I am dealing with a weak dye and a weak laser. I have previously read that the laser strength should not be more than 50%. Is it a rule or just a normal guideline? I cannot see the signal clearly at this strength however, only increasing the laser strength gives me a good signal. I need to compare cells pre and post treatment so any advice with the laser strength, amplifier gain, etc would be great.
I hope everyone here realises that percentage is a relative measure (relative to the maximum possible intensity of your particular laser). In order to compare your laser power to powers other people use you should give the laser power in an absolute measure (ie: kW/cm^2). So whether you should use 100% power depends on your particular setup.
If bleaching is your problem: Bleachingrate is not entirely linear with laser power, so instead of raising laser power, you might want to try to increase integration time to avoid bleaching and still get a fair signal.
It is always a good idea to test untreated cells (i. e., no dye) with your settings that you want to use for your experiment. Especially at high laser power values you might observe cellular autofluorescence which might result in wrong interpretation of signals.
Best regards,
Christian
Nothing wrong to work at high intensities in order to get a good signal. This is particular common for old lasers or old confocal systems. But you should consider the photo bleaching of your sample. With high intensity you might experience faster bleaching of your fluorophore and this is a problem if you want to repeat the imaging over time using the same sample. If bleaching is strong over time then try to decrease the intensity (or power of the laser) and compensate the signal loss by an increase of the gain or the pinhole.
Hello Prabodh,
If you are imaging live cells, it is not a good idea to go beyond 10-15% laser power. You could play with amplification and gain etc., For fixed cells and tissues you can go higher laser power, but as everyone above said you have to address photo-bleaching of your fluorophore.
Sometimes we may use even up to 90% with the fixed cells, but you should use good antifade medium, otherwise the signal may fade before you finish scanning. It is better to use higher intensity of laser, but less gain. Encreasing gain may affect the quality of the image.
I hope everyone here realises that percentage is a relative measure (relative to the maximum possible intensity of your particular laser). In order to compare your laser power to powers other people use you should give the laser power in an absolute measure (ie: kW/cm^2). So whether you should use 100% power depends on your particular setup.
If bleaching is your problem: Bleachingrate is not entirely linear with laser power, so instead of raising laser power, you might want to try to increase integration time to avoid bleaching and still get a fair signal.
1 microWatt on the Sample Surface seems practical for Single Molecule Experiments.
It is not really the percentage of the laser power that is a consideration for this but more the laser power in watts that is incident on the sample.
For confocal microscopy you ideally want to use the entire range of your detector and should adjust the laser power and gain of the detector accordingly.
However, for biological samples and especially for live samples which it appears yours are there is the potential for photodamage at high laser powers. As has been mentioned above it is a good idea to increase the power to where you can see the sample and image the system over a prolonged period of time. Then moving the position of the laser to another part of the sample and looking at a similar cell should give you an indication of whether you are damaging the sample by imaging or not.
Ideally for live samples you want the lowest power possible where you can still see your sample clearly. That way you will reduce any problems you may get through photodamage and phototoxicity.
For live samples I tend to use powers as low as 0.2% from a 10mW laser. It is very difficult to ascertain what you mean by a weak powered laser in relation to this.
A few more specifics and I would be able to help you even more. What is the output power of the laser at the laser head and at the microscope objective?
@wim
That is a good option assuming s/n is acceptable.
However, if the signal is too weak for your detection scheme that would be a zero sum proposition. In your case it would be better to minimize signal degradation with an antifade agent and ensure a near ideally refractive index match of your interfaces.
Again long acquisition times, from my experience, only work with a photostable marker like NQD'S. (if you are taking integrations over 10's of ms.)
Also Peter has it nailed. Do not waste time imaging until your settings are straight, or you will likely to repeat all your work.
High laser power undermines confocality - at a certain power level the fluorophores at the point of focus are saturated and emit the maximal number of photons possible, but fluorophores on either side of the focal point (a cone of light on either side) are not yet saturated and will emit more photons as the laser power is increased - more out of focus light and reduced confocality.
The optimal power varies with the objective (spot size and transmission losses) and the type of fluorophore.
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