I have some cells marked with a plasmid producing luciferin/luciferase.
If I check the cells for their luminosity, I get very little signal. In fact people use specific camera that get images with very, very long exposures.
However, if I check the same cells with a confocal microscope, where the cells are painted with a monochromatic laser, then I can see a signal rather clearly.
What is the physics behind this kind of 'enhancement'?
Can it be that the laser is exciting luciferin producing a higher signal than luciferin alone would?
Thanks
Hi, Luigi. You may want first to try turning off your laser, cover all LEDs with black tape and darkening your room to use the confocal photomultiplier(s) as detectors for the bioluminescence signal (although I guess the lightpath will be larger than the optimal—too much signal loss)
About your question: In confocals the emission is detected simultaneously with sample irradiation. Did you somehow separate the excitation from emission reading?
Thank you. Without laser excitation, I get nothing or a very, very low signal. I'll check the separation more in detail later. But in theory, would the laser emission boost the signal?
Luigi Marongiu, consider that the chemiluminescent reaction is a catalitic one. If laser irradiation can boost it, you'll have a higher intensity signal. But you'll have to somehow separate your laser irradiation from your typically very weak luminescence signal (they may appear spectrally separated, but if you draw the spectra with their absolute intensity values, most probably the signal from your laser will overlap and surpass by intensity that one from the luciferase).
Here are some conditions that may affect the output of your catalitic reaction https://www.perplexity.ai/search/what-does-the-intensity-of-a-l-nfUMjI_FS6OHhu8Q.6AKrA (ATP is not required for all luminescence reactions)
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