We measure the fluorescence value of GFP by microplate readers. I don't know what is your approach to quantify "brightness" ?

The brightness of the RFP depends on the quantum yied and the extinction coefiicient of the dye ( fluorophore) used. You have to be carefull and prepaqre a very dilute solution in order to avoid quenching due to high concentration. For example (Red fluorescent TurboFPs are fluorescent proteins with colors ranginred-orange to near-infrared characterized by superbright fluorescence and superior fast maturation).

Fluorescence depends on input (excitation) as well as output (Fl emission). The quantum yield basically quantifies the amount of incoming energy that is converted to Fl emission. In an experiment, the signal you measure depends on both the QY and the extinction coefficient, so the product is a better guide to how much Fl signal to expect. This is complicated when you express an FP in vivo, as the environment will probably be different than the "test tube" conditions used during characterization of the purified FPs, and this will effect the Fl parameters.

Maybe this helps:

"Fluorophore brightness

The brightness of a given fluorophore is determined by the molar extinction coefficient and quantum yield, both of which are specific for each fluor. The molar extinction coefficient (ε) is defined as the quantity of light that can be absorbed by a fluor at a given wavelength and is measured in M-1 cm-1. The quantum yield (Φ) is calculated as the number of photons that are emitted by the fluor divided by the number of photons that are absorbed. This calculation provides the efficiency of a fluorophore and has a maximum of 1. The brightness of a fluorophore is then calculated as the product of ε and Φ."

I think what is being lost here is that you are not trying to distinguish a biological structure in a microscopic field, but, instead, trying to view broad area fluoresence from a solution. In the former case, the concentration is fixed by the structure, so that the relative amount of light absorbed is related well by the extinction coefficient, and "brightness" of the observed structure makes sense.

In your case (if I understand your problem correctly), you need to understand how much light is absorbed per unit area, on average, over the entire visual field, and then convert that to how much light is emitted. If you are modeling the concentration, then Beer's Law gets you to Absorbance (if you define the path length). Then convert Absorbance to % light absorbed (1-%T). To do things right, you really need to convert this energy to photons, and then you are home using the quantum yield.

Once you have the emitted photons, you need to do two things: distribute them over the emission spectrum, and remember that fluorescence is isotropic, so you're only looking at a fraction of the emitted energy. If you are truly interested in luminous intensity, then you need to account for the photopic eye response as well.

Hope this helps.

thank you, everybody.

I did almost what Glenn suggested, with some stronger constraints that allowed me to get an estimate to RFP concentration right away.

However, this is not my research area, so I really can't judge if the value I got is good or just an absurd

With the approach suggested by Glenn, I've got a necessary value of, approximately, 3 to 100000 Mol of RFP per ml.

Is this a big value for concentration?

actually, what I'm trying to ask is: How much RFP is expected to be found in a solution exhibiting fluorescence??

does someone knows where to look when I'm searching this kind of information? what I found in layperson web is not satisfactory.

in order to obtain the concentration of your fluorophores in the samples using known fluorescence quantum yield and absorption cross section (exctinction) you also need to know the light detection efficiency of your microscope and excitation power density (watts/cm2). Without excitation power you cannot find the concentration. More power you use, more photons you get.

ok. the information about detection efficiency and excitation power density I didn't know. thank you.

Hi, First of all determine concentration of your protein, either by Bradford or simply by absorbance at 280 nm. The theoretical way of doing that is to use the following formula. I am calculating for mRFP1 here for you.

ε (M-1cm-1) = (5500 × no. of Trp.) + (1490 × no. of Tyr.) + (125 × no. of Cys.) = (5500 × 3) + (1490 × 12) + (125 × 1)

= 16500 + 17880 + 125

= 34505

Put it into the Beer Lambert law and you can calculate concentration of your protein. For experimental values:

Make serial dilutions of your protein and take the slope as molar extinction coefficient.

Now, the interesting part is determining Quantum Yield.

The quantum yield (QY) is defined as the the number of emitted photons relative to the number of absorbed photons by a chromophore. When a photon of light is absorbed by a fluorophore, it gives rise to an energetically excited state. The end result for this excited state is an eventual return to the ground state by loosing energy. The main deactivation processes that causes this return is fluorescence (loss of energy by emission of a photon), internal conversion and vibrational relaxation (non-radiative loss of energy as heat to the surroundings), and intersystem crossing to the triplet manifold and subsequent non-radiative deactivation. So the quantum yield gives the probability of the excited state being deactivated by fluorescence rather than by another, non-radiative mechanisms.

For quantum yield calculations, use rhodamine 101 as a reference. Make fresh stock of Rhodamine by dissolving in ethanol. Take protein samples at various concentrations. For any particular concentration, adjust the peak height so the difference in the value of absorbance peak to less than 0.01 AU. Now obtain the fluorescence spectra at those concentrations and integrate this data (basically area under the curve). Plug values into the following formulae:

phi (s)/phi (r)= As/Ar x (Abs)R/(Abs)S x ηS^2/ηR^2

where

= Quantum Yield of the samples

= Quantum Yield of the reference

AS = Area under the fluorescence spectra of the sample

AR = Area under the fluorescence spectra of the reference

(Abs)R = Absorbance maxima of the reference

(Abs)S = Absorbance maxima of the sample

ηS = Refractive index of the solvent in which sample is dissolved

ηR = Refractive index of the solvent in which reference is dissolved

Brightness is just product of MEC and QY. Better to do everything by fluorimeter than a microscope. QY for rhodamine is 0.96 (if i remember correctly).

best regards,

Dr. Muhammad Safwan Akram

Thank you, Muhammad. It's not exactly what I was looking for, but helped a lot to understand the nature of quantum yield and extinction coefficient.