Microscope Objective Lens Research Articles

Large-scale microscope with improved resolution using SRGAN

Due to the limited Space-Bandwidth Product (SBP) of optical microscopy system, the Numerical Aperture (NA) and Field of View (FOV)/ Depth of Field (DOF) of conventional optical microscopes are mutually restricted. In this paper, we propose a 20× large-scale (large FOV&DOF) microscope objective lens which is enhanced by deep neural network to achieve the improved resolution close to a 40× Olympus objective lens. Based on training a Super-resolution Generative Adversarial Network (SRGAN) model to transform low-resolution images into super-resolved ones, the resolution of input images captured by low magnification objective is greatly improved without any loss of FOV. And the network output images have better performance on DOF. We also compared SRGAN with two other deep-learning based methods to prove its superiority. Such large-scale microscope with improved resolution could serve to democratize super-resolution imaging.

Bose-Einstein Condensation by Polarization Gradient Laser Cooling.

Attempts to create quantum degenerate gases without evaporative cooling have been pursued since the early days of laser cooling, with the consensus that polarization gradient cooling (PGC, also known as "optical molasses") alone cannot reach condensation. In the present work, we report that simple PGC can generate a small Bose-Einstein condensate (BEC) inside a corrugated micrometer-sized optical dipole trap. The experimental parameters enabling BEC creation were found by machine learning, which increased the atom number by a factor of 5 and decreased the temperature by a factor of 2.5, corresponding to almost 2 orders of magnitude gain in phase space density. When the trapping light is slightly misaligned through a microscopic objective lens, a BEC of ∼250 ^{87}Rb atoms is formed inside a local dimple within 40ms of PGC after MOT loading.

The Effect of Different System Parameters on the Movement of Microbial Cells Using Light-Induced Dielectrophoresis.

The manipulation of single particles remains a topic of interest with many applications. Here we characterize the impact of selected parameters on the motion of single particles thanks to dielectrophoresis (DEP) induced by visible light, in a technique called Light-induced Dielectrophoresis, or LiDEP, also known as optoelectronic tweezers, optically induced DEP, and image-based DEP. Baker's yeast and Candida cells are exposed to an electric field gradient enabled by shining a photoconductive material with a specific pattern of visible light, and their response is measured in terms of the average cell velocity towards the gradient. The impact on cell velocity when varying the shape and color of the light pattern, as well as the distance from the cell to the pattern, is presented. The experimental setup featured a commercial light projector featuring digital light processing (DLP) technology but mechanically modified to accommodate a 40× microscope objective lens. The minimal resolution achieved on the light pattern was 8 µm. Experimental results show the capability for single cell manipulation and the possibility of using different shapes, colors, and distances to determine the average cell velocity.

Electrical effects on droplet behaviour

The effect of charge on water droplets modulates various aspects of their behaviour. These include the droplet stability, evaporation, and lifetime. Microphysical models have been developed such that a reasonably good understanding of these processes has been achieved. However, the specific effects of charge deserve further scrutiny as they are an intrinsic component of the factors controlling droplet characteristics. Describing the effects of these requires an understanding of the electrostatic pressure present in the droplet and its surface tension. One way to test these effects and assess droplet response to charge is to take an experimental approach to make observations directly. In this study, individual droplets are levitated in an acoustic wave to allow isolated measurements to be taken. The droplets are monitored using a CCD camera with a microscope objective lens. In some cases, with sufficient charge present, effects on droplet stability can be observed as Rayleigh explosions, where a sudden drop in mass is seen superimposed on the evaporation profile. These events also allow the charge on the droplet to be calculated, which is then compared with the droplet evaporation. Another factor that plays a part in droplet behaviour is droplet composition. Different substances have different surface tension, and this is explored by performing some experiments on sulphuric acid droplets. Theory predicts that the more highly charged a droplet is, the more resistant to evaporation it becomes. Experimental data collected during this study agrees with this, with more highly charged droplets observed to have slower evaporation rates. However, highly charged drops were also observed to periodically become unstable during evaporation and undergo Rayleigh explosions. Each instability of a highly charged drop removes mass, reducing the overall droplet lifetime regardless of the slower evaporation rate. The sulphuric acid droplets were observed to be much more resistant to evaporation and no Rayleigh instabilities were observed.

Effect of Laser Process Loops on the Hole Diameter and Hole Formation of Laser Micro Drilling on TC4

Laser micro drilling stands as a precise manufacturing method that employs a focused laser beam to craft accurate, small holes within a diverse array of materials. Its applications span across vital industries like aerospace, medical, and electronics, playing a pivotal role in creating components like fuel injectors, medical implants, and microelectronics. Within this context, a notable challenge emerges in obtaining a refined surface finish during laser micro drilling. This study delves into the impact of a laser loop, a crucial parameter, on the surface quality of TC4, also known as Ti6Al4V—an aerospace staple. Employing a Conventional Fiber Laser with a peak output of 30 W, the experiment meticulously directs the laser beam onto the TC4 surface via a microscope objective lens. The drilling process unfolds in controlled conditions, mitigating external variables such as temperature and humidity. Assessment of drilled hole surfaces transpires through both light and 3D microscopes. Interestingly, holes subjected to higher laser power and increased laser loop rates demonstrate enhanced surface smoothness. In essence, this inquiry demonstrates the substantial influence of laser loop on TC4’s surface finish during laser micro drilling. Elevating the laser loop factor leads to heightened surface refinement and diminished roughness in drilled holes. It was found that the diameter entry of the micro-holes was increased by 61% - 89.35% and the diameter exit of the micro-holes also increased by 55.55% - 62.79%. The outcomes of this investigation offer valuable insights for refining the laser micro drilling process to achieve premium surface quality on TC4 and comparable materials. As such, these findings extend guidance for optimal laser loop settings in the realm of laser micro drilling across various materials, benefiting future manufacturing endeavors.

Design and optimization of a conical electrostatic objective lens of a low-voltage scanning electron microscope for surface imaging and analysis in ultra-high-vacuum environment

Low-voltage scanning electron microscopy (LV-SEM) with landing energies below 5 keV has been widely used due to its advantages in mitigating the damage and charging effects to a specimen and enhancing surface information due to small interaction volume of electrons inside a specimen. Additionally, for elemental analysis of the surfaces of bulk specimens with Auger electron spectroscopy (AES) or electron energy loss spectroscopy (EELS), ultra-high-vacuum (UHV) environment is essential to maintain clean surfaces without the absorption of gas molecules during the electron beam irradiation for the acquisition of spectral data. In this study, we propose the optimal design and condition of a conical Electrostatic Objective Lens (EOL) for a UHV LV-SEM to achieve the high spatial resolution and secondary electron (SE) detection efficiency. The EOL is composed of only the three electrodes (retarding, focusing and booster electrodes) and the insulators, which is suitable for maintaining a UHV environment with less out-gassing. The cone angle of the EOL is determined as 60° to integrate a spectrometer in the UHV LV-SEM and in a large size and a higher tilt angle of the sample. Through the optimization with the simulations, the EOL achieves the minimized spherical and chromatic aberration coefficients of 0.05 and 0.03 mm at the sample side, respectively, at the landing energy of 50 eV and the shortest working distance (WD) of 1 mm for high-resolution imaging. In addition, the probe diameter of the optimized EOL is 2.3 nm at 1 keV and 5.7 nm at 50 eV with a WD of 1 mm and a probe current of 10 pA, which are comparable to previously studied compound objective lenses with magnetic and electrostatic lenses. Using a longer WD of 4 mm for analysis, the probe diameter was 5.4 nm at 1 keV and the SE detection efficiency was 83.3 % owing to the separated scintillator detector structure from the booster electrode.These results imply that the optimized EOL has the potential to be applied to a high-performance UHV LV-SEM for the surface imaging and analysis with a simple system configuration.

Evaluation of Uncertainty in Measurement for Magnification Error of Metallographic Microscope Objective Lens

Propose a method for evaluating the measurement uncertainty of magnification error in metallographic microscope objective lens. Establish a measurement uncertainty evaluation model based on calibration methods and analyse the sources of each uncertainty. Combining measurement examples, this article elaborates on the evaluation of uncertainty in the measurement of magnification error of metallographic microscope objective lens.

Microscopic Projection Lithography for Integrating Metallic Microstructures on Silk Protein for Biodevice Applications.

Precise patterning of metallic micro/nanostructures enables application of silk protein in biomedical devices with a seamless human-machine interface. However, high-quality, expensive equipment and facilities involved in micro/nanofabrication hinder rapid prototyping for explorative laboratory-based research. Here, we report cost-effective and high-resolution light-emitting diode-based projection lithography methods for fabricating a Cr photomask and metallic microstructures on a silk protein layer. After two-step photolithography performed using a commercial camera and microscopic objective lens, inkjet-printed patterns are successfully projected on the silk layers with 100× and 500× demagnification ratios. A lift-off process is conducted to integrate Au patterns on the lithographic-patterned resist/silk layer, and various Au microstructures with sizes <2 μm are generated. In all the processes, the silk protein exhibits a high resistance to chemicals for resist solvent, development, resist strip, and lift-off, as well as a strong adhesion to gold, along with low cytotoxicity. Dopamine sensing and transistor operating capabilities are proved by measuring the changes in the electrical signals through the Au patterns. The proposed method is a cost-effective and simple approach for rapid prototyping of silk-based biomedical devices.

In-situ study of laser-induced novel ripples formation on SiC surface by an oblique-illumination high-resolution imaging setup

After decades of research, formation and development of laser induced periodic surface structures (LIPSS) was proposed to be closely related to the roughness of sample surface and multipulse incubation effect. To exclude the influence of the stochastic distribution of surface roughness on ripple formation and further study the multipulse incubation mechanism, there is an urgent need to induce ripples in situ and monitor the evolution of ripples synchronously. In this paper, single-site pulse-by-pulse irradiation was carried out on single-crystalline SiC surface and the induced ripples morphologies were in-situ captured by an oblique-illumination high-resolution imaging setup. A type of novel ripples with spacing located between traditional low-spatial-frequency-LIPSS and high-spatial-frequency-LIPSS was reported. In-situ imaging shows that concentric annular ripples were first induced by the 1st pulse, which were possibly caused by the Fresnel diffraction of a slightly divergent light passing through the aperture-shaped entrance pupil of microscopic objective lens. Simulations based on finite difference time domain method (FDTD) showed that next pulse with linear polarization acting on the annular ripples induced an asymmetric light distribution, leading to the generation of ripples perpendicular to laser polarization. Further simulations showed that the local field enhancement in the grooves of ripples led to the deepening of grooves and the resulting decrease of local ripple spacing with pulse number. Comparison of the morphologies of ablation spots induced by single-site pulse-by-pulse irradiation and successive pulse irradiation demonstrated that ripples developed faster in the former, further indicating the role of the pre-formed structures in promoting growth of ripples. Our oblique-illumination imaging technology provides direct observation of ripples evolution and the novel ripples here are useful for extending the ripples application in surface tailoring.

A method for estimating magnetic field of TEM objective lens

We propose a method for calibration of magnetic field in the objective lens of transmission electron microscope. The calibration process is based on classical Fresnel imaging of Permalloy disks and measuring the displacement of the magnetic vortex core when the sample is tilted at various excitations of the objective lens. We adopted the Carl Zeiss LIBRA 200 MC transmission electron microscope for Lorentz electron microscopy using this method. The objective lens magnetic field evaluation is tested on the Co/Pt multilayered films with known magnetic properties.

Digital holographic nanoscopy for erythrocyte, nanoparticle and quantum dot characterization

Optical detection of biological cells and nanoparticles is a challenging task in optical physics due to the individual nanoparticles' low inherent scattering, size, and shape. High magnification is required for the study of such particles. In the present work, we have introduced a modified Digital Holographic Microscopy (DHM) for qualitative and quantitative optical characterization of biological cells, nanoparticles and quantum dots through phase measurement. The proposed system can measure a quantum dot of a minimum size of 6.8 nanometers with 1,53,284X magnification by placing two Microscope Objective (MO) lenses in the path of the object beam of the DHM. We have recorded digital holograms of the specimen for different combinations of MOs by varying distances between them. The system can characterize the specimen of micro to nanometer scales by varying its magnification as per requirements. The results are shown for human erythrocytes of diameter of 7.31 ± 0.08 µm & thickness of 2.3 ± 0.058 µm and gold nanoparticles (AuNPs) of different sizes (6.8 nm - 50 nm). The size of erythrocytes and AuNPs are determined experimentally using the DHM by mapping with USAF-1951 high-resolution microscope targets. The obtained results are verified by Field Emission Scanning Electron Microscopy (FESEM) and High-Resolution Transmission Electron Microscopy (HRTEM) measurements.

On the characterization of bias errors in defocusing-based 3D particle tracking velocimetry for microfluidics

In this work, we provide a systematic theoretical and experimental characterization of bias errors in defocusing particle tracking (DPT) methods based on a single calibration function, with respect to microfluidic applications, in which it is not possible to use calibration targets inside the measurement volume. This approach is widely used in microfluidic experiments, but bias errors are often neglected and to date only few works reported empirical procedures to compensate for that. A systematic characterization of the impact of such error in DPT measurements is still lacking. We show that the field curvature aberration and the refractive index mismatch are the main sources of bias error in these applications. We present a correction methodology for the bias error based on the determination of a reference surface, and in addition we propose a procedure based on a reference measurement of a Poiseuille flow to determine the reference surface on microfluidic channels with constant cross section. We discuss the impact of the refractive index mismatch and how to correctly compensate for it. We validated our methodology and quantified the bias errors on 10 different experimental setups, using different working fluids, materials, geometries, and microscope objective lenses ranging from 5times to 40times magnification. Our results indicate that the impact of this type of bias errors is in general not predictable and must be evaluated case by case. The proposed methodology allows to estimate and minimize the bias error in most microfluidic setups and is suitable for any single-camera DPT approach.

Enhancement of Raman signal of monolayer graphene films using a single optical microsphere-assisted Raman microscopic technique

For the first time, we report the enhancement of the Raman scattering signal of monolayer graphene films (MGFs) on Cu foils using a single optical microsphere-assisted Raman microscopic (SOMRM) technique. Initially, the Raman scattering spectra of MGF on Cu foil are recorded using the conventional Raman microscopic (CRM) technique, where the excitation laser is directly focused on the MGFs with the help of a different microscopic objective lens. The obtained spectra are observed to consist of only the low-intensity G and 2D bands but not the D band, known as the disorder or defect band. However, the intensity of all three bands is enhanced significantly using the SOMRM technique. Finally, the numerical investigation is performed on the SOMRM technique to understand the origin of the enhancement of the Raman scattering signal of MGF on the Cu substrates. The role of the substrate for MGF and the radius of the microsphere on the enhancement of the Raman scattering signal of MGFs is also investigated numerically in detail.

SOFFLFM: Super-resolution optical fluctuation Fourier light-field microscopy

Fourier light-field microscopy (FLFM) uses a microlens array (MLA) to segment the Fourier plane of the microscopic objective lens to generate multiple two-dimensional perspective views, thereby reconstructing the three-dimensional (3D) structure of the sample using 3D deconvolution calculation without scanning. However, the resolution of FLFM is still limited by diffraction, and furthermore, it is dependent on the aperture division. In order to improve its resolution, a super-resolution optical fluctuation Fourier light-field microscopy (SOFFLFM) was proposed here, in which the super-resolution optical fluctuation imaging (SOFI) with the ability of super-resolution was introduced into FLFM. SOFFLFM uses higher-order cumulants statistical analysis on an image sequence collected by FLFM, and then carries out 3D deconvolution calculation to reconstruct the 3D structure of the sample. The theoretical basis of SOFFLFM on improving resolution was explained and then verified with the simulations. Simulation results demonstrated that SOFFLFM improved the lateral and axial resolution by more than [Formula: see text] and 2 times in the second- and fourth-order accumulations, compared with that of FLFM.

High-resolution optical recording of bioelectric signals using electrochromic materials.

Non-invasive methods with high spatial and temporal resolution are valuable tools in the study of bioelectric signals and can help uncover how a network of interconnected neurons transmits and processes information. Optical recording using voltage-sensitive fluorescent probes is already used to measure neuronal activity. However, these methods often suffer from photobleaching and phototoxicity and are therefore limited in the long-term monitoring of these activities. We have previously reported on optical electrochromic recording (ECORE), a technique which leverages the electrochromic properties of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) in order to study electrical signals in biological cells. ECORE represents a non-invasive and highly flexible method of detecting such signals while simultaneously enabling the long-term recording of these activities. Here we report on our work to increase the spatial resolution of ECORE by incorporating a microscope objective lens into our setup. Improved spatial resolution will enable us to probe local regions of cells of interest and allow us to study these cells in more detail, adding to the benefits already presented by ECORE.

Lateral resolution enhanced interference microscopy using virtual annular apertures

The lateral resolution in microscopic imaging generally depends on both, the wavelength of light and the numerical aperture of the microscope objective lens. To quantify the lateral resolution Ernst Abbe considered an optical grating illuminated by plane waves. In contrast, the Rayleigh criterion holds for two point sources or point scatterers separated by a lateral distance, which are supposed to emit spherical waves. A portion of each spherical wave is collected by the objective lens and results in an Airy disc corresponding to a diffraction limited intensity point spread function (PSF). If incoherent illumination is employed the intensity PSFs related to different scatterers on an object are added resulting in the well-known Rayleigh resolution criterion. In interference microscopy instead of the intensity the electric field scattered or diffracted by an object will be affected by the transfer function of the optical imaging system. For a reflective object the lateral resolution of an interference microscope can be again characterized by the Abbe limit if the object under investigation is a grating. However, if two irregularities on a flat surface are being imaged the resolution no longer obeys the Rayleigh criterion. Instead, it corresponds to an optical system with an annular aperture and thus surpasses the prediction given by the Rayleigh criterion. This holds true for both, amplitude as well as phase objects, as it will be elucidated in this study by theoretical considerations, simulation results and an experimental proof of principle.

Scalable-resolution structured illumination microscopy.

Structured illumination microscopy suffers from the need of sophisticated instrumentation and precise calibration. This makes structured illumination microscopes costly and skill-dependent. We present a novel approach to realize super-resolution structured illumination microscopy using an alignment non-critical illumination system and a reconstruction algorithm that does not need illumination information. The optical system is designed to encode higher order frequency components of the specimen by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in structured illumination microscopy. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. This algorithm eliminates the need of clean peaks in illumination and the knowledge of illumination patterns, which makes instrumentation simple and flexible for use with a variety of microscope objective lenses. We present a variety of experimental results on beads and cell samples to demonstrate resolution enhancement by a factor of 2.6 to 3.4 times, which is better than the enhancement supported by the conventional linear structure illumination microscopy where the same objective lens is used for structured illumination as well as collection of light. We show that the same system can be used in SIM configuration with different collection objective lenses without any careful re-calibration or realignment, thereby supporting a range of resolutions with the same system.

Methods and instruments for the measurement of numerical aperture for microscope objective lens: A mini review.

Numerical aperture (NA) of objective lens is an important parameter for the design of microscope systems and evaluation of imaging characteristics. The present mini review presents and summarizes the methods and instruments used in the NA measurement of objective lens. Five different categories of methods are introduced, which are original versions of apertometer measurement for angular aperture, method based on the working of Abbe apertometer and its modified versions, geometry-based methods, focal-plane (FP)-imaging-based methods, and back-FP-imaging-based methods, respectively. The methodology, devices, applied scenarios, and characteristics of methods (instruments) are summarized. Finally, some issues and potential areas of application are indicated as well.

Enhancing the optical response and biosensing capabilities of bioinspired peptide micro-waveguides exploiting chromatic aberration.

Bioinspired peptide waveguides of mesoscopic length scales have established a new paradigm in photonics with possible applications in precision bioimaging, sensing, and diagnostics. Here, we improve the efficiency of coupling various constituent colors of a white light source into single self-assembled microtube-shaped passive peptide waveguides by employing chromatic aberration. Thus, we use a chromatically aberrated microscope objective lens to couple light into peptide waveguides. Using both numerical simulation and experiments, we show that the waveguide response displays higher quality factor, wavelength selectivity, and axial coupling range compared to a chromatically corrected standard plan-fluoritic objective lens. We also demonstrate absorption and refractive index-based sensing by studying the changes in the optical responses of the peptide tubes in the presence of a wide concentration range of the absorptive Congo red, and the nonabsorptive Coumarin dyes. The former understandably display a much higher response than the latter due to the low finesse of the waveguides. We obtain a detection limit of around 10nM for Congo red, and 10 mM for Coumarin. Our study opens up possibilities for deploying such peptide microtubes for various biosensing applications utilizing spectral and waveguide characteristics.

Demystifying speckle field interference microscopy

Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in terms of phase sensitivity, and spatial and temporal resolution compared to conventional light source illumination. To date, despite conspicuous advantages, it has not been extensively implemented in the field of phase imaging due to inadequate understanding of interference fringe formation, which is challenging to obtain in dynamic speckle illumination interference microscopy (DSI-IM). The present article provides the basic understanding of DSI through both simulation and experiments that is essential to build interference microscopy systems such as quantitative phase microscopy, digital holographic microscopy and optical coherence tomography. Using the developed understanding of DSI, we demonstrated its capabilities which enables the use of non-identical objective lenses in both arms of the interferometer and opens the flexibility to use user-defined microscope objective lens for scalable field of view and resolution phase imaging. It is contrary to the present understanding which forces us to use identical objective lenses in conventional IM system and limits the applicability of the system for fixed objective lens. In addition, it is also demonstrated that the interference fringes are not washed out over a large range of optical path difference (OPD) between the object and the reference arm providing competitive edge over low temporal coherence light source based IM system. The theory and explanation developed here would enable wider penetration of DSI-IM for applications in biology and material sciences.