Now I want to detect fluorescence molecules excited by laser, the power of which is 0.5 mW detected by power meter above the sample. So I want to know whether it is enough to obtain clear images?
Indeed that is a powerful laser and is likely to 'bleach' your fluorophores very rapidly. You should only use enough power to get a good image - that is determined more by the sensitivity of your detector that the power of the laser. With a very sensitive digital camera we are able to obtain good images of multiply labelled cells - without using lasers
It depends on the level of your background signal. Since Fluorescence is a stimulated process, the intensity of the fluorescence photons will necessarily be less than the total intensity of the exciting laser.
0.5 mW of laser power is very powerful. With the appropriate detection techniques (such as lock-in amplifiers) it is possible to detect single photons.
Perhaps you could consult the documentation supplied by your instrument manufacturer.
Indeed that is a powerful laser and is likely to 'bleach' your fluorophores very rapidly. You should only use enough power to get a good image - that is determined more by the sensitivity of your detector that the power of the laser. With a very sensitive digital camera we are able to obtain good images of multiply labelled cells - without using lasers
It is difficult to answer and depends on following aspects:
- the (photo-)stability of the dye and the sample matrix
- is it a CW or pulsed laser
- the power in relation to the illuminated area (power density)
- wavelength of the laser
You might want to start with a lower power to optimize the detection geometry, then find the detector settings (PMT or ICCD??) and find a balance between S/N ratio, accumulation time, laser power and photostability by changing slits, exposure time, gain, etc...depending on the detector.
For example, salicylic acid in water (2 mL) was showing degradation after approx. 200-500 shots with a pulsed Laser @290 nm, 50 mW (@ 10 Hz, 8 ns pule length) in our experiments but some dyes we use in living cells are much more affected by photobleaching. There is no general answer.
Many samples should be able to cope with your laser intensity though.
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