Many fluorescence spectrometers detect intensity in c.p.s (counts per second) but the measured intensity (in cps) will not necessarily be the same from instrument to instrument. Also the intensity of your fluorescence will be heavily dependant on your excitation and emission "slits" (on the monochromes).  For this reason, we often refer to the intensity as an arbitrary unit.  You can also use cps as your unit.

In my research I normally measure both an emission and an excitation spectrum, and an absorption spectrum. I then normalize the maximum of each spectrum to a value of 1 and place them all in the same graph.  The intensity can then be labelled as Normalized Intensity (a.u.)

I hope this helps.   

Hi Gurmeet, fluorescence arbitrary units (AU) are correctly described. They are arbitrary.

Unlike absorbance, where you can plug values into equations like Beer-Lambert, the fluorescence readings you get are dependent on fluorophore composition, instrument, ion concentrations, ion species, bleaching, quenching, FRET, etc, etc.

Therefore, most fluorescence readings of macromolecules can't be used as a reliable metric for anything.

If your instrument gives you a reading of "900" then that is your fluorescence AU.

I agree with the statement above however a spectrometer will often give you units of cps (counts per second).  A value of 900 cps is very week in intensity (just slightly over background noise).  This is assuming that a PMT (photomultiplier tube) is being used as a detector. 

Dear all,

thanks for all your answers.

1. Actually I am supposed to measure the fluorescence intensity on ethidium bromide.

2.I use a wavelength of 530nm  (excitation) and 600(emission).

3. I have tried getting this with the help of a "fluorescecne spectrophometer, HitachiL7000(FS)" and multimode mode reader (MM)(Tecan).

4.For the same sample by the FS, I get a values fluorescence like "1.00,1.012..." where as by MM I get Fluorescnecne intensity as "900,901,954.....".

5. The only difference are is a cuvette is used in the first and in latter a black 96 well plate is used.  

6,The first one FS gives fluorescence and the MM give as "fluorescent intensity".

7. Difference between Fluorescecne and fluorescence intensity???

Which I am supposed to take. 

You can use a.u for arbitrary unit but for  the reason of this unit you Please check ,Molecular Fluorescence: Principles and Applications. Bernard Valeur Page No-50.That will help you more. 

Hi Gurmeet, as far as I know "Fluorescence" and  "Fluorescence  Intensity" are equally arbitrary.

FS uses a cuvette and MM uses a plate. These employ totally different detection methods. 

Because of the different optics, pathlengths, etc, etc, these numbers cannot be correlated to each other.

Besides, these values are totally meaningless.

It is only meaningful if you use the fluorescence arbitrary units to demonstrate a difference within an experiment using the same parameters, such as the same instrument, buffer, etc.

an option is to calibrate your "signal" with the use of a calibration compound; see the following question in ResearchGate, with an answer by an expert of the Tecam company

How to calculate the quantum yield of fluorescent materials? by Joyce Dora

You can google to find it or else via the site of Joyce

and this

Abbas Mohammadi

Can anyone help me with the calculation of fluorescence quantum yield?

How can I determine fluorescent quantum yield using UV-vis spectroscopy of perylene derivatives? Which parameters do we need?

Regarding the difference between "fluorescence" and "fluorescence intensity", this is really a vocabulary issue. Every manufacturer and software package uses slightly different names, so it's all arbitrary. I agree with others, that the numbers you are getting from the spectrophotometer and multimode plate reader cannot be directly compared to each other. Those numbers are determined by the software, the detector systems, the electronics and filtering, etc. 

Although the arbitrary values are not meaningful, they can be used for relative, ratiometric quantification - to determine the fold difference or change between various samples that you analyze in parallel. You can normalize the data by designating one sample as equal to 1. Then use the ratiometric results to scale all your values relative to that sample. If you do this with both the spectrophotometer and the multimode reader datasets, the results should be very similar. This is one form of calibration; or, as Harry suggested above, you could calibrate the signals against an appropriate fluorescent compound.

I would recommend that you analyze all samples at the same time, in parallel, with the same conditions to minimize variability. If you really need to compare samples assayed at different times, the later experiment should include one or more samples from the previous experiment to allow for normalization and improve accuracy. 

The principle reasons for variations between experiments are changes in lamp intensity and photomultiplier response over time.  Measuring quantum yield using a standard fluorophore and quantum counter is one way to solve the problem but is awkward.   In my lab, we prepare a standard solution of the fluorophore in an appropriate solvent that has been flushed with N2 and Ar and carefully seal it in a cuvette.  Protect this from over exposure to light and O2 and it can be used to normalize data from a set of experiments performed on different days over a week or two.  Beyond that, it is wise to prepare a new standard and relate this to the first.  You can link multiple data sets in this way.  It is easy to prove to yourself that this is the case.  We have done so by reproducing and superimposing data over experiments for several days.  

Thanks all . Then what is the concept of relative fluorescence???

Hi Gurmeet, I have seen the use of "relative fluorescence" in fluorescence data that has been normalized to either the lowest or the highest value of a fluorimeter readout.

When results are expressed as "relative fluorescence", it usually means that the data was normalized by designating one sample as equal to 1, and then applying that adjustment factor to all the data. So, relative fluorescence refers to the fluorescence intensity of that sample relative to the sample designated as equal to 1.

Densitometry, fluorometry, phosphorimaging, and other techniques also use "arbitrary units", and are often normalized in this way so that relative values can be expressed. If precise, purified controls are available, you may occasionally see "absolute quantification" rather than relative quant, but that's the exception.  

Anybody is having paper in which Fluorescence Intensity unit shows in (cps)

Hi

In the attached article (J. Fluoresc. (2012) 22:1493 –1500), cps is the unit of the measured intensity.