Today, biomedical researchers have more tools to study intracellular activities to understand processes within the cell. That can lead to better testing, diagnosis and treatment of disease.
These scientists strive to develop chemical markers or sensors to indicate changes in cellular activity and cellular function. Lars-Olof Pålsson, Ph.D. is one such scientist.
Pålsson, an assistant professor in the Department of Chemistry at Durham University pursues a line of research that involves sensing of the biological environment through optical spectroscopy. He is a bit of a fluorescence spectroscopy expert.
“We are using luminescence imaging to study bio-active materials using emissive probes,” he said. “The excited state decay of organic fluorescence probes and organo metallic complexes is monitored using time-resolved luminescence microscopy.”
He also uses time-gated detection and conventional photon counting techniques to study the organic fluorescence.
But his use of microscopy detection enable single cells to be studied. He can study the excited state decay of many different types of emissive probes over a very wide dynamic range over 100 orders of magnitude.
An example of some of the research that we have pursued is related to metal sensing. Some transition metals are key for enzymatic reactions but there is a very tight concentration range for some of these metals in cells. Furthermore, the type of transition metals is also important, as some could be carcinogenic in mammalian cells but important for biological function in plant cells.
“We were able to track from the fluorescence decay, when levels of saturation had been reached, using a fluorescent metal sensing complex. This information is very difficult to obtain from a normal confocal fluorescence microscopy experiment as the variations in intensity can be due other effects. The time resolved fluorescence is simply a concentration independent measurement.”
Another example is the work using lanthanide complexes for sensing an imaging.
“We actually have some examples from Durham where some of the work has actually moved into pre preclinical medical screening,” he said. “One example is, work on lanthanide complexes led by my colleague Professor David Parker FRS, which we can use to screen against prostate cancer. And this is actually now moving into the clinical environment state too.”
In the early stages of prostate cancer, the acidity of body fluids in the bladder is changing. And this is something that in principle and can be checked then by taking a urine sample mixing in some lanthanide complex and look at the emission spectrum of the lanthanide emissions. Researchers have actually developed a small device standard. That can be introduced into say a doctor's practice. That preclinical screening is relatively straightforward compared to a test which is more widely used called PSA test. A prostate specific antigen (PSA) test measures the level of PSA in the blood. PSA is a substance made by the prostate. The levels of PSA in the blood can be higher in men who have prostate cancer.[i]
Pålsson says the PSA test can leads to false, positives. There’s also the time delay between the moment the test is taken and the days it takes to return results from an outside lab. And of course a false positive can bring anxiety and the need for further clinical testing.
How does it work?
“Lanthanides complexes have sharp emission bands and there are some bands that are sensitive to the environment here. And one, one key factor here is essentially acidity. There's one band that will hold its level in the lanthanide emission, but then there's another band that will change intensity. So the relative intensity of these bands actually leaves a fingerprint. And what they did here then was to calibrate to construct the calibration curve earlier. It turns out that this variation can be due to the acidity.”
“They have quite a respectable body of data and statistical material here and I believe this is a fairly robust method by now.”
Pålsson also uses fluorescence spectroscopy to develop better display technologies and electronics based on organic materials. After all, organic display materials have, in the end, better display properties, are more environmentally friendly and cheaper to produce.
The quest in display technology is still for materials that can emit in the blue range, Pålsson said. If you want to have a display like a computer screen, a mobile phone, or television screen, you need to be able to generate all the colors of the rainbow. Many of these congregate materials that came say 20 years ago in the 1990s, and these were relatively robust and easy to produce. Obviously spectral characterization here is important. But to reach the blue, he said, has been a bit more of a challenge.
“The band gap, the energy gap peer is larger, Pålsson said. “There's more photo degradation of the material. So, so it's, it's a feedback loop here where the synthetic chemists, they make some kind of modification which will hopefully will lead to better stability of them hitting material for a longer life, and then the device gains higher brightness. And then it comes to us then for testing. So we need to do a proper spectral characterization of the material layer. Where does the emission come in? The visible spectrum? How broad is the emission that we observed?”
The company supplying the materials may dictate different requirements.
“And then of course, the brightness sort of quantum efficiency here, that is the other key aspect that, that we then characterize,” he said. “Where does the emission come from? There of course we have another aspect, which is solution inside the cell, which we call the physiological environment in a cuvette. We basically call it the dielectric medium media, how it’s going to have an impact on the fluorescence whether it’s going to quench, red shift or blue shift.”
Pålsson’s work revolves around building better sensors to detect fluorescence properties that contribute to superior display materials.
Durham uses four HORIBA Fluorolog Modular Research Fluorometers in its labs for Lifetime and Steady State Measurements. The institution has progressive generations of the Fluorolog, from the very early ones to later versions. The instruments’ modularity has allowed the department to add several accessories and components, including two emission channels, an iHR imaging spectrometer with an array detector and a Quanta-Phi integrating sphere, expanding the fluorometer into a complete quantum yield system.
His lab also operates a DeltaFlex TCSPC system with NIR PMT detector.
“Some of the materials that we are testing now also have a short past in display technology,” he said. “So, it's a new application on thin films, thin fluorescent film. This is why I like the Fluorolog system that we have at the minute here in Durham, because the sensitivity is phenomenal.”
 Prostate Specific Antigen (PSA) Test, Centers for Disease Control
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