I would not suggest to normalise to a weak background intensity, as you should find in fluorescence techniques, because it could get quite unstable. (If measuring is done as in spectroscopy you should see nearly no background- depening on filters or monochromators stray light supression)  

May be, F0 means not the Background fluorescence, it may mean a kind of excitation intensity reading?

I would not suggest to normalise to a weak background intensity, as you should find in fluorescence techniques, because it could get quite unstable. (If measuring is done as in spectroscopy you should see nearly no background- depening on filters or monochromators stray light supression)  

May be, F0 means not the Background fluorescence, it may mean a kind of excitation intensity reading?

Hi Jan and Sudarson,

Thank you for your responses.  I apologize for not including the details.  I'm currently working with a 96 well plate of cultured cells that I am staining with fluorescent stain Calcien AM.  Calcien AM fluoresces once its AM esters are cleaved and the dye is trapped in the cell. The fluorescence intensity correlates with cell number, and the background in this case is the signal from wells that have dye but no cells.  The manufacturer of the plate kit recommends subtracting the signal from no-cell wells from the raw fluorescence of sample wells.

The detector is a SpectraMax plate reader from Molecular Devices.

Thank you again.

in this I would recommend subtracting, too. I guess, the SpectraMax is a filterbased Reader, isnt it?

I think,in the protocoll with normalisation F0 means an Absorption reading or a fluorescence reading at a different wavenlength. This could minimise the influence of uncertainty of the amount of dye in the well.

Depending on wavelength and plate you will may a background signal, Independent from dye in the well, coming from plastics fo the plate.

Hi Jan,

Thank you for your reply.  I think the SpectraMax is a monochromator-based reader.  I'll try subtracting Fo from average raw sample F as you suggest. 

Hi Sudarson,

Yes, F0 is the fluorescence signal in the absence of cells.  I'm using fluorescence intensity as a way to measure cell number.  Wells with dye alone give a background signal, which I subtract from wells with cells and dye.  I'm only measuring fluorescence, not absorbance.  The dye alone does not fluoresce strongly.  It requires cleavage inside of cells to become fluorescent. 

Thanks for your comments.

You are right- I was thinking of another device.

Hi there, your F0 is a background that occurs in all well, and adds to the fluroescecne intensity coming from xou biological reaction. Therefore, it is useful and legit to subtract the F0 value (measured from well without cells) from all wells. It's like a baseline correction by subtracting a fixed offset.