Fluorescence “Lifetime” Microscopy Technique Uses Frequency Combs and No Mechanical Parts to Observe Dynamic Biological Phenomena

Comb of a Lifetime: A New Method for Fluorescence Microscopy

Fluorescence microscopy is extensively utilized in biochemistry and life sciences due to the fact that it permits researchers to straight observe cells and specific substances around them. Fluorescent particles soak up light within a particular wavelength variety and after that re-emit it at the longer wavelength variety. However, the significant constraint of standard fluorescence microscopy strategies is that the outcomes are really challenging to examine quantitatively; fluorescence strength is considerably impacted by both speculative conditions and the concentration of the fluorescent compound. Now, a brand-new research study by researchers from Japan is set to transform the field of fluorescence life time microscopy. Read on to comprehend how!

A method around the standard issue is to concentrate on fluorescence life time rather of strength. When a fluorescent compound is irradiated with a brief burst of light, the resulting fluorescence does not vanish instantly however in fact “decays” gradually in a manner that specifies to that compound. The “fluorescence lifetime microscopy” method leverages this phenomenon — which is independent of speculative conditions — to properly measure fluorescent particles and modifications in their environment. However, fluorescence decay is very quick, and common video cameras cannot catch it. While a single-point photodetector can be utilized rather, it needs to be scanned throughout the sample’s location to be able to rebuild a total 2D image from each determined point. This procedure includes motion of mechanical pieces, which significantly restricts the speed of image capture.

Fortunately, in this current research study released in Science Advances, the previously mentioned group of researchers established an unique technique to get fluorescence life time images without demanding mechanical scanning. Professor Takeshi Yasui, from Institute of Post-LED Photonics (pLED), Tokushima University, Japan, who led the research study, describes, “Our method can be interpreted as simultaneously mapping 44,400 ‘light stopwatches’ over a 2D space to measure fluorescence lifetimes — all in a single shot and without scanning.” So, how was this accomplished?

This brand-new fluorescence microscopy method will determine both fluorescence strength and life time and it will not need mechanical scanning of a centerpiece; rather, it will produce images from all points in the sample all at once, allowing a more quantitative research study of vibrant biological and chemical procedures. Credit: Suana Science YMY

One of the primary pillars of their approach is making use of an optical frequency comb as the excitation light for the sample. An optical frequency comb is basically a light signal made up of the amount of numerous discrete optical frequencies with a consistent spacing in between them. The word “comb” in this context describes how the signal looks when outlined versus optical frequency: a thick cluster of equidistant “spikes” increasing from the optical frequency axis and looking like a hair comb. Using unique optical devices, a set of excitation frequency comb signals is broken down into specific optical beat signals (dual-comb optical beats) with various intensity-modulation frequencies, each bring a single modulation frequency, and irradiated on the target sample. The secret here is that each beam strikes the sample on a spatially unique area, producing a one-to-one correspondence in between each point on the 2D surface area of the sample (pixel) and each modulation frequency of the dual-comb optical beats.

Because of its fluorescence homes, the sample re-emits part of the caught radiation while still maintaining the previously mentioned frequency-position correspondence. The fluorescence produced from the sample is then merely focused utilizing a lens onto a high-speed single-point photodetector. Finally, the determined signal is mathematically changed into the frequency domain, and the fluorescence life time at each “pixel” is quickly determined from the relative stage hold-up that exists in between the excitation signal at that modulation frequency versus the one determined.

Thanks to its exceptional speed and high spatial resolution, the microscopy approach established in this research study will make it much easier to make use of the benefits of fluorescence life time measurements. “Because our technique does not require scanning, a simultaneous measurement over the entire sample is guaranteed in each shot,” remarks Prof. Yasui, “This will be helpful in life sciences where dynamic observations of living cells are needed.” In addition to supplying much deeper insight into biological procedures, this brand-new technique might be utilized for synchronised imaging of several samples for antigen screening, which is currently being utilized for the medical diagnosis of COVID-19.

Perhaps most notably, this research study showcases how optical frequency combs, which were just being utilized as “frequency rulers,” can discover a location in microscopy strategies to forge ahead in life sciences. It holds pledge for the advancement of unique healing choices to deal with intractable illness and boost life span, consequently benefitting the entire of mankind.

Reference: “Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats” by T. Mizuno, E. Hase, T. Minamikawa, Y. Tokizane, R. Oe, H. Koresawa, H. Yamamoto and T. Yasui, 1 January 2021, Science Advances.
DOI: 10.1126/sciadv.abd2102

Funding: Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Japan Society for the Promotion of Science (JSPS), Cabinet Office – Government of Japan and others.