Wataru Inami

We formulate and numerically evaluate a direct, non-iterative algebraic estimator for multicomponent fluorescence lifetimes using harmonic phase measurements in a two-photon excitation scheme. The method operates on the complex harmonic responses available at the repetition frequency of a femtosecond pulse train and its harmonics. Rather than performing iterative nonlinear lifetime fitting, we map the measured harmonic responses through a linear coefficient-reconstruction step to the denominator polynomial of a rational representation of the multiexponential decay and obtain the decay constants from its roots. This procedure requires neither initial lifetime estimates nor iterative nonlinear minimization. Numerical simulations were performed for a three-component system, with photon-limited performance evaluated using Poisson counting statistics. For the closely spaced lifetime set of 0.5, 1.5, and 2.5 ns, the mean relative lifetime error decreased from 14.49% at a peak expected count of 100 photons per time bin to 5.94% at 1000 photons and 1.31% at 10 000 photons, with each condition evaluated by averaging 500 statistically independent acquisitions. In a matched 500-trial Monte Carlo comparison at 1 × 10 7 expected photons per pixel, the success probabilities were 0.484 for the unconstrained algebraic estimator, 0.854 for the DC-constrained algebraic estimator, and 0.898 for single-start nonlinear least-squares (NLLS). Thus, imposing the DC constraint substantially improved the photon-noise robustness of the algebraic reconstruction relative to the minimally determined unconstrained formulation, while single-start NLLS remained slightly more robust than the DC-constrained algebraic estimator. In a separate matched 500-sample computational benchmark at 1 × 10 8 expected photons per pixel, the median parameter-estimation times were 0.138, 0.095, and 5.552 ms per pixel for the unconstrained algebraic, DC-constrained algebraic, and single-start NLLS estimators, respectively. Relative to the DC-constrained algebraic median, the unconstrained algebraic and single-start NLLS calculations required approximately 1.45 and 58.35 times longer processing time, respectively. These results identify both a constraint-induced robustness improvement within the algebraic formulation and a robustness–computation trade-off relative to iterative fitting. The present work should therefore be regarded as a theoretical and numerical algebraic implementation within the established multifrequency frequency-domain framework. Experimental validation and harmonic-dependent instrument-response calibration remain necessary for practical two-photon FLIM implementation.

Modern correlative microscopy and imaging techniques, such as inverted Scanning Electron Microscopy (SEM) and its super-resolution application, Direct Electron Beam Excitation Assisted SEM (D-EXA SEM), are rapidly advancing. In an inverted SEM, particularly one employing an open atmospheric sample chamber with a Silicon Nitride thin window, the ability to accommodate non-standard samples and auxiliary instrumentation is constrained by a fundamental trade-off. Specifically, increasing the working distance (WD) between the electron beam source (beam forming assembly) and the specimen. The offset required for larger samples or non-standard setups significantly deteriorates the spatial resolution and signal-to-noise ratio of the image collected from the vacuum side (secondary electron image). To systematically evaluate and overcome this dependence, we developed a novel adjustable test instrument. This tool integrates a tungsten dot array target whose position can be varied in-situ and in-vacuum under continuous SEM operation. The instrument is designed to simulate specified geometrical constraints and allows researchers to pre-set optimal electron beam parameters (voltage, current). We demonstrate the tool's use in quantifying the relationship between WD and achievable spatial resolution, providing a rapid method for assessing and calibrating high-resolution capability across various experimental setups.

Scanning electron microscopy (SEM) is widely used for nanoscale imaging and the study of surface fine structure. However, its image quality is often limited by low secondary electron (SE) yield and surface charging, especially on insulating or micro-structured materials. In this study, we introduce a non-destructive technique that significantly improves SEM image acquisition by irradiating the specimen surface with ultraviolet (UV) light during observation. This approach leverages the photoelectric effect to enhance SE emission, resulting in a higher signal-to-noise ratio and improved image contrast in SEM imaging. Experiments were conducted using an in situ UV irradiation module on three representative samples: a 1 µm thick gold film (Au) deposited on a 525 µm thick silicon (Si) substrate, a black silicon (b-Si) sample, and a GaN substrate. The results demonstrate clear SE signal enhancement and effective charge mitigation under UV illumination. This UV-assisted SEM technique offers a simple and practical approach to improving electron-beam-based imaging and is expected to expand capabilities for high-contrast observation of nanoscale materials.

We report on the utilization of Si in plasmonics research, specifically targeting deep ultraviolet (DUV) applications. By incorporating periodic submicron gratings on a Si surface, we demonstrate a photoconductive Si detector with significantly enhanced responsivity to DUV light, which is achieved via surface plasmon resonance (SPR). Notably, even without a metal, the detector exhibits improved responsivity only under TM-polarized light, supporting the interpretation that Si excites SPR. Our demonstration not only validates the effectiveness of Si plasmonics for enhancing photodetector performance but also introduces innovative techniques with potential applications in various research fields. Published by the American Physical Society 2025

Crystallography has been the routine technique for studying high-resolution structures of proteins for over five decades. A major bottleneck in structure determination of macromolecules is obtaining crystals of a size and quality suitable for single-crystal X-ray crystallography experiments. Many challenging proteins either fail to grow into crystals or fail to grow into crystals of a size suitable for obtaining high-resolution structures using conventional X-ray crystallography. When it comes to smaller crystals, they can be used either for seeding to get larger crystals or for serial crystallography and electron diffraction for obtaining the structures. For both purposes, a limiting step is to non-invasively image such small crystals of sub-micrometre dimensions and to screen the conditions where such crystals prevail. Here we use cathodoluminescence-based (CL-based) nanoscopy to image protein nanocrystals. We show that crystals of micrometre and submicrometre dimensions can be non-invasively imaged by the CL-based nanoscope. The results presented here demonstrate the feasibility of non-invasive imaging of protein crystals with sub-100 nm resolution.

Autofluorescence in living cells on aluminum thin film was excited with deep-ultraviolet surface plasmon resonance (deep-UV SPR). Deep-UV SPR under aqueous medium was excited with Kretschmann configuration by using a sapphire prism. Deep-UV SPR is one of the promising techniques for high-sensitive autofluorescence imaging of living cells without staining. Label-free observation is significant for the structural analysis of living cells. We demonstrated the high-sensitive autofluorescence imaging of living yeast cells with deep-UV SPR. We applied a high refractive index prism, such as sapphire, which is suitable for the observation of specimens in aqueous medium, to excite deep-UV SPR. Although typical autofluorescence from living cells is buried in background noise, deep-UV SPR enhances the autofluorescence signal. The deep-UV SPR excitation of an aluminum thin film through a sapphire prism was investigated theoretically and experimentally. It showed that the fluorescence intensities are increased 2.8-fold. Deep-UV SPR enhanced the autofluorescence of cell structures, and yeast cells were found to be very sensitive. As a result, for water-immersed specimens, the sapphire-prism-based Kretschmann configuration excited SPR in deep-UV. Findings from this study suggest that deep-UV SPR can be considered an effective technique for attaining high-sensitivity observation of biological samples.

We developed a high spatially-resolved ion-imaging system using focused electron beam excitation. In this system, we designed a nanometric thin sensor substrate to improve spatial resolution. The principle of pH measurement is similar to that of a light-addressable potentiometric sensor (LAPS), however, here the focused electron beam is used as an excitation carrier instead of light. A Nernstian-like pH response with a pH sensitivity of 53.83 mV/pH and linearity of 96.15% was obtained. The spatial resolution of the imaging system was evaluated by applying a photoresist to the sensing surface of the ion-sensor substrate. A spatial resolution of 216 nm was obtained. We achieved a substantially higher spatial resolution than that reported in the LAPS systems.

Optical fields enhanced by surface plasmon polaritons (SPPs) on metal-dielectric interfaces are useful for increasing several light-matter interactions, and application to nonlinear optics (NLO) is one of the most important uses of this type of structure. Most previous studies on this subject took advantage of the nonlinear susceptibilities of metal surfaces. However, the limited NLO light-matter interaction lengths prevented the transfer of the technologies to practical use. In the present paper, we tried to overcome this problem by growing NLO polymer thin films on metal surfaces. The NLO responses of the system were characterized by second-harmonic generation (SHG) spectroscopy. Our experimental results suggested that SPP-enhanced optical fields induced not only surface nonlinearities in Ag but also bulk nonlinearities in NLO polymer. There was an optimal polymer thickness for the SHG conversions, and a greater thickness did not always result in higher conversion. The maximum conversion efficiency was approximately 40 times higher than that of the nonpolymer-coated bare Ag surface. The growth and propagation of the SHG waves were addressed with a numerical approach combining the transfer matrix method and Green's function analysis. The SHG conversion efficiencies were determined by three factors, the SPP-field enhancement factor, the nonlinear light-matter interaction length, and the degree of interference between the forward- and backward-propagating SHG waves. The latter two factors predominantly determined the optimal SHG conversion efficiencies. The proposed strategy of hybridizing NLO polymers brings us closer to practical uses for nonlinear plasmonics.