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Abstract As a basic component of the versatile semiconductor devices, metal oxides play a critical role in modern electronic information industry. However, ultra-high precision nanopatterning of metal oxides often involves multi-step lithography and transfer process, which is time-consuming and costly. Here, we report a strategy, using metal-organic compounds as solid precursor photoresist for multi-photon lithography and post-sintering, to realize ultra-high precision additive manufacturing of metal oxides. As a result, we gain metal oxides including ZnO, CuO and ZrO2 with a critical dimension of 35 nm, which sets a benchmark for additive manufacturing of metal oxides. Besides, atomic doping can be easily accomplished by including the target element in precursor photoresist, and heterogeneous structures can also be created by multiple multi-photon lithography, allowing this strategy to accommodate the requirements of various semiconductor devices. For instance, we fabricate an ZnO photodetector by the proposed strategy.

Stimulated emission depletion microscopy (STED) holds great potential in biological science applications, especially in studying nanoscale subcellular structures. However, multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation. Here, we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity. By separating live-cell fluorescent probes with similar spectral properties using phasor analysis, our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures. The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.

Abstract Mask-free multi-photon lithography enables the fabrication of arbitrary nanostructures low cost and more accessible than conventional lithography. A major challenge for multi-photon lithography is to achieve ultra-high precision and desirable lateral resolution due to the inevitable optical diffraction barrier and proximity effect. Here, we show a strategy, light and matter co-confined multi-photon lithography, to overcome the issues via combining photo-inhibition and chemical quenchers. We deeply explore the quenching mechanism and photoinhibition mechanism for light and matter co-confined multiphoton lithography. Besides, mathematical modeling helps us better understand that the synergy of quencher and photo-inhibition can gain a narrowest distribution of free radicals. By using light and matter co-confined multiphoton lithography, we gain a 30 nm critical dimension and 100 nm lateral resolution, which further decrease the gap with conventional lithography.

Confocal laser scanning microscopy is one of the most widely used tools for high-resolution imaging of biological cells. However, the imaging resolution of conventional confocal technology is limited by diffraction, and more complex optical principles and expensive optical-mechanical structures are usually required to improve the resolution. This study proposed a deep residual neural network algorithm that can effectively improve the imaging resolution of the confocal microscopy in real time. The reliability and real-time performance of the algorithm were verified through imaging experiments on different biological structures, and an imaging resolution of less than 120 nm was achieved in a more cost-effective manner. This study contributes to the real-time improvement of the imaging resolution of confocal microscopy and expands the application scenarios of confocal microscopy in biological imaging.

Observing subcellular structural dynamics in living cells has become the goal of super-resolution (SR) fluorescence microscopy. Among typical SRM techniques, structured illumination microscopy (SIM) stands out for its fast imaging speed and low photobleaching. However, 2D-SIM requires nine raw images to obtain a SR image, leading to undesirable artifacts in the fast dynamics of live-cell imaging. In this paper, we propose a single-frame structured illumination microscopy (SF-SIM) method based on deep learning that achieves SR imaging using only a single image modulated by a hexagonal lattice pattern. The SF-SIM method used the prior knowledge to complete the structure enhancement of SR images in the spatial domain and the expansion of the Fourier spectrum through deep learning, achieving the same resolution as conventional 2D-SIM. Temporal resolution is improved nine times, and photobleaching is reduced by 2.4 times compared to conventional 2D-SIM. Based on this, we observed the fast dynamics of multiple subcellular structures and the dynamic interaction of two organelles. The SF-SIM methods provide a powerful tool for live-cell imaging.

Structured illumination microscopy (SIM) achieves super-resolution (SR) by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction. The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain, it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary, besides, the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts. Here, we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets, and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets (the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function (OTF)). Experiments on reconstructing raw datasets including nonbiological, biological, and simulated samples demonstrate that our method has SR capability, high reconstruction speed, and high robustness to aberration and noise.

Spectral confocal sensors are effective for measuring displacements. The core of the spectral confocal measurement system is a dispersive objective lens that uses optical dispersion to establish a one-to-one correspondence between the focusing position and wavelength, achieving high-resolution measurements in the longitudinal direction. Despite significant progress in dispersive objective lenses for spectral confocal sensor systems, challenges such as a limited dispersion range, high cost, and insufficient measurement accuracy persist. To expand the measurement range and improve the accuracy of the spectral confocal sensor, we designed a compact, long-axial dispersion objective lens. This lens has a simple structure that requires only six lens elements, two of which form cemented doublets. The system length is 58 mm, with a working distance of 46 ± 6 mm and a dispersion range of 12 mm within the wavelength range of 450–656 nm. The lens has an object-side numerical aperture (NA) of 0.22 and an image-side NA between 0.198 and 0.24, ensuring high light energy utilization. Finally, a spectral confocal measurement system was constructed based on the designed dispersive objective lens, and performance evaluation tests were conducted. The test results showed that the system achieved a resolution of 0.15 μm and a maximum linear error of ±0.7 μm, demonstrating high-precision measurement capabilities. The proposed lens design enables the development of more portable and cost-effective spectral confocal sensors.

As a supplement to optical super-resolution microscopy techniques, computational super-resolution methods have demonstrated remarkable results in alleviating the spatiotemporal imaging trade-off. However, they commonly suffer from low structural fidelity and universality. Therefore, we herein propose a deep-physics-informed sparsity framework designed holistically to synergize the strengths of physical imaging models (image blurring processes), prior knowledge (continuity and sparsity constraints), a back-end optimization algorithm (image deblurring), and deep learning (an unsupervised neural network). Owing to the utilization of a multipronged learning strategy, the trained network can be applied to a variety of imaging modalities and samples to enhance the physical resolution by a factor of at least 1.67 without requiring additional training or parameter tuning. Given the advantages of high accessibility and universality, the proposed deep-physics-informed sparsity method will considerably enhance existing optical and computational imaging techniques and have a wide range of applications in biomedical research.

The high dark current of perovskite photodetectors hinders the full potential of perovskites as active material for X-ray detectors. Here, Jin et al. provide a strategy to reduce the dark current to zero and massively enhance the signal-to-noise ratio of perovskite X-ray detectors and photodetectors.

Abstract Advances in direct laser writing to attain super-resolution are required to improve fabrication performance and develop potential applications for nanophotonics. In this study, a novel technique using single-color peripheral photoinhibition lithography was developed to improve the resolution of direct laser writing while preventing the chromatic aberration characteristics of conventional multicolor photoinhibition lithography, thus offering a robust tool for fabricating 2D and 3D nanophotonic structures. A minimal feature size of 36 nm and a resolution of 140 nm were achieved with a writing speed that was at least 10 times faster than existing photoinhibition lithography. Super-resolution and fast scanning enable the fabrication of spin-decoupled metasurfaces in the visible range within a printing duration of a few minutes. Finally, a subwavelength photonic crystal with a near-ultraviolet structural color was fabricated to demonstrate the potential of 3D printing. This technique is a flexible and reliable tool for fabricating ultracompact optical devices.

Since the 1990s, continuous technical and scientific advances have defied the diffraction limit in microscopy and enabled three-dimensional (3D) super-resolution imaging. An important milestone in this pursuit is the coherent utilization of two opposing objectives (4Pi geometry) and its combination with super-resolution microscopy. Herein, we review the recent progress in 4Pi nanoscopy, which provides a 3D, non-invasive, diffraction-unlimited, and isotropic resolution in transparent samples. This review includes both the targeted and stochastic switching modalities of 4Pi nanoscopy. The schematics, principles, applications, and future potential of 4Pi nanoscopy are discussed in detail.

Quantum tunneling electrical probes, consisting of a pair of nanoelectrodes with a gap width of less than 5 nm, can be used as a robust electrical sensing platform for the detection of various nanoscale objects. To achieve this, stable and gap-width-controllable electrodes are essential. Although various methods, including lithography and electrochemical strategies, have been proposed for the fabrication of tunneling electrodes, the ability to precisely control the gap width and ensure reproducibility is still lacking. Here, we report a feedback-controlled electrochemical etching approach to fabricate the tunneling electrodes with a controlled nanogap. The connected nanoelectrodes, derived from a dual-barrel nanopipette, were subjected to a controlled electrochemical etching process from a short-circuited state to a tunneling gap. The resulting tunneling electrodes exhibited solvent-response current–voltage electrical behavior, which was well fitted with the Simons model, indicating the formation of tunneling electrodes. Overall, a success rate of more than 60% could be achieved to obtain the tunneling gaps. Furthermore, to verify the function of tunneling electrodes, we used the etched-tunneling electrodes for free-diffusing protein detection, showing the potential of etched-tunneling electrodes as single-molecule sensors.

IntroductionAtherosclerotic cardiovascular disease is the leading cause of death worldwide. The Edgeworthia gardneri (Wall.) Meisn is a Tibetan medicine commonly used to prepare herbal tea to alleviate the local people's metabolic diseases. However, the anti-atherosclerotic effect of ethanol extract of the flower of E. gardneri (Wall.) Meisn (EEEG) and its underlying mechanism remain unknown.MethodsEEEG was used to treat low-density lipoprotein (ox-LDL)-induced macrophages to detect macrophage foaminess, cholesterol binding and uptake, and lipid transport-related gene expression. eEEG treated ApoE−/− mice fed a high-fat diet for 16 weeks to detect atherosclerotic plaque area, macrophage infiltration, and liver and small intestine lipid transport-related gene expression.ResultsEEEG inhibited macrophage-derived foam cell formation induced by oxidized low-density lipoprotein (ox-LDL) by reducing CD36-mediated lipoprotein uptake. EEEG significantly alleviated atherosclerosis in ApoE−/− mice fed a high-fat diet for 16 weeks. EEEG treatment significantly decreased atherosclerotic plaque area, macrophage infiltration, and increased collagen content. Moreover, EEEG treatment significantly downregulated mRNA expression of hepatic Srb1 and intestinal Npc1l1 and increased expression of hepatic Cyp7a1.ConclusionOur study highlighted that EEEG played a role in attenuating atherosclerotic plaque formation by reducing macrophage foam cell formation.

In this paper, we propose a new fluorescence emission difference microscopy (FED) technique based on polarization modulation. An electro-optical modulator (EOM) is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency, which leads to solid- and donut-shaped beams after spatial light modulation. Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve [Formula: see text] spatial resolution. Using the proposed system, the dynamic imaging of subcellular structures in living cells over time is achieved.

Micromachines, as a platform for manipulation, assembling, detection and imaging, is a typical interdisciplinary field related to broad areas, e [...]

Two-photon lithography (TPL) plays a vital role in microstructure fabrication due to its high processing accuracy and maskless characteristics. To optimize the manufacturing quality deteriorated by the defocus of the substrate, an autofocus approach based on improved differential confocal microscopy (IDCM) is proposed in this paper. Through analyzing the intensity response signals from two detectors with symmetrical axial offset, the defocus amount is measured and compensated for with high precision and noise immunity to stabilize the substrate. The verification experiments on the coverslip reported a detection sensitivity of 5 nm, a repetitive measurement accuracy of less than 15 nm, and a focusing accuracy reaching around 5 nm. The consistency between simulation and characterization demonstrated the effectiveness and superior performance of the autofocus system for the high production quality of the metalens array. The proposed autofocus method shows promise for further application to the fabrication of complex structures on various substrates.

Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues. To break the limit of resolution caused by diffraction, researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s. These contributions, however, still make sub-10[Formula: see text]nm imaging an obstacle. Here, we name a series of technologies as modulated illumination localization microscopy (MILM), which makes ultra-high-resolution imaging practical. Besides, we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up development of MILM. This review divides MILM into two types: point-scanning and wide-field. The schematics, principles and future research directions of MILM are discussed elaborately.

In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the conventional bench-top microscopic imaging equipment is limited. The comprehensive multi-mode (bright/dark field imaging, fluorescence excitation imaging, polarized light imaging, and differential interference microscopy imaging, etc.) biomedical microscopy imaging systems are generally large in size and expensive. They also require professional operation, which means high labor-cost, money-cost and time-cost. These characteristics prevent them from being applied in bio-medical mobile workstations. The bio-medical mobile workstations need microscopy systems which are inexpensive and able to handle fast, timely and large-scale deployment. The development of lightweight, low-cost and portable microscopic imaging devices can meet these demands. Presently, for the increasing needs of point-of-care-test and tele-diagnosis, high-performance computational portable microscopes are widely developed. Bluetooth modules, WLAN modules and 3G/4G/5G modules generally feature very small sizes and low prices. And industrial imaging lens, microscopy objective lens, and CMOS/CCD photoelectric image sensors are also available in small sizes and at low prices. Here we review and discuss these typical computational, portable and low-cost microscopes by refined specifications and schematics, from the aspect of optics, electronic, algorithms principle and typical bio-medical applications.

Beams with optical vortices are widely used in various fields, including optical communication, optical manipulation and trapping, and, especially in recent years, in the processing of nanoscale structures. However, circular vortex beams are difficult to use for the processing of chiral micro and nanostructures. This paper introduces a multiramp helical–conical beam that can produce a three-dimensional spiral light field in a tightly focused system. Using this spiral light beam and the two-photon direct writing technique, micro–nano structures with chiral characteristics in space can be directly written under a single exposure. The fabrication efficiency is more than 20 times higher than the conventional point-by-point writing strategy. The tightly focused properties of the light field were utilized to analyze the field-dependent properties of the micro–nano structure, such as the number of multiramp mixed screw-edge dislocations. Our results enrich the means of two-photon polymerization technology and provide a simple and stable way for the micromachining of chiral microstructures, which may have a wide range of applications in optical tweezers, optical communications, and metasurfaces.

X-ray phase contrast imaging is a promising technique in X-ray biological microscopy, as it improves the contrast of images for materials with low electron density compared to traditional X-ray imaging. The spatial resolution is an important parameter to evaluate the image quality. In this paper, simulation of factors which may affect the spatial resolution in a typical 2D grating–based phase contrast imaging system is conducted. This simulation is based on scalar diffraction theory and the operator theory of imaging. Absorption, differential phase contrast, and dark-field images are retrieved via the Fourier transform method. Furthermore, the limitation of the grating-to-detector distance in the spatial harmonic method is discussed in detail.

Singlet microscopy is very attractive for the development of cost-effective and portable microscopes. In contrast to conventional microscope objectives, which consist of multiple lenses, the manufacturing process for singlet lenses is done without extensive assembling and aligning. In this manuscript, we report a novel singlet virtual Zernike phase contrast microscopy setup for unstained pathological tumor tissue slides. In this setup, the objective consists of only one lens. There is no need for the inset Zernike phase plate, which is even more expensive than a whole brightfield microscopy setup. The Zernike phase contrast is virtually achieved by the deep learning computational imaging method. For the practical virtual Zernike phase contrast microscopy setup, the computational time is less than 100 ms, which is far less than that of other computational quantitative phase imaging algorithms. With a conceptual demo experimental setup, we proved our proposed method to be competitive with a research-level conventional Zernike phase contrast microscope and effective for the unstained transparent pathological tumor tissue slides. It is believed that our deep learning singlet virtual phase contrast microscopy is potential for the development of low-cost and portable microscopes and benefits resource-limited areas.

Single-molecule surface-enhanced Raman spectroscopy (SM-SERS) has the potential to detect single molecules in a non-invasive, label-free manner with high-throughput. SM-SERS can detect chemical information of single molecules without statistical averaging and has wide application in chemical analysis, nanoelectronics, biochemical sensing, etc. Recently, a series of unprecedented advances have been realized in science and application by SM-SERS, which has attracted the interest of various fields. In this review, we first elucidate the key concepts of SM-SERS, including enhancement factor (EF), spectral fluctuation, and experimental evidence of single-molecule events. Next, we systematically discuss advanced implementations of SM-SERS, including substrates with ultra-high EF and reproducibility, strategies to improve the probability of molecules being localized in hotspots, and nonmetallic and hybrid substrates. Then, several examples for the application of SM-SERS are proposed, including catalysis, nanoelectronics, and sensing. Finally, we summarize the challenges and future of SM-SERS. We hope this literature review will inspire the interest of researchers in more fields.

Singlet lenses are free from precise assembling, aligning, and testing, which are helpful for the development of portable and low-cost microscopes. However, balancing the spectrum dispersion or chromatic aberrations using a singlet lens made of one material is difficult. Here, a novel method combining singlet lens microscopy and computational imaging, which is based on deep learning image-style-transfer algorithms, is proposed to overcome this problem in clinical pathological slide microscopy. In this manuscript, a singlet aspheric lens is used, which has a high cut-off frequency and linear signal properties. Enhanced by a trained deep learning network, it is easy to transfer the monochromatic gray-scale microscopy picture to a colorful microscopy picture, with only one single-shot recording by a monochromatic CMOS image sensor. By experiments, data analysis, and discussions, it is proved that our proposed virtual colorization microscope imaging method is effective for H&E stained tumor tissue slides in singlet microscopy. It is believable that the computational virtual colorization method for singlet microscopes would promote the low-cost and portable singlet microscopy development in medical pathological label staining observing (e.g., H&E staining, Gram staining, and fluorescent labeling) biomedical research.

Despite the urgent need to image living specimens for cutting-edge biological research, most existing fluorescent labeling methods suffer from either poor optical properties or complicated operations required to realize cell-permeability and specificity. In this study, we introduce a method to overcome these limits—taking advantage of the intrinsic affinity of bright and photostable fluorophores, no matter if they are supposed to be live-cell incompatible or not. Incubated with living cells and tissues in particular conditions (concentration and temperature), some Atto and BODIPY dyes show live-cell labeling capability for specific organelles without physical cell-penetration or chemical modifications. Notably, by using Atto 647N as a live-cell mitochondrial marker, we obtain 2.5-time enhancement of brightness and photostability compared with the most commonly used SiR dye in long-term imaging. Our strategy has expanded the scientist's toolbox for understanding the dynamics and interactions of subcellular structures in living specimens.

The properties of optical vortex provide new insights into a wide range of optical and physical phenomena, giving rise to numerous applications in multiple disciplines. Notably, as much as the associated technologies have become increasingly flexible, it is still challenging to simultaneously generate multiple longitudinal polarization optical vortices with random topological charges (l) and tunable intensities at arbitrary positions. Herein, a novel approach that, by manipulating phase and amplitude at the back pupil of a high numerical‐aperture (NA) objective, realizes arbitrary longitudinal polarization vortices within the tightly focused light field is developed. Building upon the same model, the impacts of polarization on the optical vortex field and its potential of tuning the ratio of the longitudinal polarization component are further investigated.

Three-dimensional (3D) imaging using dual-lens fluorescence microscopies is popular in observing fluorescently labeled biological samples, such as mammalian/model animal cells, tissues, and embryos. Specifically, dual-lens super-resolution fluorescence microscopy methods using two opposing objective lenses allow significantly higher axial resolution and better signal to noise ratio than traditional single-lens counterparts, and thus distinguish more details in 3D images of fine intracellular structures. For 3D imaging of thick tissues and entire embryos, dual-lens light-sheet fluorescence microscopy methods using two objective lenses, either orthogonal or non-orthogonal, to achieve selective plane illumination, can meet the requirements, and thus can be used to observe embryo development and structures of interest in thick tissues. This review summarizes both dual-lens fluorescence microscopy methods, including their principles, configurations, and 3D imaging applications, providing a guideline for biological laboratories with different 3D imaging needs.

Super-resolution microscopy enables images to be obtained at a resolution higher than that imposed by the diffraction limit of light. Structured illumination microscopy (SIM) is among the fastest super-resolution microscopy techniques currently in use, and it has gained popularity in the field of cytobiology research owing to its low photo-toxicity and widefield modality. In typical SIM, a fluorescent sample is excited by sinusoidal patterns by employing a linear strategy to reconstruct super-resolution images. However, this strategy fails in cases where non-sinusoidal illumination patterns are used. In this study, we propose the least-squares SIM (LSQ-SIM) approach, which is an efficient super-resolution reconstruction algorithm in the framework of least-squares regression that can process raw SIM data under both sinusoidal and non-sinusoidal illuminations. The results obtained in this study indicate the potential of LSQ-SIM for use in structured illumination microscopy and its various application fields.

In super-resolution optical microscopes, aberrations often compromise the image performances by reducing its resolution and contrast. In previous works, the aberrations in stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM) have been well-investigated, while the research on the aberrations in structured illumination microscopy (SIM) is not sufficient, the researchers always poured attention into aberrations only in the detection path. In this paper, we investigate the aberrations in SIM in a comprehensive manner, and their causes and effects on both the illumination and the detection paths are discussed. The aberrations in the illumination path may distort illumination patterns, and deteriorate the final images, together with the aberrations in the detection path. In addition, several non-aberration-related factors, especially the misalignment of the incident beams with respect to the objective pupil, can also dramatically influence the performances of SIM. The analysis provides the theoretical basis and for optimizing a SIM system.

3D super-resolution imaging of dynamic processes in live cells is still challenging, especially in a large field of view. Here the authors combine SIM with multi-angle evanescent light illumination and achieve improved lateral and axial resolution, with stack acquisition time in the range of 1–2 s.

We report a quasi T-channel electrokinetics-based micromixer with electrically conductive sidewalls, where the electric field is in the transverse direction of the flow and parallel to the conductivity gradient at the interface between two fluids to be mixed. Mixing results are first compared with another widely studied micromixer configuration, where electrodes are located at the inlet and outlet of the channel with electric field parallel to bulk flow direction but orthogonal to the conductivity gradient at the interface between the two fluids to be mixed. Faster mixing is achieved in the micromixer with conductive sidewalls. Effects of Re numbers, applied AC voltage and frequency, and conductivity ratio of the two fluids to be mixed on mixing results were investigated. The results reveal that the mixing length becomes shorter with low Re number and mixing with increased voltage and decreased frequency. Higher conductivity ratio leads to stronger mixing result. It was also found that, under low conductivity ratio, compared with the case where electrodes are located at the end of the channel, the conductive sidewalls can generate fast mixing at much lower voltage, higher frequency, and lower conductivity ratio. The study of this micromixer could broaden our understanding of electrokinetic phenomena and provide new tools for sample preparation in applications such as organ-on-a-chip where fast mixing is required.

Image scanning microscopy based on pixel reassignment can improve the confocal resolution limit without losing the image signal-to-noise ratio (SNR) greatly [C. J. R. Sheppard, “Super-resolution in confocal imaging,” Optik 80(2) 53–54 (1988). C. B. Müller, E. Jörg, “Image scanning microscopy, “Phys. Rev. Lett. 104(19) 198101 (2010). C. J. R. Sheppard, S. B. Mehta, R. Heintzmann, “Superresolution by image scanning microscopy using pixel reassignment,” Opt. Lett. 38(15) 2889–2892 (2013)]. Here, we use a tailor-made optical fiber and 19 avalanche photodiodes (APDs) as parallel detectors to upgrade our existing confocal microscopy, termed as parallel-detection super-resolution (PDSR) microscopy. In order to obtain the correct shift value, we use the normalized 2D cross correlation to calculate the shifting value of each image. We characterized our system performance by imaging fluorescence beads and applied this system to observing the 3D structure of biological specimen.

Pixel reassignment image scanning microscopy (PRISM) is a useful tool to improve the resolution of confocal laser scanning microscopy (CLSM) only equipped with a detector array. However, while it can improve the lateral resolution, it has little effect on the axial resolution. Here, new microscopy has been proposed which combines three-dimension fluorescence emission difference microscopy (3D FED) with PRISM to further improve three-dimension resolution. We call this method three-dimension pixel reassignment fluorescence emission difference microscopy (3D-PRFED). Detailed theoretical analysis and simulation are presented in this paper. Additionally, the performance of lateral and axial resolution improvement of this method has been demonstrated by imaging 100 nm fluorescent beads and nuclear pore complexes samples. Experiment results show that this method in our system can improve lateral resolution by a factor of 1.85 and axial resolution by a factor of 1.48 compared with CLSM.

Numerous advances have been made in X-ray technology in recent years. X-ray imaging plays an important role in the nondestructive exploration of the internal structures of objects. However, the contrast of X-ray absorption images remains low, especially for materials with low atomic numbers, such as biological samples. X-ray phase-contrast images have an intrinsically higher contrast than absorption images. In this review, the principles, milestones, and recent progress of X-ray phase-contrast imaging methods are demonstrated. In addition, prospective applications are presented.

Fourier ptychographic microscopy (FPM) is a newly developed imaging technique which stands out by virtue of its high-resolution and wide FOV. It improves a microscope’s imaging performance beyond the diffraction limit of the employed optical components by illuminating the sample with oblique waves of different incident angles, similar to the concept of synthetic aperture. We propose to use an objective lens with high-NA to generate oblique illuminating waves in FPM. We demonstrate utilizing an objective lens with higher NA to illuminate the sample leads to better resolution by simulations, in which a resolution of 0.28μm is achieved by using a high-NA illuminating objective lens (NA=1.49) and a low-NA collecting objective lens (NA=0.2) in coherent imaging (λ=488nm). We then deeply study FPM’s exact relevance of convergence speed to spatial spectrum overlap in frequency domain. The simulation results show that an overlap of about 60% is the optimal choice to acquire a high-quality recovery (520*520 pixels) with about 2 min’s computing time. In addition, we testify the robustness of the algorithm of FPM to additive noises and its suitability for phase objects, which further proves FPM’s potential application in biomedical imaging.

We report three-dimensional fluorescence emission difference (3D-FED) microscopy using a spatial light modulator (SLM). Zero phase, 0–2π vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid, doughnut and z-axis hollow excitation spot, respectively. Our technique achieves super-resolved image by subtracting three differently acquired images with proper subtractive factors. Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED. Also, the improvement of lateral and axial resolution is demonstrated by imaging 100nm fluorescent beads. The experiment yields lateral resolution of 140nm and axial resolution of approximate 380nm.

We propose a method theoretically to break the diffraction limit and to improve the resolution in all three dimensions for fluorescence emission difference microscopy. We produce two kinds of hollow focal spot by phase modulation. By incoherent superposition, these two kinds of focal spot yield a 3D hollow focal spot. The optimal proportion of these two kinds of spot is given in the paper. By employing 3D hollow focal spot, super-resolution image can be yielded by means of fluorescence emission difference microscopy, with resolution enhanced both laterally and axially. According to computation result, size of point spread function of three-dimensional super-resolution imaging is reduced by about 40% in all three spatial directions with respect to confocal imaging.