Fluorescence spectroscopy

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halynkaf
halynkaf's picture
Fluorescence spectroscopy

Hi
I want to know how can I determining a scaling factor for  fluorescence plate reader. I can't understand what kind of chemicals reference and blank solution must contain due to such protocol:

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Digestive gland tissue was ice-cold homogenized (1:10) in 0.32 M sucrose, 20 mM HEPES (pH 7.4), 1 mM MgCl2, 0.5 mM PMSF, and 2 µM leupeptine using a Potter-Elvehjem glass/Teflon homogenizer. Homogenates were centrifuged at 20,000 g for 20 min at 4°C. Aliquots of the supernatant (166 µg protein) were incubated with 30 mM HEPES (pH 7.2), 200 mM KCl, 1 mM MgCl2, and 16 µM DHR in a total volume of 2 ml. DHR is a nonfluorescent derivative that is converted to the fluorescent dye rhodamine 123 on reaction with reactive oxygen species. Probe fluorescence signal was detected by using a Perkin Elmer LS 50B spectrofluorometer [excitation (ex.) = 505 nm, emission (em.) = 534 nm, slit = 2.5 nm].

Thanks for your assistance!

Sami Tuomivaara
Sami Tuomivaara's picture
halynkaf,

halynkaf,

You need to be more specific about your question(s). Do you have two separate questions, (1) what is a scaling factor and (2) how to prepare blank solution?

First of all, what do you mean by scaling factor? If you are talking about scaling factor between different path lengths, for example 1 cm path length cuvette reading and a microplate reading, the scaling factor is simply the ratio of the path lengths. For microplate reading, you have to calculate the path length (distance between the bottom of the well and the meniscus) from the geometry of the well (manufacturer gives the diameter etc. of the wells) and the volume of the solution in the well. But this is not trivial calculation because the meniscus of the solution is curved in the microplate well, and the curvature depends on the surface tension of the solution. So you have to approximate by assuming flat meniscus...

About your blank solution (=reference solution, = zero solution). The blank solution composition has to be as close as possible to the actual measurement solution composition and it has to include all the components that have some background fluorescence or that can affect the fluorescence of other molecules.

Thus, you should leave out the DHR (from your description it is non-fluorescent) and add all other components in correct concentrations, this is the blank solution. Then you make another fresh solution where you add all the components including DHR, this is the measuring solution. Hence, you need to prepare two separate solutions and use twice the amount of tissue fluid (this could be a problem if you don't have much tissue fluid at your disposal). Because DHR has low solubility in water, you can't just add concentrated aqueous DHR stock solution to the blank (after measuring the it) and use the new solution that as a measuring solution.

But because of high solubility of DHR in organic solvents, you could also make concentrated stock of DHR in ethanol for example. Here you measure the blank without DHR and then add very tiny volume (microliters) of the DHR stock to the blank solution and then use that as a measuring solution, Here, you save some tissue fluid but you add a bit of organic solvent to the solution. There is no fluorescence from the ethanol so that is not a concern but I don't know what secondary effect the organic solvent has to the measurement (may affect enzyme activity, quantum yield of the fluorophore, etc.).

The final fluorescence value is then Fluorescence_measurement minus Fluorescence_blank.

Eventually, you, as a scientist, have to make a judgement call to best of your ability which of the abovementioned options is the best way to go.

Cheers,

halynkaf
halynkaf's picture
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I am grateful for your support. This type of analysis is absolutely new for me and due to this your answer is very useful.
 

ALEXANDROS PAPA...
ALEXANDROS PAPAGEORGIOU's picture
Humic substances

Hi everyone,I am currently investigating the fluorescence mission of humic acids with synchronous fluorescence spectroscopy.Does anyone knows which are the main peaks of a humic acid standard solution?I am trying to figure out in which chemical structures correspond the peak at λex=240 nm of a humic acids standard.Thanks,Alexandros