Cancer cells and healthy cells show differing biological characteristics in their metabolic patterns. This is reflected in their level of oxygen consumption. The ability to measure the oxygen level of cells with fluorescence microscopy could be of great potential use in diagnosing the presence of cancer.
However, the measurement of oxygen content using the effect of collisions with oxygen molecules, which last only a few microseconds, is made difficult by the nanosecond-range lifetimes of conventional fluorophores in the excited state.
TRAST Application in Oxygen Monitoring
Transient State Imaging (TRAST) is a novel method of measuring cell oxygen content. It is based upon the occurrence of transitions between the T1 and excited states of fluorophores. The T1 state is a term which represents the dark lowest triplet state of a fluorophore that occurs in response to light. It has a relatively long duration and is not characterized by fluorescence. The T1 state is present with almost every type of fluorescent molecule.
The use of a modulated laser source for fluorescence microscopy enables systematic changes to be made in laser characteristics, in order to analyze the kinetic data using the T1 state. The duration of this stage varies with the oxygen concentration. If fluorescence intensity is imaged, it is seen to depend upon the laser modulation. This type of imaging yields direct information about the local oxygen concentration around the fluorophore molecules.
TRAST imaging is thus important in providing a method of detection of oxygen concentration at high sensitivity by a fluorescent readout. It also responds with high sensitivity to the environmental state by virtue of the long dark state lifetimes. TRAST is also suitable for a wide array of fluorophores, including auto-fluorophore molecules which show weak emissions.
Figure 1. (A) Fluorescence intensity images of cells undergoing a cancer cell specific metabolism (left) and a normal cellular metabolism (right). (B) Corresponding TRAST images, showing the T1 decay rate in the cells. The decay rates are lower in cells with normal metabolism, indicating lower local oxygen concentrations, and thus higher oxygen consumption
Cancer-specific Cell Metabolism
In the current experiment, cell cultures were studied using TRAST imaging with the use of Eosin Y, a dye with a high triplet state. The cells were from several breast cancer and fibroblast lines. One line was the breast cancer cell line MCF-7, with cells being cultured in different media to drive the cells in two directions, either a metabolic pattern that is specific to malignant cells or one that is characteristic of normal cells.
Cells from both cultures were then subjected to TRAST imaging to compare the rates of oxygen consumption. The results, as shown in Figure 1, shows the increased rate of oxygen use by the MCF-7 cells that show a normal metabolic pattern compared to those which have a malignant cell-specific metabolism.
Figure 2. Typical modulation waveforms (A) and noise performance (B) of lasers from the Cobolt 06-01 Series
Lasers for TRAST and Fluorescence Microscopy
The use of TRAST has great potential in oxygen content measurement within living cells. Cells that are diseased manifest changes in metabolism as well as oxygen consumption. These are picked up by TRAST which thus distinguishes normal cells from diseased ones, whether cancerous, infected or sick in any other way.
An advantage of TRAST is the suitability of compact and less expensive lasers, thus reducing the cost of instrumentation for potential applications such as identification and analysis of malignant tumors. Any laser used for TRAST must offer highly reliable and sturdy imaging performance, as well as having superior power characteristics. The laser beam must conform to high TEM00 stability and quality. The ability to directly modulate the laser beam is also preferable.
The Cobolt-06-01 Series of diode lasers meets all these requirements. They have plug-and-play build, can be fully modulated, and offer a power spectrum up to 250 mW. The Cobolt DPSS Lasers form a second series which incorporate AOM.
Figure 3. Typical beam profile of the Cobolt 06-MLD laser (top), Cobolt 06-MLD laser (bottom)
TRAST has shown itself to be a suitable option to measure oxygen content within living cells, thus providing an early method of detecting cancerous cells with higher sensitivity by identifying the altered metabolic patterns. Cobolt lasers are available in many different models and are excellent for this type of application.
(1) Thiemo Spielmann , et al. ‘Transient state microscopy probes patterns of altered oxygen consumption in cancer cells’ FEBS Journal 281 (2014) 1317–1332
(2) Hevekerl H, Tornmalm J, Widengren J “Fluorescence-based characterization of non-fluorescent transient states of tryptophan – prospects for protein conformation and interaction studies” Scientific Reports 6, 35052, 2016
(3) Tornmalm J, Widengren J “Label-free monitoring of ambient oxygenation and redox conditions using the photodynamics of flavin compounds and transient state (TRAST) spectroscopy”, Methods, in press 2018
This information has been sourced, reviewed and adapted from materials provided by Cobolt AB.
For more information on this source, please visit Cobolt AB.