Comb of life: a new way for a fluorescence microscope

A fluorescence microscope is widely used in biochemistry and life sciences because it allows scientists to directly monitor cells and some compounds in and around them. Fluorescent molecules absorb light within a specific wavelength range and then repel it at the longer wavelength range. However, the main limitation of conventional fluorescence microscopy methods is that the results are very difficult to quantify orally; the intensity of fluorescence has a significant effect on both experimental conditions and the density of the fluorescent material. Now, a new study by scientists from Japan is going to dramatically change the field of fluorescence microscopy. Read on to understand how!

A way around the usual problem is to focus on fluorescence life instead of intensity. When a fluorescent material is irradiated by a short burst of light, the resulting fluorescence does not disappear immediately but actually “rots” over time in a way specific to that material. The “fluorescence microscopy” technique reduces this phenomenon – which is independent of experimental conditions – to accurately measure fluorescent molecules and changes in their environment. However, fluorescence decays very quickly, and normal cameras cannot capture it. Although a single-point photodetector can be used instead, it must be scanned across the sample area to be able to reconstruct a complete 2D image from each measured point. This process involves the movement of mechanical pieces, which significantly limits the speed of image capture.

Fortunately, in this recent study published in Advances in science, the previously mentioned team of scientists developed a modern approach to obtaining fluorescence longevity images without the need for mechanical scanning. Professor Takeshi Yasui, from the Institute of Post-LED Photonics (pLED), Tokushima University, Japan, who led the study, explains, “Our approach can be described as mapping at the same time. time 44,400 ‘light stopwatches’ over 2D space to measure fluorescence times -all in a single image and 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 a light signal made up of a sum of many separate optical frequencies with a constant spacing between them. The word “comb” in this context refers to how the signal looks when plotted against optical frequency: a dense collection of symmetrical “spikes” rising from the frequency axis optical and like a hair comb. Using special optical equipment, a pair of excitation frequency comb signals are decomposed into individual optical pulse signals (dual-excitation pulses) with different intensity modulation frequencies, each carrying one change frequency, and irradiation of the target sample. The key here is that each light carrier hits the sample at a specific location, creating one-to-one communication between each point on the 2D surface of the sample (pixels) and each frequency of change. of the dual-comb optical pulses.

Due to its fluorescence properties, the sample redistributes part of the captured radiation while still maintaining the stated frequency setting contact. The fluorescence emitted from the sample is then focused using a lens on a high-speed single-point photodetector. Finally, the measured signal is mathematically converted into the frequency range, and the fluorescence time at each “pixel” is easily measured from the relative phase delay between the excitation signal at that change frequency. compared to the one measured.

Thanks to its higher speed and high spatial resolution, the microscopy method developed in this study makes it easier to exploit the life-saving benefits of fluorescence. “Because our approach does not require scanning, simultaneous measurement over the entire sample is guaranteed in all cases,” says Dr. Yasui, “This will be helpful in life sciences where dynamic ideas of living cells are needed.” In addition to providing a deeper insight into biological processes, this new method could be used for simultaneous multiple-sample imaging for antigen testing, which is already used for to detect COVID-19.

Perhaps more importantly, this study demonstrates how optical frequency combs, used only as “frequency controllers,” can find a place in microscopy techniques to push the envelope in life sciences. It promises to develop novel therapeutic options to treat intractable diseases and increase life expectancy, thus benefiting all of humanity.

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About Tokushima University, Japan

Founded in 1949 by combining multiple educational facilities, Tokushima University has grown to become one of the most prestigious universities in Japan. The present vision is to seek truth, knowledge creation, and the development of distinguished sciences and cultures with a spirit of independence and independence, all for the peaceful development of humanity and the solution of social issues. Tokushima University counts with seven faculties, eight graduate schools, and an institute of liberal arts and sciences spread across three major campuses, serving 5,900 undergraduate students and more than 2,000 graduate students. The university also counts with more than 200 international students from 29 countries. Tokushima University is open to the whole world and works hard to create a prosperous and peaceful society for the future.

Website: https: //www.tokushima-u.ac.jp /English/

About the Institute of Post-LED Photonics (pLED), Tokushima University, Japan

This institute was established at Tokushima University in March 2019 to open a new range of invisible regenerative light, i.e., deep ultraviolet, infrared, and terahertz. Research in pLED involves the development and use of the practical light source in these waves. PLED also develops innovative medical methods by combining optical science with medical science. All researchers with different experiences pursue advanced optical science, while sharing the same insights and directions. PLED will develop interdisciplinary research beyond one specific field through close communication and interaction between researchers with different backgrounds.

Website: https: //www.promised.tokushima-u.ac.jp /wp-content /topics / subjectsplp /pdf /pamph_english.pdf

About Professor Takeshi Yasui from Tokushima University

Professor Takeshi Yasui graduated from Tokushima University, Japan, in 1992 and went on to obtain two doctoral degrees: one in Engineering from Tokushima University in 1997 and one in Medical Science from Tokushima University. Nara healed in 2013. Since 2019, he has been an Institute Director. of Post-LED Photonics (pLED), Tokushima University. He has published more than 100 peer-reviewed papers and is currently interested in research on optical frequency comb, terahertz instrument, and non-conventional optical microscope.

https: //femto.me.tokushima-u.ac.jp /eng /index.html

Funding information

The study was supported by grants for Research in Advanced Technology (ERATO), Japan Science and Technology Organization (MINOSHIMA Intelligent Optical Synthesizer Project, JPMJER1304), Japan Society for the Advancement of Science (18H01901, 18K13768, 19H00871), Cabinet Office, Government Japan (Funding for Regional University and Regional Business Creation), Nakatani Foundation for the Advancement of Measurement Technologies in Biomedical Engineering, and Research Clusters program at Tokushima University (1802003).