Cytometry literally means measuring cells. Although many people may be familiar with traditional flow cytometry, an emerging area called chemical and metabolic cytometry examines the composition of single cells. “While flow and image cytometry analyze a few components, chemical and metabolic cytometry can characterize thousands of components such as nucleic acids, proteins, and even metabolites. Indeed, it is now possible to detect down to the yoctomole levels of analytes within cells,” said Norman Dovichi, Ph.D., professor of chemistry and biochemistry, University of Notre Dame.
Chemical and metabolic cytometry can provide new layers of cellular information. “These types of analyses are important because single cells can differ dramatically from their neighbors; classical analytical methods average the composition of cells, but this masks cell-to-cell differences. Traditional flow cytometry measures physical properties such as cell size and the forward and side light-scatter patterns. However, only a few components can be measured utilizing affinity probes against known targets. Thus, the unexpected is invisible to the analysis.”
Chemical cytometry lyses cells, labels proteins and biogenic amines, and employs capillary-based separation of components (e.g., capillary electrophoresis or microbore liquid chromatography). Signals are generated typically from laser-induced fluorescent readings to describe cellular composition.
Another arm of chemical cytometry is metabolic cytometry, which measures with exquisite sensitivity selected metabolic pathways in single cells. It differs from chemical cytometry in that cells are first treated with a fluorescent substrate that is taken up and enzymatically processed within the cell. Then labeled cells are aspirated into a capillary, lysed, and components electrophoretically separated. The metabolites are detected using laser-induced fluorescence.
Dr. Dovichi is applying the technology to better understand the glycolipid metabolism of neurons. “We see that there are dramatic differences among individual neurons that could be important for understanding their functional roles and how this may impact illnesses such as Tay-Sach’s disease.”
For the future, Dr. Dovichi is working on interfacing the power of mass spectrometry with chemical and metabolic cytometry methodologies. “This would be especially powerful because of the great detail afforded by mass spec.”
The workhorse technology of flow cytometry continues to evolve and grow. As technologies advance, generating increasingly complex datasets, quick and accurate analysis will remain a challenge.