Microscope Optics Research Articles

Plug-and-play adaptive optics microscopy with full-field correction with isoplanatic patch estimation and field segmentation

Plug-and-play adaptive optics microscopy with full-field correction with isoplanatic patch estimation and field segmentation

Detectability of unresolved particles in off-axis digital holographic microscopy.

Off-axis digital holographic microscopy (DHM) provides both amplitude and phase images, and so it may be used for label-free 3D tracking of micro- and nano-sized particles of different compositions, including biological cells, strongly absorbing particles, and strongly scattering particles. Contrast is provided by differences in either the real or imaginary parts of the refractive index (phase contrast and absorption) and/or by scattering. While numerous studies have focused on phase contrast and improving resolution in DHM, particularly axial resolution, absent have been studies quantifying the limits of detection for unresolved particles. This limit has important implications for microbial detection, including in life-detection missions for space flight. Here we examine the limits of detection of nanosized particles as a function of particle optical properties, microscope optics (including camera well depth and substrate), and data processing techniques and find that DHM provides contrast in both amplitude and phase for unresolved spheres, in rough agreement with Mie theory scattering cross-sections. Amplitude reconstructions are more useful than phase for low-index spheres and should not be neglected in DHM analysis.

Comparative Optics of the Surgical Microscope and Rigid Endoscopes in Neurosurgery.

This chapter is intended to provide a brief overview of the optics of surgical microscopes and rigid endoscopes, with the aim of providing the reader with the principles dictating the nature of surgical visualization when either of the visual control systems is used. It is not by any means geared toward elaborating on the detailed optical physics of these systems, which is beyond the scope and objective of this chapter.

Adaptive Optics Microscopy with Wavefront Sensing Based onNeighbor Correlation.

Complex structures in living cells and tissues induce wavefront errors when light waves pass through them, and images observed with optical microscopes are undesirably blurred. This problem is especially serious for living plant cells because images are strikingly degraded even within a single cell. Adaptive optics (AO) is expected to be a solution to this problem by correcting such wavefront errors, thus enabling high-resolution imaging. In particular, scene-based AO involves wavefront sensing based on the image correlation between subapertures in a Shack-Hartmann wavefront sensor and thus does not require an intense point light source. However, the complex 3D structures of living cells often cause low correlation between subimages, leading to loss of accuracy in wavefront sensing. This paper proposes a novel method for scene-based sensing using only image correlations between adjacent subapertures. The method can minimize changes between subimages to be correlated and thus prevent inaccuracy in phase estimation. Using an artificial test target mimicking the optical properties of a layer of living plant cells, an imaging performance with a Strehl ratio of approximately 0.5 was confirmed. Upon observation of chloroplast autofluorescence inside living leaf cells of the moss Physcomitrium patens, recovered resolution images were successfully obtained even with complex biological structures. Under bright-field illumination, the proposed method outperformed the conventional method, demonstrating the future potential of this method for label- and damage-free AO microscopy. Several points for improvement in terms of the effect of AO correction are discussed.

Comparative Analysis of Cooling Methods for Dynamic Infrared Thermography (DIRT)-Based Skin Cancer Diagnosis

Skin cancer is a significant global health issue, placing a growing burden on individuals and society. Conventional diagnostic methods like visual examination and biopsy have limitations in invasiveness and accuracy. As a result, alternative tools such as infrared thermography have gained attention in skin cancer diagnosis. Tissue-mimicking phantoms have been instrumental in facilitating research in this field, offering controlled environments. While they do not fully replicate human skin complexity, physical skin models provide stability, ease of fabrication, and control over properties. Agarose phantoms are employed in this study. This research focused on testing and comparing cooling techniques for human skin in the context of skin cancer diagnosis using dynamic infrared thermography. Six cooling methods were investigated: a cool pack, an aluminum medal, ice, alcohol, a vortex cooler and a Zimmer Cryo 6 cooler. The experimental setup involved an infrared camera (Optris Xi400) with microscope optics positioned above an agar phantom mimicking flat skin and an ulcerating skin lesion. Based on experiments conducted on the skin phantom, it was observed that convective cooling methods offered more consistent and uniform cooling. Conversely, conductive methods proved effective for flat objects but posed challenges in achieving uniform cooling for bulging skin or ulcerated lesions. Ice or alcohol were deemed unsuitable due to artifacts influencing the infrared radiation and thermal camera view. A decision matrix assessed cooling techniques based on criteria such as uniformity, repeatability, view obstruction, efficiency, workload, patient comfort, clinical suitability, noise exposure, consumables, additional equipment, and price. The Zimmer Cryo 6 cooler emerged as the most suitable cooling method after evaluating various factors.

Valley photonic crystal waveguides fabricated with CMOS-compatible process

Valley photonic crystal (VPhC) waveguides have attracted much attention because of their ability to enable robust light propagation against sharp bends. However, their demonstration using a CMOS-compatible process suitable for mass production has not yet been reported at the telecom wavelengths. Here, by tailoring the photomask to suppress the optical proximity effect, VPhC patterns comprising equilateral triangular holes were successfully fabricated using photolithography. We optically characterized the fabricated VPhC devices using microscopic optics with NIR imaging. For comparison, we also fabricated and characterized line-defect W1 PhC waveguides, in which the transmission intensities decreased at some regions within the operating bandwidth when sharp turns were introduced into the waveguide. In contrast, the developed VPhC waveguides can robustly propagate light around the C-band telecommunication wavelengths, even in the presence of sharp bends. Our results highlight the potential of VPhC waveguides as an interconnection technology in silicon topological photonic ICs.

Deflectometry for specular surfaces: an overview

Deflectometry as a technique to assess reflective surfaces has now existed for some 40 years. Its different aspects and variations have been studied in multiple theses and research articles; reviews are available for certain subtopics. Still a field of active development with many unsolved problems, deflectometry now encompasses a large variety of application domains, hardware setup types, and processing workflows for different purposes, and spans a range from qualitative defect inspection of large vehicles to precision measurements of microscopic optics. Over these years, many exciting developments have accumulated in the underlying theory, in the systems design, and in the implementation specifics. This diversity of topics is difficult to grasp for experts and non-experts alike and may present an obstacle to a wider acceptance of deflectometry as a useful tool for research and industrial applications. This paper presents an attempt to summarize the status of deflectometry and to map relations between its notable branches. Its aim is to provide a communication basis for experienced practitioners and also to offer a convenient entry point for those interested in learning about the method. The list of references introduces some prominent trends and established research groups in order to facilitate further self-directed exploration.

Voxelotor does not inhibit sickle hemoglobin fiber formation upon complete deoxygenation

The drug voxelotor (commercially known as Oxbryta) has been approved by the US Food and Drug Administration for the treatment of sickle cell disease. It is known to reduce disease-causing sickling by inhibiting the transformation of the non-polymerizing, high-oxygen-affinity R quaternary structure of sickle hemoglobin into its polymerizing, low-affinity T quaternary structure. It has not been established whether the binding of the drug has anti-sickling effects beyond restricting the change of quaternary structure. By using a laser photolysis method that employs microscope optics, we have determined that fully deoxygenated sickle hemoglobin will assume the T structure. We show that the nucleation rates essential to generate the sickle fibers are not significantly affected by voxelotor. The method employed here should be useful for determining the mechanism of sickling inhibition for proposed drugs.

Realizing the Full Potential of Advanced Microscopy Approaches for Interrogating Plant-Microbe Interactions

Microscopy has served as a fundamental tool for insight and discovery in plant-microbe interactions for centuries. From classical light and electron microscopy to corresponding specialized methods for sample preparation and cellular contrasting agents, these approaches have become routine components in the toolkit of plant and microbiology scientists alike to visualize, probe and understand the nature of host-microbe relationships. Over the last three decades, three-dimensional perspectives led by the development of electron tomography, and especially, confocal techniques continue to provide remarkable clarity and spatial detail of tissue and cellular phenomena. Confocal and electron microscopy provide novel revelations that are now commonplace in medium and large institutions. However, many other cutting-edge technologies and sample preparation workflows are relatively unexploited yet offer tremendous potential for unprecedented advancement in our understanding of the inner workings of pathogenic, beneficial, and symbiotic plant-microbe interactions. Here, we highlight key applications, benefits, and challenges of contemporary advanced imaging platforms for plant-microbe systems with special emphasis on several recently developed approaches, such as light-sheet, single molecule, super-resolution, and adaptive optics microscopy, as well as ambient and cryo-volume electron microscopy, X-ray microscopy, and cryo-electron tomography. Furthermore, the potential for complementary sample preparation methodologies, such as optical clearing, expansion microscopy, and multiplex imaging, will be reviewed. Our ultimate goal is to stimulate awareness of these powerful cutting-edge technologies and facilitate their appropriate application and adoption to solve important and unresolved biological questions in the field. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Non-Decimated Wavelet Transform and Vector Quantization for Lossy Medical Images Compression

This study presents a new approach for lossy medical image compression using vector quantization. Recently, the digital image has been a reliable replacement for a hard copy of medical images, therefore, an effort has been made to ensure maintaining high-quality images to use for archiving, classification, or automated diagnostics support. Although the medical application contains all sorts of the images like microscopic, X-rays, tomography, and fiber optics imaging by angioplasty, all of this comes at the cost of using digital storage that needs to be regularly backed up and maintained and to help minimize the need for larger storage media, this study is focusing on applying Non-Decimated Wavelet Transform (NDWT) and combined lossy and lossless compression techniques that will allow the images to take much smaller storage space while maintaining the high level of quality for these images. This study is focusing on chest X-ray images compression using a combination of lossy compression techniques using two Vector Quantization (VQ) algorithms such as k-means clustering and Linde, Buzo, and Gray (LBG) algorithm, and three lossless compression techniques such as Arithmetic Coding (AC), Run Length Encoding (RLE) and Huffman Coding (HC) and choose the optimum combination of them. Then, the performance is measured using Compression Ratio (CR), processing time, or called run time, Peak Signal to Noise Ratio (PSNR), and Bit Rate.

Low-aberration ExB deflector optics for scanning electron microscopy.

To suppress aberrations in the signal electron optics of a scanning electron microscope, we propose ExB deflector (deflector with superimposed electric and magnetic fields) optics that cancel the aberrations generated during large-angle deflection. This improves the resolution of the angle or position of the signal electrons on the sample surface, allowing them to be discriminately detected. The proposed optics consist of two ExB deflectors and a transfer system with two 4-f systems, or systems that have four times the focal length, placed between them. This configuration maintains the symmetry of the electron beam trajectory throughout the transfer system such that aberrations generated by the first ExB deflector are negated by the second. The effect of the proposed optics was confirmed using a ray-tracing simulation of the electron beam, and the aberration was reduced to at most one-tenth of that in the case with only one ExB deflector. Furthermore, as an example, we examined the implementation of the proposed ExB deflector optics to resolve the signal electron angle and found that the sample emission angle range of 80° can be resolved with an angular resolution of 1°. Therefore, the proposed ExB deflector optics can be applied to the signal electron optics of a scanning electron microscope to improve the resolution of the signal electrons.

Synthetic Micrographs of Bacteria (SyMBac) allows accurate segmentation of bacterial cells using deep neural networks

BackgroundDeep-learning–based image segmentation models are required for accurate processing of high-throughput timelapse imaging data of bacterial cells. However, the performance of any such model strictly depends on the quality and quantity of training data, which is difficult to generate for bacterial cell images. Here, we present a novel method of bacterial image segmentation using machine learning models trained with Synthetic Micrographs of Bacteria (SyMBac).ResultsWe have developed SyMBac, a tool that allows for rapid, automatic creation of arbitrary amounts of training data, combining detailed models of cell growth, physical interactions, and microscope optics to create synthetic images which closely resemble real micrographs, and is capable of training accurate image segmentation models. The major advantages of our approach are as follows: (1) synthetic training data can be generated virtually instantly and on demand; (2) these synthetic images are accompanied by perfect ground truth positions of cells, meaning no data curation is required; (3) different biological conditions, imaging platforms, and imaging modalities can be rapidly simulated, meaning any change in one’s experimental setup no longer requires the laborious process of manually generating new training data for each change. Deep-learning models trained with SyMBac data are capable of analysing data from various imaging platforms and are robust to drastic changes in cell size and morphology. Our benchmarking results demonstrate that models trained on SyMBac data generate more accurate cell identifications and precise cell masks than those trained on human-annotated data, because the model learns the true position of the cell irrespective of imaging artefacts. We illustrate the approach by analysing the growth and size regulation of bacterial cells during entry and exit from dormancy, which revealed novel insights about the physiological dynamics of cells under various growth conditions.ConclusionsThe SyMBac approach will help to adapt and improve the performance of deep-learning–based image segmentation models for accurate processing of high-throughput timelapse image data.

4D Direct Laser Writing of Submerged Structural Colors at the Microscale.

Biomimetic stimuli-responsive structure colors (SCs) can improve the visualization and identification in the micro functional structure field such as information encryption/decryption and smart actuators. However, it is still challenging to develop the ability to 4D print arbitrary submerged colorful patterns with stimuli-responsive materials at the microscale. Herein, a hydrogel photoresist with feature resolution (98nm) for the fabrication of 4D microscopic SCs by the femtosecond direct laser writing method is developed. The 4D printed woodpile SCs are grouped as pixel palettes with various laser parameters and they spanned almost the entire color space. The coloring mechanism of diffraction gratings is not only investigated by optics microscopy and spectroscopy but also supported by simulation. Moreover, the 4D printed hydrogel-integrated amphichromatic fish constructions and pixelated painting can visually discolor reversibly by regulating the solution pH. This finding promises an ideal coloring method for sensors, anti-counterfeiting labels, and transformable photonic devices.

Optical encryption in spatial frequencies of light fields with metasurfaces

Optical encryption has attracted attention recently as information security becomes important in modern society. For most encryption methods based on metasurfaces, however, information is encrypted into the metasurfaces of physical forms, limiting information sharing and compatibility with digital information processing technology. Here, we demonstrate an alternative scheme in which information is encrypted on optical images other than metasurfaces, releasing the constraints mentioned above. This is achieved by modulating the spatial frequencies of optical images with metasurfaces. Specifically, information encryption and decryption based on the intensity or phase of spatial frequency have been demonstrated with the developed microscopic Fourier optics technique. The abundance of spatial frequency also promises a flexible multiplexing platform for increasing information capacity, which has been verified by a 4-bit encryption experiment. These findings could enable high-security and high-capacity optical encryption platforms.

Pilot study of fluorescence imitating brightfield imaging for rapid, slide-free dermatopathology.

Fluorescence imitating brightfield imaging (FIBI) is a novel alternative microscopy method that can image freshly excised, non-sectioned tissue. We examine its potential utility in dermatopathology by examining readily available specimens embedded in paraffin blocks. Nine skin samples embedded in paraffin blocks were superficially deparaffinized using xylene and ethanol and stained with H&E. FIBI captured tissue surface histopathology images using simple microscope optics and a color camera. We then applied deep-learning-based models to improve resemblance to standard H&E coloration and contrast. FIBI images were compared with corresponding standard H&E slides and concordance was assessed by two dermatopathologists who numerically scored epidermal and dermal structure appearance and overall diagnostic utility. Dermatopathologist scores indicate that FIBI images are at least equivalent to standard H&E slides for visualizing structures such as epidermal layers, sweat glands, and nerves. Images acquired with FIBI are comparable to traditional H&E-stained slides, suggesting that this rapid, inexpensive, and non-destructive microscopy technique is a conceivable alternative to standard histopathology processes especially for time-sensitive procedures and in settings with limited histopathology resources.

810 FIBI (Fluorescence Imitating Brightfield Imaging) for rapid, slide-free dermatopathology

Background: Fluorescence Imitating Brightfield Imaging (FIBI) is a novel alternative microscopy method that can image freshly excised, non-sectioned tissue.We examine its potential utility in dermatopathology. Methods: Five skin samples embedded in paraffin blocks were superficially deparaffinized using xylene and ethanol and then briefly stained with hematoxylin and eosin (H&E). FIBI captured tissue surface histology images using simple microscope optics and a color camera. Subsequently, we applied deep learning-based models to improve resemblance to standard H&E coloration and contrast.

Does Oncocalyxone A (oncoA) have intrinsic fluorescence?

BackgroundOncocalyxone A (oncoA) is a quinone extracted from the Cordia oncocalyx plant. This compound has pharmacological properties, such as anti-inflammatory, analgesic, and cytotoxic activities, among others. OncoA presents a similar chemical structure to doxorubicin, a drug used in cancer treatment, which possesses an intrinsic fluorescence explored in various studies, including those using doxorubicin-loaded nanoparticles. Thus, due to the chemical structural similarity, the question arose whether oncoA could also show autofluorescence. Therefore, this study proposed to characterize the absorption and emission spectral profiles of oncoA and analyze if this compound could be used as a fluorescent probe. MethodsFor this, fucoidan-coated polyisobutylcyanoacrylate (PIBCA) nanoparticles containing oncoA were prepared, and an uptake study was performed using a human metastatic breast cancer cell line (MDA-MB-231 cells). ResultsOncoA presented a maximum emission wavelength in the blue region, near 430 nm, at 350 nm excitation, compatible with standard microscope optics. Fluorescence microscopy analyses showed that oncoA-loaded PIBCA nanoparticles were internalized by MDA-MB-231 cells under incubation times as shorter as 15 min. ConclusionAccording to these findings, oncoA-encapsulated nanoparticles are promising fluorescent probes and could be useful for cellular uptake studies.

Digital 3D "Heads-up" Cataract Surgery: Safety Profile and Comparison with the Conventional Microscope System.

The aim of this study was to compare the complication rates between surgery performed using digital heads-up 3Dsystem (3Dgroup) and a conventional binocular microscope-based system (BM group) in a large series of cataract operations performed by the same surgeon. This retrospective analysis included a consecutive series of 2,000 cataract operations. The 3Dgroup included n = 1,000 operations performed immediately following the introduction of a 3Dsystem (Alcon Ngenuity). For comparison, the last n = 1,000 operations performed with a binocular microscope were included in the BM group. The 3Dsystem was adapted to the existing microscope so that the microscope optics remained unchanged. The In both groups, the surgical techniques used were either phacoemulsification or femtosecond laser cataract surgery. Complications were recorded and analyzed retrospectively. The proportion of femto-laser cataract operations was 19.8% in the 3Dgroup and 18.6% in the BM group. Capsule rupture occurred in 10 eyes (3D: n = 4 (0.4%), anterior vitrectomy: n = 2, pars plana vitrectomy: n = 1; BM: n = 6 cases (0.6%), anterior vitrectomy: n = 4, pars plana vitrectomy: n = 1). A short-term iris prolapse occurred in 3 eyes (3D: n = 2, BM: n = 1). Zonulolysis occurred in 2 eyes (3D: n = 1, BM: n = 1). Overall, there was no statistically significant difference between the two groups (p > 0.5). There was no significant increase in the duration of surgery following the switch to the 3Dtechnique. In a large series of 2000 eyes, there was no significant difference between 3D and BM surgery in terms of the safety profile during cataract surgery. 3D surgery can, therefore, be used for cataract operations without additional risk.

Fusion enhancement of hollow tungsten arc coaxially assisted by fiber laser

Bead-on-plate experiment was performed on a continuous-mode fiber-laser hollow tungsten coaxial welding platform designed and built by us. The weld bead cross-section was observed by using a optics microscope, and the welding arc image was recorded by using a high-speed camera to track the welding process. The changes in the arc physical field after laser addition were analyzed by conducting numerical simulations to explore the mechanism of increasing the welding depth. The results showed that the coaxial hybrid of fiber laser and hollow tungsten arc can significantly increase the weld depth and thermal efficiency of hybrid arc. When the welding current remained constant at 70 A, the weld penetration increased with an increase in the laser power. However, the growth rate was different under different laser power settings and there were the upper and lower critical values of laser power which were 450 W and 60 W. When the laser power remained constant at 400 W, the weld penetration first increased and then remained constant with an increase in the current, and there was a current critical value of 170 A. The main reasons for the increase in the penetration depth were the increase in the arc temperature, arc pressure and thermal efficiency of hybrid welding.

Microfluidic flow assembly system with magnetic clamp for unlimited geometry in millimetric hydrogel film patterning

Polymeric sheets with geometrically selective patterns and functions can be used for many labeling and sensing applications. Conventional fabrication techniques such as templated-based printing require cumbersome, multiple and intensive steps, pose difficulty towards automation, and thus are mostly applied for simple array patterned sheets. Alternative microfluidics-based methods, such as flow lithography is limited to creating parallel patterns. We develop a microfluidic assembly fabrication based on flow lithography to make millimetric polymeric sheets with geometrically complex and selective patterns. This new fabrication is empowered by integrating a magnetically actuated clamp in the microfluidic channel. The integration of this magnetic clamp in combination of flow lithography allows rapid flow-assisted exchanges of multiple compositions during sequence patterning. Resolution is limited only by the quality of the UV photolithography source and the microscope optics. Using our method, we demonstrate the synthesis of enclosed geometrically complex hydrogels film with the inclusion of choice chemical functionalizations for pH and temperature sensing, fluorescent markers and for biomolecule sensing, fluorescence and enzymatic activity.