I'm measuring plastics with NileRed but the blank on the coverslip is already giving a lot of signals. I have reduced all sorts of contamination sources and cleaned the cover slips with HNO3 and acetone and ozone. Yet the background fluorescence is still existing.
Can you post a spectrum or give a bit more description? Without that we can't really say anything.
The glass itself may be fluorescent. Check with the manufacturer whether the type of glass is compatible with fluorescence microscopy.
Jürgen Weippert I observed these diffraction-limited fluorescent particles sticking on the glass slides (with above mentioned cleaning it gets less but does not disappear) and also in the solution (diffusing) around when HPLC water and Nile Red (NR) together is used as sample (Blank). I can't extract any spectra from them because of their low concentrations for bulk spectroscopy.
I am assuming these are dirt particles that fluoresce in presence of NR.
Dear Swarupa Chatterjee thank you for posting this very interesting technical question. First of all I would clarify the point raised by Adam B Shapiro. If the glass isn't the source of fluorescence itself, you could try cleaning it with piranha solution, i.e. a mixture of sulfuric acid and H2O2 (https://www.youtube.com/watch?v=cLpSapjKcxM).
In this context it might also be worth having a look at the answers given to the following closely related RG question:
Does anyone know how to eliminate fluorescence impurities on the coverslip?
https://www.researchgate.net/post/Does_anyone_know_how_to_eliminate_fluorescence_impurities_on_the_coverslip
(14 answers)
I hope this helps. Good luck wth your research and best wshes, Frank Edelmann
Dear Swarupa Chatterjee P.S. Several recipes for cleaning coverslips with piranha solution are described in the following link:
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/piranha
A typical example is:
"Clean the coverslips with piranha solution (H2SO4:H2O2 volume ratio of 2:1) at 120 °C for 30 min. Rinse the cover glass with de-ionized H2O and dry in 100% EtOH."
We normally used 30% H2O2 for making the piranha solution. In any case be very careful when preparing this mixture and follow a published procedure! Always keep the piranha solution away from acetone as this could form highly explosive mixtures!
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