A new method for fluorescence microscopy

Fluorescence microscopy is widely used in biochemistry and life sciences because it enables scientists to observe cells and certain compounds in and around them directly. Fluorescent molecules absorb light within a specific wavelength and then emit it again at the longer wavelength. However, the main limitation of conventional fluorescence microscopy techniques is that the results are very difficult to quantify; fluorescence intensity is significantly affected by both experimental conditions and the concentration of the fluorescent substance. Now a new study by scientists from Japan is a revolution in the field of lifelong microscopy with fluorescence.

One way around the conventional problem is to focus on fluorescence lifetime instead of intensity. When a fluorescent substance is irradiated with a short ray of light, the resulting fluorescence does not disappear immediately, but “decays” over time in a specific way for the substance. The fluorescence-lifelong microscopy technique uses this phenomenon, which is independent of experimental conditions, to quantify fluorescent molecules and changes in their environment. However, the decay of fluorescence is very fast and ordinary cameras cannot capture it. While a single-point photodetector can be used instead, it must be scanned throughout the sample to be able to reconstruct a complete 2-D image from each measuring point. This process involves the shifting of mechanical pieces, which greatly limits the speed of image capture.

In this recent study, published in Scientific progress, the team of scientists developed a new approach to obtain lifelong images of fluorescence without the need for mechanical scanning. Professor Takeshi Yasui, of the Institute of Post-LED Photonics (pLED), Tokushima University, Japan, who led the study, said: “Our method can be interpreted as the simultaneous mapping of 44,400 light-based ‘stopwatches’ over a 2-D space measures the lifespan of fluorescence – all in one shot and without scanning. ”

One of the most important pillars of their method is the use of an optical frequency comb as the generating light for the sample. An optical frequency comb is essentially a light signal consisting of the sum of very discrete optical frequencies with a constant space in between. The word ‘comb’ in this context refers to what the signal looks like when plotted against the optical frequency: a dense group of equidistant points that rise from the optical frequency axis and look like a hair comb. Using special optical equipment, a few generating frequency comb signals are decomposed into individual optical measuring signals (double comb optical beats) with different intensity modulation frequencies, each carrying a single modulation frequency and irradiated on the target sample. The key here is that each ray of light hits the sample in a spatial location, creating a one-to-one correspondence between each point on the 2-D surface of the sample (pixel) and each modulation frequency of the dual comb optical beats.

Due to the fluorescence properties, the sample again emits a portion of the determined radiation while maintaining the frequency position correspondence. The fluorescence released from the sample is then simply focused with a lens on a high-speed single-point photodetector. Finally, the measured signal is mathematically converted into the frequency domain, and the fluorescence lifetime at each “pixel” is easily calculated from the relative phase delay that exists between the generation signal at the modulation frequency versus the measurement point.

Thanks to the superior speed and high spatial resolution, the microscopy method developed in this study will make it easier to take advantage of fluorescence life measurements. “Because our technique does not need to be scanned, a simultaneous measurement over the entire sample in each survey is guaranteed,” says prof. Yasui, “it will be useful in life sciences where dynamic observations of living cells are required.” In addition to deeper insight into biological processes, this new approach can be used for simultaneous imaging of multiple samples for antigen testing, already used for the diagnosis of COVID-19.

Most importantly, this study shows how optical frequency combs, used only as ‘frequency rulers’, can find a place in microscopy techniques to print the envelope in life sciences. It holds promise for the development of new therapeutic options to treat unacceptable diseases and increase life expectancy, which benefits all of humanity.

Provided by Tokushima University