However, the main limitation of traditional fluorescence microscopy techniques is that the results are very difficult to evaluate quantitatively. Fluorescence intensity is greatly affected by both experimental conditions and the concentration of fluorescent material.
The research team has developed a new approach for obtaining fluorescence lifetime images without the need for mechanical scanning. ‘
The way to avoid traditional problems is to focus on fluorescence lifetime rather than intensity. When a fluorescent substance is exposed to a short burst of light, the resulting fluorescence does not disappear immediately, but it actually “attenuates” over time in a way that is unique to that substance.
“Fluorescence lifetime microscopy” technology takes advantage of this experimental condition-independent phenomenon to accurately quantify changes in fluorescent molecules and their environment. However, the fluorescence decay is so fast that a normal camera cannot capture it.
A single-point photodetector can be used instead, but the entire area of the sample must be scanned to be able to reconstruct a complete 2D image from each measurement point. This process involves moving mechanical parts, which significantly limits the speed of image capture.
Professor Takeshi Yasui of the Institute for Post-LED Photonics (pLED), Tokushima University, who led the research, explained as follows. -Everything in one shot, without scanning. So how was this achieved?
One of the main pillars of their method is to use an optical frequency comb as the excitation light for the sample. An optical frequency comb is essentially an optical signal consisting of the sum of many individual optical frequencies with regular intervals between them.
The word “comb” in this context refers to what the signal looks like when plotted against the optical frequency. A dense cluster of equidistant “spike” that rises from the optical frequency axis and resembles a hair comb. Using specialized optics, a pair of excitation frequency comb signals are decomposed into individual optical beat signals (dual comb optical beats) with different intensity modulation frequencies, each carrying a single modulation frequency, into the target sample. It will be irradiated.
The important thing here is that each ray hits the sample in a spatially different location, creating a one-to-one correspondence between each point on the sample’s 2D surface (pixels) and each modulation frequency of the dual comb light beat. ..
Due to its fluorescence properties, the sample re-emits some of the captured radiation while maintaining the frequency-positional correspondence described above. The fluorescence emitted from the sample is then easily focused on a fast single point photodetector using a lens.
Finally, the measured signal is mathematically transformed into the frequency domain, and the fluorescence lifetime at each “pixel” is easily calculated from the relative phase delay between the excitation signal at that modulation frequency and the measured one. Will be done.
Thanks to its excellent speed and high spatial resolution, the microscopy developed in this study makes it easy to take advantage of fluorescence lifetime measurements. “Our method does not require scanning, which guarantees simultaneous measurement of the entire sample on each shot,” says Professor Yasui.
“This is useful in life sciences that require dynamic observation of living cells.” In addition to providing deeper insight into biological processes, this new approach is already in the diagnosis of COVID-19. It can be used for simultaneous imaging of multiple samples for the antigen test being used.
Perhaps most importantly, this study shows how optical frequency combs, which were used only as “frequency rulers,” can find a place for microscopy technology to push the boundaries of life science. I will.
It is promising for the development of new treatment options that treat intractable diseases, prolong life expectancy, and thereby benefit humanity as a whole.