Laser nanoprocessing via an enhanced longitudinal electric field of a radially polarized beam.

Single-shot laser ablation is performed on the surface of a transparent glass material using a radially polarized femtosecond beam. Theoretical and experimental investigation revealed the significant role of the material interface under high-numerical-aperture conditions. The longitudinal electric field at the focus was remarkably enhanced due to the total reflection on the interface when a radially polarized beam was focused on the back surface of the glass from the inside using an immersion lens. This focusing condition enabled the fabrication of a small ablation hole sized 67 nm. This study offers a novel, to the best of our knowledge, approach to realize laser nanoprocessing with radially polarized beams.

Graded arc beam in light needle microscopy for axially resolved, rapid volumetric imaging without nonlinear processes.

High-speed three-dimensional (3D) imaging is essential for revealing the structure and functions of biological specimens. Confocal laser scanning microscopy has been widely employed for this purpose. However, it requires a time-consuming image-stacking procedure. As a solution, we previously developed light needle microscopy using a Bessel beam with a wavefront-engineered approach [Biomed. Opt. Express13, 1702 (2022)10.1364/BOE.449329]. However, this method applies only to multiphoton excitation microscopy because of the requirement to reduce the sidelobes of the Bessel beam. Here, we introduce a beam that produces a needle spot while eluding the intractable artifacts due to the sidelobes. This beam can be adopted even in one-photon excitation fluorescence 3D imaging. The proposed method can achieve real-time, rapid 3D observation of 200-nm particles in water at a rate of over 50 volumes per second. In addition, fine structures, such as the spines of neurons in fixed mouse brain tissue, can be visualized in 3D from a single raster scan of the needle spot. The proposed method can be applied to various modalities in biological imaging, enabling rapid 3D image acquisition.

Multibeam continuous axial scanning two-photon microscopy for in vivo volumetric imaging in mouse brain.

This study presents an alternative approach for two-photon volumetric imaging that combines multibeam lateral scanning with continuous axial scanning using a confocal spinning-disk scanner and an electrically focus tunable lens. Using this proposed system, the brain of a living mouse could be imaged at a penetration depth of over 450 µm from the surface. In vivo volumetric Ca2+ imaging at a volume rate of 1.5 Hz within a depth range of 130-200 µm, was segmented with an axial pitch of approximately 5-µm and revealed spontaneous activity of neurons with their 3D positions. This study offers a practical microscope design equipped with compact scanners, a simple control system, and readily adjustable imaging parameters, which is crucial for the widespread adoption of two-photon volumetric imaging.

Nanoprocessing of Self-Suspended Monolayer Graphene and Defect Formation by Femtosecond-Laser Irradiation.

We demonstrate the femtosecond-laser processing of self-suspended monolayer graphene grown by chemical vapor deposition, resulting in multipoint drilling with holes having a diameter of <100 nm. Scanning transmission electron microscopy revealed the formation of many nanopores on the laser-irradiated graphene. Furthermore, atomic-level defects as well as nanopores were found in the graphene membrane by high-resolution transmission electron microscopy, while the overall crystal structure remained intact. Raman spectroscopy showed an increase in the defect density with an increase in the number of laser shots, suggesting that the nanopore formation triggered the creation of the <100 nm holes. The approach presented herein can offer an experimental insight into the simulation of atomic dynamics in graphene under femtosecond-laser irradiation. The thorough examination of the atomic defect formation and secondary effect of surface cleaning observed in this study would help develop engineering methods for graphene and other two-dimensional materials in the future.

Single-scan volumetric imaging throughout thick tissue specimens by one-touch installable light-needle creating device.

Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 µm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 µm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.

Wavefront engineered light needle microscopy for axially resolved rapid volumetric imaging.

Increasing the acquisition speed of three-dimensional volumetric images is important-particularly in biological imaging-to unveil the structural dynamics and functionalities of specimens in detail. In conventional laser scanning fluorescence microscopy, volumetric images are constructed from optical sectioning images sequentially acquired by changing the observation plane, limiting the acquisition speed. Here, we present a novel method to realize volumetric imaging from two-dimensional raster scanning of a light needle spot without sectioning, even in the traditional framework of laser scanning microscopy. Information from multiple axial planes is simultaneously captured using wavefront engineering for fluorescence signals, allowing us to readily survey the entire depth range while maintaining spatial resolution. This technique is applied to real-time and video-rate three-dimensional tracking of micrometer-sized particles, as well as the prompt visualization of thick fixed biological specimens, offering substantially faster volumetric imaging.

Single-path single-shot phase-shifting digital holographic microscopy without a laser light source.

We propose single-path single-shot phase-shifting digital holographic microscopy (SSP-DHM) in which the quantitative phase information of an object wave is acquired without a laser light source. Multiple phase-shifted holograms are simultaneously obtained using a linear polarizer, a liquid crystal on a silicon spatial light modulator (LCoS-SLM), and a polarization-imaging camera. Complex amplitude imaging of a USAF1951 test target and phase imaging of transparent HeLa cells are performed to show its quantitative phase-imaging ability. We also conduct an experiment for the motion-picture imaging of transparent particles to highlight the single-shot imaging ability of SSP-DHM.

Roadmap on Recent Progress in FINCH Technology.

Fresnel incoherent correlation holography (FINCH) was a milestone in incoherent holography. In this roadmap, two pathways, namely the development of FINCH and applications of FINCH explored by many prominent research groups, are discussed. The current state-of-the-art FINCH technology, challenges, and future perspectives of FINCH technology as recognized by a diverse group of researchers contributing to different facets of research in FINCH have been presented.

Two-step phase-shifting interferometry for self-interference digital holography.

We propose a phase-shifting interferometry technique using only two in-line phase-shifted self-interference holograms. There is no requirement for additional recording or estimation in the measurement. The proposed technique adopts a mathematical model for self-interference digital holography. The effectiveness of the proposed technique is demonstrated by experiments on incoherent digital holographic microscopy and color-multiplexed fluorescence digital holography with computational coherent superposition. Two-color-multiplexed four-step phase-shifting incoherent digital holography is realized for the first time, to the best of our knowledge, using the proposed technique.

Adaptive Optical Two-Photon Microscopy for Surface-Profiled Living Biological Specimens.

We developed adaptive optical (AO) two-photon excitation microscopy by introducing a spatial light modulator (SLM) in a commercially available microscopy system. For correcting optical aberrations caused by refractive index (RI) interfaces at a specimen's surface, spatial phase distributions of the incident excitation laser light were calculated using 3D coordination of the RI interface with a 3D ray-tracing method. Based on the calculation, we applied a 2D phase-shift distribution to a SLM and achieved the proper point spread function. AO two-photon microscopy improved the fluorescence image contrast in optical phantom mimicking biological specimens. Furthermore, it enhanced the fluorescence intensity from tubulin-labeling dyes in living multicellular tumor spheroids and allowed successful visualization of dendritic spines in the cortical layer V of living mouse brains in the secondary motor region with a curved surface. The AO approach is useful for observing dynamic physiological activities in deep regions of various living biological specimens with curved surfaces.

Laser microprocessing of metal surfaces using a tightly focused radially polarized beam.

Tight focusing of a radially polarized beam is used for single-shot laser ablation of metals. The strong longitudinal field is generated at the focus, and its contribution to the ablation process is comprehensively examined for various metal materials. In the presence of the longitudinal field at the focus, a fabricated crater at the surface exhibits either a spot shape or a doughnut shape, depending on the material. The experimental results indicate that the strong longitudinal electric field on metal surfaces is capable of promoting material removal, which may provide a novel processing scheme in ultrafast laser microprocessing with enhanced spatial resolution.

Ultrafast laser ablation of 10-nm self-supporting membranes by two-beam interference processing.

Ultrafast laser ablation was applied to process 10-nm self-supporting membranes. The membranes were processed over tens of square micrometers by single-shot irradiation of two visible laser pulses, followed by the realization of periodic sub-microstructures. The fabricated geometry is dependent on the intensity distribution of the superposed input pulses, providing flexibility and facilitating practical micro- and nanoengineering. Ease of designing the processing parameters and speed of processing are the significant advantages of this method compared to focused ion beam (FIB) milling.

Imaging with a longitudinal electric field in confocal laser scanning microscopy to enhance spatial resolution.

The longitudinal electric field produced by focusing a radially polarized beam is applied in confocal laser scanning microscopy by introducing a higher-order transverse mode, combined with a technique of polarization conversion for signal detection. This technique improves signal detection corresponding to the longitudinally polarized field under a small confocal pinhole, enabling full utilization of the small focal spot characteristic of the longitudinal field. Detailed numerical and experimental studies demonstrate the enhanced spatial resolution in confocal imaging that detects a scattering signal using a higher-order radially polarized beam. Our method can be widely applied in various imaging techniques that detect coherent signals such as second-harmonic generation microscopy.

Direct generation of the lowest-order vortex beam using a spot defect mirror in the ultraviolet region.

The creation of ultraviolet optical vortex beams with the topological charge of $ \vert l \vert = 1 $|l|=1 at the wavelength of 325 nm was demonstrated from a He-Cd metal vapor laser with a spot defect mirror. The measured $ {{\rm M}^2} $M2 factor was close to the theoretical value of two of the $ {{\rm LG}_{01}} $LG01 Laguerre-Gaussian mode. Some interference experiments showed that the obtained vortex beams were stable enough for practical applications such as holographic lithography.

Light needle microscopy with spatially transposed detection for axially resolved volumetric imaging.

The demand for rapid three-dimensional volumetric imaging is increasing in various fields, including life science. Laser scanning fluorescence microscopy has been widely employed for this purpose; however, a volumetric image is constructed by two-dimensional image stacking with a varying observation plane, ultimately limiting the acquisition speed. Here we propose a method enabling axially resolved volumetric imaging without a moving observation plane in the framework of laser scanning microscopy. A scanning light needle spot with an extended focal depth provides excitation, which normally produces a deep focus image with a loss of depth information. In our method, the depth information is retrieved from transposed lateral information on an array detector by utilising non-diffracting and self-bending characteristics imposed on fluorescent signals. This technique, implemented in two-photon microscopy, achieves truly volumetric images constructed from a single raster scan of a light needle, which has the capability to significantly reduce the acquisition time.

Subtraction imaging by the combination of higher-order vector beams for enhanced spatial resolution.

We demonstrate that the spatial resolution in confocal laser scanning microscopy is remarkably improved by simple image subtraction between images acquired by higher-order transverse modes of a cylindrical vector beam, referred to as radially and azimuthally polarized Laguerre-Gaussian mode beams. Two types of combinations of vector mode beams suitable for subtraction imaging are derived by a systematical study based on numerical calculations. The spatial resolution of about 100 nm is experimentally achieved without the degradation of image quality.

Non-diffracting linear-shift point-spread function by focus-multiplexed computer-generated hologram.

An Airy beam can be used to implement a non-diffracting self-bending point-spread function (PSF), which can be utilized for computational 3D imaging. However, the parabolic depth-dependent spot trajectory limits the range and resolution in rangefinding. In this Letter, we propose a novel pupil-phase-modulation method to realize a non-diffracting linear-shift PSF. For the modulation, we use a focus-multiplexed computer-generated hologram, which is calculated by multiplexing multiple lens-function holograms with 2D sweeping of the foci. With this method, the depth-dependent trajectory of the non-diffracting spot is straightened, which improves the range and resolution in rangefinding. The proposed method was verified by numerical simulations and optical experiments. The method can be applied to laser-based microscopy, time-of-flight rangefinding, and so on.

Vector beam generation from vertical cavity surface emitting lasers.

Radially and azimuthally polarized beams in a single transverse mode are generated from a commercially available vertical cavity surface emitting laser (VCSEL) in an external cavity with a birefringent rutile lens, of which the c axis is parallel to the optical axis of the cavity, to select favorable polarization. Additionally, a vector Bessel-Gaussian beam is generated from a VCSEL, which is fabricated to oscillate with a linear polarization in a fixed direction in free running, in the same way. These results clearly show the potential ability of VCSELs to generate vector beams, which will be essential to space-division multiplexing in the future optical communication.

Micro-hole drilling by tightly focused vector beams.

We demonstrated that azimuthally polarized beams offer high-speed laser micro-hole drilling compared with radially and linearly polarized beams under a tight focusing condition. The speed was evaluated by piercing time by vector beams for several objective lenses with different numerical apertures (NAs). As a result, in the case of NA=0.63, the piercing time of an azimuthally polarized beam was 2.4 to 3.2 times shorter than that of a linearly polarized beam for all materials tested. Surprisingly, for NA=0.85, the difference is expanded to about 7.7 times for copper. This indicates that the number of reflections on the side wall of the hole played a significant role to give rise to the difference in the piercing time, depending on the polarization of the light beam.

STED microscopy--super-resolution bio-imaging utilizing a stimulated emission depletion.

One of the most popular super-resolution microscopies that breaks the diffraction barrier is stimulated emission depletion (STED) microscopy. As the optical set-up of STED microscopy is based on a laser scanning microscopy (LSM) system, it potentially has several merits of LSM like confocal or two-photon excitation LSM. In this article, we first describe the principles of STED microscopy and then describe the features of our newly developed two-photon excitation STED microscopy. On the basis of our recent results and those of other researchers, we conclude by discussing future research and new technologies in this field.