3D imaging can be performed by Confocal Microscopy and Optical Coherence Tomography. Only confocal microscopy is fluorescence based.

Which technique are you referring to??? Is it Confocal Microscopy???

I mean 3D technique for plotting excitation, emission and intensity in contour or colored map.

Hello Muhammad,

the importance of these experiments is to find out the position of fluorescence activity quickly. If you define steps large enough between your single excitation measurements, you can scan an unknown sample for fluorescence automatically in a limited amount of time. If you have identified your spectral region of interest, you can follow up with finer 3D scans or single excitation measurements. Additionally if you perform 3D scans on a series of differently treated samples you can determine whether quenching, shifting or new fluorescence peaks are a result of your treatment.

Best regards,

Moritz

Thanks for ur detailed answer. but how to resolve the problem of blank subtraction ?!!!!!

Since I don't have any experience with Perkin-Elmer spectrometers, I'm afraid, I can give no advice. The instrument I use allows to record raw data (no blank substraction or other corections) as well as using the detector-specific calibration and/or the lamp intensity curve. Maybe it is a possibility to perform a measurement with an empty cell and one with a sample and subtract the blank data.

3D experiments are very similar to what was being done earlier. Instead of doing many expts earlier, now the same is being done in just one expt. It reduces manual effort. The software also overlays all the spectra and presents it, so comparison becomes easy.

The recent version of optical spectroscopy software Spekwin32 has quite some functions for handling EEM data. It is also able to subtract a Blank EEM dataset. This might be what you are searching for. Please start from http://spectroscopy.ninja For details on the needed functions, read the online manual, starting from here: http://effemm2.de/spekwin/spekwin_man_2d_en.html

See Journal of Physical Chemistry 10/1988; 92(21). DOI:10.1021/j100332a038, for historical technique, but this kind of experiment is now automated as described by Mr. Menges. If you only have access to old fashioned equipment as I used 27 years ago,then you should be able to put all the data into an Xcel file and subtract out the blank that way. 

for reading:

https://scholar.google.de/scholar?q=eem+fluorescence+review

Thanks all very much!

Dear Friedrich,

i installed Spekwin, but when i tried to subtract the blank (background) from 40 EEM spectra i got the message "the current data is not of EEM type, Blank EEM is NOT possible". please, advise what i did wrong.

Thanks in advance,

Dear Muhammad, the EEM emission spectra must first have their excitation wavelengths assigned, this can be done with the function "Excitation Wavelength Assignment" from the "Fluorescence/ EEM" menu. All functions from this menu are explained here: http://www.effemm2.de/spekwin/spekwin_man_2d_en.html

There is a dramatic advantage in using 3D EEMs as compared with excitation, emission and synchronous spectra. This advantage is the use of parallel factor analysis (PARAFAC) which allows separating the whole fluorescence map into the components. The feature is that, specifically regarding EEM of fluorescence, this separation does not involve any assumptions on the shape of the peaks. Ideally, if you had a right guess about the number of fluorescing components, you are supposed to get their excitation and emission spectra and scores quantifying the changes in concentrations ina series. Here is the difference as compared with regular PCA etc analysis. Currently, EEM (+PARAFAC) becomes almost a standard tool in analyzing the complexed systems containing natural organic matter or during water treatment etc. However, it should have much greater potential (when used with PARAFAC!) in analyzing more traditional issues, e.g., acid-base equilibria and complex formation reaction in "simple" systems. By some reasons, the number of studies using PARAFAC for such a resolving is much more limited in 'simple" syastems as compared with natural ones. Anyway, this puts EEM analysis as a fingerprinting method on its important place.