND filter rather than a power setting or detector reading. You can use the latter if you are sure they are linear.
As others have said a standard control is always a good idea in any experiments involving intensity. To avoid artifacts apply Murphy's Law .... "whatever can go wrong will go wrong" then you understand why a controlled standard is never a bad idea.
You will need to confirm with a calibration experiment if the to conditions are equivalent. Try to re-create a calibration curve.
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