New Method for Fluorescence Microscopy

Conventional fluorescence micro­scopy provides poor quantitative information of the sample because it only captures fluorescence intensity, which changes frequently and depends on external factors. Now, scientists from Japan have developed a new fluorescence microscopy technique to measure both fluores­cence intensity and lifetime. Their method does not require mechanical scanning of a focal point; instead, it produces images from all points in the sample simul­taneously, enabling a more quanti­tative study of dynamic biological and chemical processes.

Schematic view of the experiment: 2D arrangement of 44,400 light stopwatches enables scan-less fluorescence lifetime imaging. (Source: Tokushima U.)

Fluorescence microscopy is widely used in bio­chemistry and life sciences because it allows scientists to directly observe cells and certain compounds in and around them. Fluorescent molecules absorb light within a specific wavelength range and then re-emit it at the longer wavelength range. However, the major limitation of conventional fluorescence micro­scopy techniques is that the results are very difficult to evaluate quanti­tatively; 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 set to revo­lutionize the field of fluorescence lifetime microscopy.

A way around the conven­tional problem is to focus on fluorescence lifetime instead of intensity. When a fluorescent substance is irradiated with a short burst of light, the resulting fluorescence does not disappear immediately but actually decays over time in a way that is specific to that substance. The “fluorescence lifetime microscopy” technique leverages this phenomenon to accurately quantify fluorescent molecules and changes in their environment. However, fluorescence decay is extremely fast, and ordinary cameras cannot capture it. While a single-point photo­detector can be used instead, it has to be scanned throughout the sample’s area to be able to reconstruct a complete 2D picture from each measured point. This process involves movement of mechanical pieces, which greatly limits the speed of image capture.

Fortunately, the team of scientists developed a novel approach to acquire fluorescence lifetime images without necessi­tating mechanical scanning. Takeshi Yasui from Institute of Post-LED Photonics (pLED), Tokushima University, explains, “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.” One of the main pillars of their method is the use of an optical frequency comb as the excitation light for the sample. An optical frequency comb is essentially a light signal composed of the sum of many discrete optical frequencies with a constant spacing in between them.

Using special optical equipment, a pair of excitation frequency comb signals is decomposed into indi­vidual optical beat signals (dual-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 light beam hits the sample on a spatially distinct location, creating a one-to-one corres­pondence between each point on the 2D surface of the sample and each modulation frequency of the dual-comb optical beats. Because of its fluores­cence properties, the sample re-emits part of the captured radiation while still preserving the frequency–position corres­pondence. The fluorescence emitted from the sample is then simply focused using a lens onto a high-speed single-point photo­detector. 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 that exists between the excitation signal at that modulation frequency versus the one measured.

Thanks to its superior speed and high spatial resolution, the micro­scopy method developed in this study will make it easier to exploit the advantages of fluorescence lifetime measure­ments. “Because our technique does not require scanning, a simultaneous measurement over the entire sample is guaranteed in each shot,” remarks Yasui, “This will be helpful in life sciences where dynamic obser­vations of living cells are needed.” In addition to providing deeper insight into biological processes, this new approach could be used for simul­taneous imaging of multiple samples for antigen testing, which is already being used for the diagnosis of COVID-19. Perhaps most impor­tantly, this study showcases how optical frequency combs, which were only being used as frequency rulers, can find a place in micro­scopy techniques to push the envelope in life sciences. It holds promise for the development of novel thera­peutic options to treat intractable diseases and enhance life expectancy, thereby bene­fitting the whole of humanity. (Source: U. Tokushima)

Reference: T. Mizuno et al.: Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats, Sci. Adv. 7, eabd2102 (2021); DOI: 10.1126/sciadv.abd2102

Link: Institute of Post-LED Photonics (pLED), Tokushima University, Tokushima, Japan

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