A-TEEM–A Fantastic Spectroscopy that Rivals Raman

This month, I am going to talk about a different spectroscopy that rivals Raman in its usefulness. A-TEEM stands for Absorption, Transmission, Excitation/Emission Matrix Fluorescence. While it does not give direct information on the structure of a molecule, its value lies in its ability to detect subtle changes in structure due to changes in conformation of large organic molecules or changes in interactions with other molecules. While this is not a technology with which I have been directly involved, I have been impressed with its potential and thought this column would be a good place to share my thoughts.

I was first exposed to emission matrix spectra (EEM) while still at the Johnson Foundation during the dark ages of my career. I thought it was neat that you could simultaneously visualize the excitation and emission of the fluorescence by plotting on a two-dimensional figure. But, thinking about this measurement, I realized that there is an essential problem to the measurement.

Normally, the measurement is done in a cuvette where the exciting light travels through in one direction (from left to right in Figure 1), and the emitting light comes out at 90° (moving in the upwards direction on the page). Now, let’s think about what is happening as the light goes through the sample. Because we are doing a fluorescence measurement, the sample is absorbing light at the excitation wavelengths. That means that, as the light passes through the cuvette, excitation intensity decreases, and consequently, the fluorescence intensity will decrease. There is the additional possibility that the emitted light can also be absorbed before exiting the sample, depending on whether there is overlap between the absorption and emission spectra. What can the analyst do? In the past, the recommendation was to measure dilute samples so these effects would be small. But there is now a correction for the inner filter effect (IFE) where the absorption and transmission spectra can be measured on the same instrument to compensate for these effects. That is A-TEEM, and both hardware and software are currently available to make the corrections (1).

A-TEEM: An Enabling Technology

For a start, by correcting for the IFE, the profiles are independent of concentration–they are linear over 2 absorbance units (AU)! And, from the corrected EEM, one can measure the quantum efficiency (QE) of the fluorescence process. From the absorbance, one can do multicomponent analysis. and from transmission, one can measure colorimetry (Commission Internationale de l’Eclairage; CIE). In addition, because it is intrinsically a fluorescence technique, A-TEEM benefits from sensitivity beyond that of traditional vibrational techniques and without interference from water.So what do A-TEEM spectra look like? Figure 2 shows the spectra of the three aromatic amino acids. Clearly, the spectra are different (which is not unexpected), and when examining a protein. it will be possible to quantify these molecules and to observe changes when the protein structure is affected by its environment.

Figure 3 shows a three-dimensional plot of the A-TEEM fingerprint that enables insight into the intensities.

Cell Culture Media

There are numerous recipes for cell culture media in which specific cells are grown for producing specific biomolecules. Figure 4 shows A-TEEM plots for three similar cell culture media. Fluorescence methods offer advantages over traditional approaches like mass spectrometry and chromatography; advantages include rapid testing, minimal sample handling, and relatively lower costs. Examination of Figure 4 indicates that the differences are small, but when the spectra are subjected to Parallel Factor Analysis (PARAFAC) and principal component analysis (PCA) the various species separate quite well. Figure 5 shows the Scores plot for eight different cell media. Clearly similar media cluster indicating similar chemistry. Having established that A-TEEM 3-D spectra can differentiate similar media, it becomes of interest to determine if differences can be seen when the materials are aged. Figure 6 shows the Scores plot for the EXCELL medium that had been aged for several days at either a cold temperature or at ambient temperature. The Scores plot shows that the scores for the samples held at cold temperature overlaid with the fresh sample, but the samples held at ambient temperature deviated, and the longer the samples were held at ambient, the farther the scores were from the fresh sample! Clearly, there is information in these A-TEEM spectra.

Environmental Water Research

One of the first applications where A-TEEM’s have been employed is in environmental water research. One can monitor dissolved organic matter (DOM) in water and by following it as a function of time and location one can follow water pollution and its remediation. A group in China followed the DOM from multiple sites on the Heilongjiang River after they treated with micro-organisms and followed the fluorescence evolution as a function of treatment time (2). They measured the EEM fluorescence on a system on which they also measured the Absorption to perform the inner filter effect correction. Analysis was applied to identify DOM components as they changed with incubation time. PARAllel FACtor analysis (PARAFAC) is used to decompose trilinear data arrays and facilitate the identification and quantification of independent underlying signals, termed components. It is an extension of multivariate analysis used on spectroscopic data with one variable, and is necessary to analyze EEM data because there are two variables (excitation and emission spectra). The results of PARAFAC analysis on these A-TEEM spectra were analogous in the various samples with the characteristics listed in Table I (the A-TEEM spectra are not shown in the publication, so I list the characteristics in Table I). What is interesting about this study is that the analogous Parafac factors were then treated with 2D-COS. The wavelengths of the spectra can be used to suggest the chemical changes occurring, and then the 2D-COS determines the order in which they occur. One thing that the authors found is that the shorter wavelength substances are transformed into longer wavelength substances. In summary, they found that shorter wavelength tryptophan-like substances were converted before the microbial humic-like substances at shorter wavelengths. And microbial humic-like substances at longer wavelengths were produced before terrestrial humic-like substances at shorter wavelengths. In addition, the authors found that terrestrial humic-like substances were produced more when nutrition was poor in the aquatic ecosystem, because shorter wavelength microbial humic-like substances were utilized.

There is much more information that I am unable to include in this short article, but my hope is that the reader has seen enough to appreciate the knowledge to be gained about the evolution of the DOM in the biogeochemical environment.

Summary

I have tried to describe what A-TEEM is, how it is done, and why it is useful. Even Andrew Whitley (HORIBA Scientific) has partially jumped ship and is working on A-TEEM for life science applications in addition to Raman! If you are working on any kind of organic material that is fluorescent, A-TEEM should provide you with knowledge that is not available otherwise.

References

1. US Patent # US 8,901,513. https://www.horiba.com/usa/scientific/products/fluorescence-spectrometers/a-teem/a-teem-downloads/ (accessed 2025-11-04)
2. Shi, J.; Zhao, Y.; Wei, D.; Zhang, D.; Wei, Z.; Wu, J. Insight into Transformation of Dissolved Organic Matter in the Heilongjiang River. Environ. Sci. Pollut. Res. 2019, 26 (4), 3340–3349. DOI: 10.1007/s11356-018-3761-9