If the emission you are detecting is not responding to a change in concentration of the fluorophore, then you are probably not detecting it. The small signal you are seeing is something else, such as Raman scattering from the solvent.

You may be using an inappropriate excitation wavelength, or the fluorophore may be non-fluorescent in the solvent you are using, or it may be insoluble in the solvent. It may have weak fluorescence and require a higher concentration. The excitation light intensity may be insufficient. The detector may be insensitive in the near-IR.

Make sure the spectrophotometer is actually working by taking spectra of fluorescein in dilute NaOH, or use some other handy fluorophore.

Thanks a lot for your answer. I dissolved it in DMSO/Buffer solution (1:1), pH 7.4 as reported in literature and it was soluble, and also I used the reported excitation wavelength. However the equipment is working with other fluorophore (CTG), but it did not work with DCM. I'm not sure if the buffer type could be a reason or not and also I thought the same that may be I need to increase the concentration, then I increased it to 10 mM but there is was no change at all.

Plz, could you tell me how to know the excitation light intensity is sufficient or not. especially I went through many references using the same fluorophore and works well but i don't know why it does not work with me.

Thanks again for your help.

I never worked with this (DCM-OH) fluorphore but according to literature (Article Ion-responsive fluorescent compounds. 2. Cation-steered intr...

I'm sorry for late reply and thanks a lot for your answer, I'm using Cary Eclipse fluorescence spectrophotometer. The excitation wave length I used was 560 nm. I did this experiment twice, first I tried three concentration 50, 100 uM, 10 mM and adjust the excitation at 560 nm but unfortunately, the emission was week and almost the same at the three concentration ( Fig. 1). I used phosphate buffer solution and DMSO ( 1:1) to dissolve the fluorophore.

The next time I used 50 uM and changed the absorbance, I found it absorb and emit at the same wave lenght 699 nm ( Fig. 2). it look weird but that what was happened with me. I changed the buffer into phosphate buffer saline to be the same conditions as literature and also used (1:1) ratio.

Thanks again for you help.

Thank you for sharing the data. The 700 nm excitation data is obviously Rayleigh scattering. As I mentioned before increasing the concentration wont help you. For a 50 uM concentration, using a standard 1 cm cuvette, the absorbance is more the 2 (considering absorption coefficient of 45000 M-1cm-1), which is very high. So I suggest you to dilute you to 1 uM. If you cannot dilute it take a cuvette of smaller path length. In short, decrease the absorbance at the excitation wavelength to around 0.1 (I strongly suspect that inner filter effect is diminishing the emission).

I also wonder why you are exciting at 560 nm. According to literature the peak is around 470 nm (with emission around 630 nm) in methanol. Can you please share the absorption spectra?

I do not think the buffer has any role to play here. As a control measure you can check the emission in methanol. Some dyes are strongly emissive in alcohol but weakly emissive in water. But I think you should dilute the solution first.

Thanks a lot for your attention. I used DCM-OH and the structure is attached ( it is reported to be excited at 560 nm and emit at 690: 700 nm) but when I excite it at 560 nm I did not find the excitation peak at 560 nm as reported. instead it excite at 700 nm and emit at the same wave length ( Fig.2, it is the same figure i got for both emission and excitation). so I just wonder why it does not give the same reported excitation peak.

I will dilute it to 1uM and measure the emission again but i think it will not fix the problem of the absence of excitation peak at 560 nm.

Do you think that is the conditions i'm using ( pH, Temp, buff type) could be the reason for shifting the absorption peak wavelength.

Then I don't think we are talking about the same molecule (the references I attached have amino group). DCM is a very popular solvatochromic dye which is partly due to this amino group (Article Ground state and singlet excited state of laser dye DCM: Dip...

Can you please show us the absorption spectra?

Please record the spectra in methanol to confirm it. As I mentioned earlier many dyes are weakly emissive in water.

"when I excite it at 560 nm I did not find the excitation peak at 560 nm"

I am not sure what do you mean by this.

"Do you think that is the conditions i'm using ( pH, Temp, buff type) could be the reason for shifting the absorption peak wavelength."

I think pH will have significant effect on the emission not buffer type.

Can you please the reference in which they have excited at 560 nm and found the emission around 700 nm?

Here are the references and the absorption spectra ( it showed there is absorption peak at 700 nm instead of 560 nm).

"when I excite it at 560 nm I did not find the excitation peak at 560 nm" Sorry if not clear. I mean that i did not find the absorption peak at 560 nm and when i adjust the excitation to 560 nm, I got very weak emission.

Do you suggest to use methanol alone to dissolve the dye as it need pH 7.4 to give the pink colour or you suggest to use mixture of buffer and Methanol.

That is not an absorption spectrum. If I understand correctly, it is an excitation spectrum monitored at 700 nm. It clearly shows there is no emitting species (at 700 nm) present in your system. Absorption spectra and excitation spectra are not the same thing though for a pure sample (only one chromophore) absorption and excitation spectra are identical.

Since this molecule (DCM-OH) is completely new I don't think I could of any help here. The authors of the papers are in the best position to solve your problems. Though in none of the papers I could find the spectra of pure DCM-OH (like the spectra of DCM-1, DCM-B1, DCM-B2). It is shown to be product of a reaction. Spectroscopic properties of DCM-OH (absorption coefficient, quantum yield) is not mentioned in the papers. So I don't think it would be wise for me to comment any further.

Thanks a lot for sharing your ideas. It was helpful for me.