The University of Tokushima

Optical nano-imaging techniques find immense applications in nanotechnology for visualizing nanoscale defects in samples. However, it is challenging to image large micron-sized samples at nanoscale resolution owing to signal distortions resulting from unavoidable thermal and mechanical drifts of the system over time. Now, researchers from Japan have developed an ultrastable nano-imaging system that successfully detects unique nanoscale defects not observed in conventional nano-imaging in micron-scale tungsten disulfide samples, widening the technique's scope to biological samples. Raman spectroscopy, an optical microscopy technique, is a non-destructive chemical analysis technique that provides rich molecular fingerprint information about chemical structure, phase, crystallinity, and molecular interactions. The technique relies on the interaction of light with chemical bonds within a material. However, since light is a wave, optical microscopes are unable to resolve distances less than half the wavelength of the light incident on the sample. This is known as the "diffraction limit," which prevents Raman spectroscopy and other optical microscopy techniques from reaching nanoscale resolutions. To improve the spatial resolution, another technique called "tip-enhanced Raman spectroscopy" (TERS) was invented, which can reach spatial resolutions below the diffraction limit. In TERS, a metallic nano-sized tip confines the light within a nano-sized volume just above the sample. The light interacts with the sample molecules on the surface and the imaging is performed by analyzing the scattered light. TERS has been successfully used to analyze chemical compositions and surface defects in sample at nanoscale resolutions. However, during imaging, the nanotip tends to drift due to unavoidable thermal and vibrational fluctuations under ambient conditions, causing either the sample to be out-of-focus or misalignment between the nanotip and focal spot, or both. This causes considerable distortions in the scattered signals. To avoid this, TERS imaging needs to be completed within a time window of 30 minutes, a restriction that prevents imaging of any sample larger than 1 µm2 with nanoscale resolution. In a new study published in Science Advances, a research team from Japan, led by Dr. Ryo Kato, a designated Assistant Professor at the Institute of Post-LED Photonics at Tokushima University, and Associate Professor Takayuki Umakoshi and Professor Prabhat Verma from Osaka University, has now developed, for the first time, a stable TERS system that is not limited to a short imaging time window. The team demonstrated its capability by successfully imaging nanoscale defects over a period of 6 hours in a micrometer-sized, two-dimensional (2D) tungsten disulfide (WS2) film -- a material commonly used in optoelectronic devices. "Our new optical nano-imaging system enables characterization of defect analysis in large-sized WS2 layers at a high pixel resolution down to 10 nm without any significant loss of optical signal," says Dr. Kato. To compensate for the drifts over extended periods, the team developed a feedback system that tracks the displacement of the focused light source and readjusts the position of the focus plane accordingly. The focal position of the light source is tracked by measuring the displacement of a reflected laser guide beam directed into the microscope. The focus is then stabilized with a piezo-controlled objective scanner whenever the system senses a drift or a change in the focal position of the light source. To stabilize the nanotip, the team designed a laser-scanning-assisted tip drift compensation system. In this case, galvano-scanners take images of the laser spot around the metallic nanotip just as it approaches the sample surface. This image appears as a bright spot and indicates the position of the nanotip. Once the measurement at a particular pixel has been carried out, the image of the laser spot around the nanotip is captured again. The laser spot is then moved to match the new position of the nanotip in this image. The process continues throughout the imaging process, ensuring the nanotip remains at a constant position. By implementing these corrections, the team was able to image a 2D sheet of WS2 with a scan area of 1 × 4 µm2. With a 12 times longer imaging time window than that of conventional imaging, they could detect unique defects missed in conventional TER imaging. They also showed that the defect density on a larger WS2 sample (comparable to device scales) was higher than that reported for smaller samples. The study could open doors to precise, high-resolution imaging of not only optoelectronic devices but also biological samples. "Our new drift-compensated TERS microscopy could not only evaluate surface properties of device materials better but also allow us to study biological processes such as the mechanism underlying the development of diseases. This, in turn, could help develop novel clinical methods and therapies," speculates Dr. Umakoshi. These are certainly some exciting possibilities to contemplate!

A new fluorescence lifetime microscopy breakthrough led by scientists from Tokushima University holds potential in several areas within life sciences The recent breakthrough was achieved by simultaneously mapping 44,400 "light stopwatches" to measure fluorescence lifetime (Credit: Tokushima University) Scientists have developed a new microscopy technique that harnesses the lifetime of fluorescence, as well as its intensity, to capture more comprehensive images. The breakthrough, which was made by a team from Tokushima University, Japan, is likely to be “helpful” in life sciences, where dynamic observations of living cells and chemical processes are key. As well as holding promise in the development of novel therapeutic options, this new approach could also be used for simultaneous imaging of multiple samples in antigen testing – which is currently being deployed widely to diagnose Covid-19. Conventional microscopy with fluorescence – the absorption and re-emission of light by molecules – provides poor quantitative information on samples because it only captures intensity of the light emitted, which changes frequently and depends on external factors. But, using light sources known as frequency combs, a team from Tokushima University in Japan has developed a way of measuring both fluorescence intensity and lifetime, and producing images from all points in the sample simultaneously, without the need for mechanical parts. Professor Takeshi Yasui from the Institute of Post-LED Photonics (pLED) at Tokushima University, who led the study, said: “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. “Because our technique does not require scanning, a simultaneous measurement over the entire sample is guaranteed in each shot. “This will be helpful in life sciences, where dynamic observations of living cells are needed.” Challenges in fluorescence lifetime microscopy Fluorescence microscopy is widely used in the biochemistry and life sciences sectors as it allows scientists to directly observe cells, and certain compounds in and around them. However, the major limitation of conventional fluorescence microscopy techniques is that the results they produce are difficult to evaluate quantitatively, because fluorescence intensity is significantly affected by experimental conditions and the concentration of the fluorescent substance. One way around this is to focus on fluorescence lifetime instead of intensity, as this factor is independent of experimental conditions. When a fluorescent substance is irradiated with a short burst of light, the re-emitted light does not disappear immediately but ‘decays’ over time in a way that is specific to the substance in question. However, fluorescence lifetime microscopy – a technique that leverages this phenomenon, and is able to accurately quantify fluorescent molecules and changes in their environment – presents its own difficulties. The decay of fluorescence is extremely fast, and the single-point photodetectors that are traditionally used involve the movement of mechanical pieces, greatly limiting the speed of the image capturing. How did scientists develop a new technique to overcome these difficulties? In a recent study published in online scientific journal Science Advances, the team from Tokushima University outlined how they developed a novel approach for acquiring fluorescence lifetime images without the need for mechanical scanning. One of the main pillars of the method they used is an optical frequency comb – which is essentially a light signal composed of many discrete, evenly-spaced optical frequencies that resembles a hair comb when plotted against optical frequency. Using special optical equipment, a pair of excitation frequency comb signals are decomposed into individual optical beat signals 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 optical beats. Because of its fluorescent properties, the sample re-emits part of the captured radiation while still preserving the aforementioned frequency-position correspondence. The fluorescence emitted from the sample is then simply focused using a lens onto a high-speed single-point photodetector, which – thanks to this method’s superior speed and high spatial resolution – produces an image that takes into account fluorescence lifetime measurements.