Commercial Microscope Research Articles

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

We discuss the implementation and performance of a plug-play adaptive optics (AO) module for commercial microscopes comprising indirect wavefront sensing, and a deformable phase plate (DPP) located directly between the objective and the turret. With the DPP at this location, the system closely resembles a pupil-AO scheme, in which effective aberration correction is only possible within the isoplanatic patch. We overcome this limitation by estimating the aberration profiles at multiple field points in parallel and correcting them in sequence to obtain a 2D array of high-quality sub-aperture images. These are then stitched together to form a corrected full-field image. To minimize the measurement time without compromising correction quality, we propose an empirical method to identify the size of the isoplanatic patch, which is both sample and system dependent. Matching the field segment size to that of the isoplanatic patch provides the best compromise between consistent correction quality across the image and measurement time. We demonstrate the performance of the developed system in a commercial microscope using synthetic samples and discuss the performance and limitations of the system.

Adaptive optical correction in in vivo two-photon fluorescence microscopy with neural fields.

Adaptive optics (AO) techniques are designed to restore ideal imaging performance by measuring and correcting aberrations originating from both the microscope system and the sample itself. Conventional AO methods require additional hardware, such as wavefront sensors and corrective devices, for aberration measurement and correction, respectively. These methods often necessitate microscopes to adhere to strict design parameters, like perfect optical conjugation, to ensure the accurate delivery of corrective patterns for wavefront correction using corrective devices. However, in general microscope systems, including commercially available ones, conjugation errors are more prone to arise due to incomplete conjugation among optical components by design and misalignment of the components, coupled with their limited access and adjustability, which hinders the rigorous integration of AO hardware. Here, we describe a general-purpose AO framework using neural fields, NeAT, that is applicable to both custom-built and commercial two-photon fluorescence microscopes and demonstrate its performance in various in vivo imaging settings. This framework estimates wavefront aberration from a single 3D two-photon fluorescence image stack, without requiring external datasets for training. Additionally, it addresses the issue of incomplete optical conjugation by estimating and correcting any conjugation errors, which enables more accurate aberration correction by the corrective device. Finally, it jointly recovers the sample's 3D structural information during the learning process, potentially eliminating the need for hardware-based AO correction. We first carefully assess its aberration estimation performance using a custom-built two-photon fluorescence microscope equipped with a wavefront sensor which provides the ground truth aberration for comparison. We further characterize and assess the robustness of the aberration estimation to image stacks with low signal-to-noise ratios, strong aberration, and motion artifacts. As practical applications, using a commercial microscope with a spatial light modulator, we first demonstrate NeAT's real-time aberration correction performance in in vivo morphological imaging of the mouse brain. We further show its performance in in vivo functional activity imaging of glutamate and calcium dynamics within the mouse brain.

Portable, low-cost, desktop microscope

Light microscopes became essential tools in everyday lab work a long time ago. However, most commercial microscopes are costly, and they are often bulky and heavy. Therefore, microscopes are rarely seen in mobile applications or used by interested amateurs. Here, we present an affordable, portable single-lens microscope. It essentially uses a Raspberry Pi single-board computer, a camera, a touchscreen display, and an LED ring at its core. Apart from brightfield microscopy, contrast-enhancing methods by oblique, dark-field, and Rheinberg illumination are possible, as well. The microscope is ideal for applications that do not require high-end optical components. Due to its low cost and flexible use, it is also suitable for hands-on experiences of the fascinating world not visible by the human eye.

Super-resolved fluorescence imaging utilising accessible stochastic optical reconstruction microscopy (easySTORM) implemented on a low-cost, modular open-source (openFrame) microscope

Abstract Optical super-resolution microscope is a powerful tool for the life sciences, including cell biology and pathology yielding information on, e.g., biological structure and protein distribution in the nano-scale range. In recent years, there has been an exponential rise in the interest to apply super-resolution microscopes in diverse areas, but its wider utility is hindered by the cost of purchasing and maintaining super-resolved microscopes. In this paper we present the implementation of easySTORM, an accessible implementation of stochastic optical reconstruction microscopy (STORM) at Indian Institute of Technology Guwahati (IITG), India, in a flexible, user friendly and cost-effective manner using a state-of-the-art, modular, open-source, optical microscope platform called: "openFrame". Providing comparable imaging performance to commercial optical super-resolution microscopes, the openFrame-based implementation of easySTORM uses in-expensive multimode diode lasers and industry grade CMOS cameras, and the open-source and modular nature of the instrument makes it easy to maintain and to upgrade. To demonstrate its successful implementation at IITG, we image quantum dots and actin-tubulin structure in both normal and cancer cells, resolved features separated by a few tens of nanometers. This work demonstrates that openFrame-enabled easySTORM instrumentation can be widely accessible to provide affordable, research grade super-resolution microscopy capability for academic and medical research.

Conjugate adaptive optics extension for commercial microscopes

Conjugate adaptive optics extension for commercial microscopes

Full-field optical coherence microscopy enables high-resolution label-free imaging of the dynamics of live mouse oocytes and early embryos

High quality label-free imaging of oocytes and early embryos is essential for accurate assessment of their developmental potential, a key element of assisted reproduction procedures. To achieve this goal, we propose full-field optical coherence microscopy (FF-OCM), constructed as a compact module fully integrated with a commercial wide-field fluorescence microscope. Our system achieves optical sectioning in wide-field, high in-plane resolution of 0.5 µm, and high sensitivity to backscattered light. To demonstrate its imaging capabilities, we study live mouse oocytes and embryos at all important stages of meiotic maturation and early embryogenesis. Our system enables visualization of intracellular structures, which are not visible in common bright-field microscopy, i.e., internal structure of nuclear apparatus, cytoskeletal filaments, cellular cortex, cytoplasmic protrusions, or zona pellucida features. Additionally, we visualize and quantify intracellular dynamics like cytoplasmic stirring motion, nuclear envelope fluctuations and nucleolus mobility. Altogether, we demonstrate that FF-OCM is a powerful tool for research in developmental biology that also holds great potential for non-invasive time-lapse monitoring of oocyte and embryo quality in assisted reproduction.

A customizable digital holographic microscope

We propose a compact, portable, and low-cost holographic microscope designed for the characterization of micrometric particles suspended in a liquid. This system is built around a commercial optical microscope by substituting its illumination source (a light-emitting diode) with a collimated laser beam. Similarly, a quartz flow cell replaces the microscope glass slide using a 3D-printed custom mount. With the hardware presented in this paper, the holographic imaging of the electromagnetic fields emitted by the particles that intercept the laser beam achieves a resolution close to that of optical microscopes but with a greater depth of field. Several morphological and optical features can be extracted from the holograms, including particle projected section, aspect ratio, and extinction cross-section. Additionally, we introduce a remote system control that enables users to process the acquired holograms on a remote computational device. This work provides a comprehensive description of the methodology of image processing in holographic microscopy and a series of validation measurements conducted using calibrated particles. This technique is suitable for the characterization of airborne particles found in snow, firn, and ice; here we report experimental results obtained from Alpine ice cores.

Correlative Microscopy Analysis for Battery Materials

Lithium batteries (LB) are in high demand for a diverse range of applications, such as automotive industry, consumer electronics, wearable and medical devices, grid storage, etc. Each application demands a specific set of performance advantages, such as high capacity and/or energy density, long cycle life, superior electrochemical stability. Therefore, there is no one-size-fits-all for a LB chemistry and its form factor. Battery performance advantages rely on optimization of the battery components, including active materials, electrolyte, separator, binder system, and current collectors. For meaningful device optimization, electrochemical battery testing needs to be combined with a deep understanding of the material and device microstructure. Physical and chemical characterization on the material- and electrode-level is needed to establish root-cause relationships with battery performance. Properties of the interfaces between layers of the battery components often define the device characteristics. Critical information is usually buried beneath the surface of a material specimen or encapsulated in a closed-form device. Microstructural defect identification in the battery needs to be followed by a targeted defect study. Different battery form factors and chemistries make it hard to streamline root-cause analysis.Recently, a commercial focused-ion beam scanning electron microscope was developed with an integrated fs-laser mill attached to the load lock of the microscope, opening the door to new and more powerful analysis approaches in energy materials research. Applications include rapid access to deeply buried structures for high resolution imaging and analytics, large area cross-section preparation, and massive material ablation for sample preparation of structures; e.g. FIB-SEM tomography, TEM, APT, or X-ray nanoCT. Additionally, each of these methods may be correlatively guided by prior imaging, with techniques like 3D X-ray microscopy (XRM), to enable targeted analysis and preparation. The non-destructive nature of 3D XRM makes it an ideal tool for microstructural defect identification in LB. Laser mill allows researchers to combine non-destructive X-ray defect identification with electron microscopy methods for targeted defect characterization. This approach can be used for various battery types and form factors.By combining the laser mill with 3D X-ray microscopy, this platform enables comprehensive analysis of energy materials at the nanoscale. We illustrate these concepts with application examples, and use cases demonstrating the utility of the approach.

Segmented fluorescence correlation spectroscopy (FCS) on a commercial laser scanning microscope

Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.

Photoluminescence studies of different types of feldspars and the implications to the dating application using a Raman system

In recent years, the infrared photoluminescence (PL) of feldspar has been explored for geological dating because it offers a great increase in signal intensity with increasing radiation dose. However, a comprehensive understanding of the PL emission spectra of different types of feldspar minerals remains poorly understood and the uncommonly available instruments have hindered its research. In this study, we investigated PL characteristics (e.g., emission spectra and the dependence of PL emission on irradiation dose) of single-crystal alkali and plagioclase feldspars using a commercial Raman microscope instrument configured to excite samples with 532 and 785 nm lasers. The results show that despite being sample-dependent, 2–3 PL bands peaking at ∼2.10 eV (590 nm), 1.75 eV (710 nm), and 1.43 eV (865 nm) under 532 nm laser excitation, and 0–2 peaks ranging from ∼1.44 eV (863 nm) to ∼1.34 eV (925 nm) under 785 nm laser excitation, are broadly observed. These variations in PL bands may be attributed to subtle differences in chemical composition and crystal structure among the feldspar minerals. The sensitivity of PL emission to irradiation dose varies greatly depending on feldspar types and peak positions. The PL intensities of the peaks at ∼1.43 eV (865 nm) are particularly sensitive in K-feldspar, demonstrating their potential applicability for dating applications. Additionally, solar bleaching experiments demonstrate that PL signals of these peaks can be effectively reset by 1.5 h of solar exposure. The dose-response curves obtained using 860–870 nm PL signals of K-feldspar conform to a relationship of a single saturating exponential function between the signal and irradiation dose. Furthermore, this study demonstrates that a commonly available Raman system can be utilized for PL measurements of single grains.

Determination of the Localized Surface Plasmon Resonance Alteration of AgNPs via Multiwavelength Evanescent Scattering Microscopy for Pb(II) Detection.

We developed multiwavelength evanescent scattering microscopy (MWESM), which can acquire plasmonic nanoparticle images at the particle level using the evanescent field as the incident source and distinguish different LSPR (localized surface plasmon resonance) spectral peaks among four wavelengths. Our microscope could be easily and simply built by modifying a commercial total internal reflection fluorescence microscope (TIRFM) with the substitution of a beamsplitter and the addition of a semicircular stop. The ultrathin depth of illumination and rejection of the reflected incident source together contribute to the high sensitivity and contrast of single nanoparticle imaging. We first validated the capability of our imaging system in distinguishing plasmonic nanoparticles bearing different LSPR spectral peaks, and the results were consistent with the scattering spectra results of hyperspectral imaging. Moreover, we demonstrated high imaging quality from the aspects of the signal/noise ratio and point spread function of the single-particle images. Meaningfully, the system can be utilized in rapidly determining the concentration of toxic lead ions in environmental and biological samples with good linearity and sensitivity, based on single-particle evanescent scattering imaging through the detection of the alteration of the LSPR of silver nanoparticles. This system holds the potential to advance the field of nanoparticle imaging and foster the application of nanomaterials as sensors.

Step height measurement of monoatomic silicon crystal lattice steps with a commercial atomic force microscope

Abstract Precise control of advanced materials relies on accurate dimensional metrology at the sub-nanometre scale. At this scale, the accuracy of scanning probe microscopy (SPM) has been limited by the lack of traceable transfer standard artefacts with calibration structures of suitable dimensions. With the adoption in 2019 of the silicon crystal lattice spacing as a secondary realization of the metre in the International System of Units (SI), SPM users have direct access to a realization of the SI metre at the sub-nanometre level by means of the step height of self-assembled monatomic lattice steps that can form on the surface of silicon crystals. A key challenge of successfully adopting this pathway is establishing the need for established protocols to minimize measurement errors and artifacts in routine laboratory use.
In this study, step height measurements of monoatomic lattice steps in an ordinal/staircase structure on a Si(111) crystal surface have been derived from images acquired with a commercially available, research-level atomic force microscope (AFM). Measurement results derived from AFM images using three different SPM image processing and analysis software packages are compared. Significant sources of measurement uncertainty are identified; principally the contribution from the dependence on scan direction. The calibration of the
AFM derived from this measurment was used to traceably measure the sub-nanometre lattice steps on a silicon carbide crystal surface to demonstrate the viability of this calibration pathway.

Feasible and economic DIY procedures of a hand-held fluorescence detector set-FluorDetector to assist laboratory course development.

Fluorescence-related experimental techniques play an important role in biochemistry, molecular biology, and cell biology. However, fluorescence-related experiments are rarely included in the laboratory courses of most Chinese universities. This is mainly due to the conflict between large class size (50-60 students in one room) and funding/space limitations to purchase and accommodate enough fluorescence detection equipment. Here, we proposed feasible and economical Do It Yourself (DIY) procedures of a hand-held fluorescence detector set-FluorDetector to support the development of laboratory courses. Tested on several samples, clear fluorescence signals could be directly observed by FluorDetector and photographed with a smartphone. In addition, FluorDetector was able to turn a conventional stereomicroscope into a fluorescence stereomicroscope, detecting fluorescence signals with clean background. FluorDetector is easy to make with a 3D printer, with an extremely low cost ($200 each) when compared with a commercial fluorescence microscope or fluorescence stereomicroscope, and almost as sensitive as a microplate reader in measuring fluorescence. Therefore, FluorDetector is a possible strategy to solve the problem and help to integrate fluorescence-related experimental modules in laboratory courses.

Web-based diagnostic platform for microorganism-induced deterioration on paper-based cultural relics with iterative training from human feedback

PurposePaper-based artifacts hold significant cultural and social values. However, paper is intrinsically fragile to microorganisms, such as mold, due to its cellulose composition, which can serve as a microorganisms’ nutrient source. Mold not only can damage papers’ structural integrity and pose significant challenges to conservation works but also may subject individuals attending the contaminated artifacts to health risks. Current approaches for strain identification usually require extensive training, prolonged time for analysis, expensive operation costs, and higher risks of secondary damage due to sampling. Thus, in current conservation practices with mold-contaminated artifacts, little pre-screening or strain identification was performed before mold removal, and the cleaning techniques are usually broad-spectrum rather than strain-specific. With deep learning showing promising applications across various domains, this study investigated the feasibility of using a convolutional neural network (CNN) for fast in-situ recognition and classification of mold on paper.MethodsMolds were first non-invasively sampled from ancient Xuan Paper-based Chinese books from the Qing and Ming dynasties. Strains were identified using molecular biology methods and the four most prevalent strains were inoculated on Xuan paper to create mockups for image collection. Microscopic images of the molds as well as their stains situated on paper were collected using a compound microscope and commercial microscope lens for cell phone cameras, which were then used for training CNN models with a transfer learning scheme to perform the classification of mold. To enable involvement and contribution from the research community, a web interface that actuates the process while providing interactive features for users to learn about the information of the classified strain was constructed. Moreover, a feedback functionality in the web interface was embedded for catching potential classification errors, adding additional training images, or introducing new strains, all to refine the generalizability and robustness of the model.Results & ConclusionIn the study, we have constructed a suite of high-confidence classification CNN models for the diagnostic process for mold contamination in conservation. At the same time, a web interface was constructed that allows recurrently refining the model with human feedback through engaging the research community. Overall, the proposed framework opens new avenues for effective and timely identification of mold, thus enabling proactive and targeted mold remediation strategies in conservation.

Electrodynamic manipulator for commercial fluorescence microscope

We have developed a simple electrodynamic manipulator extension for a commercial fluorescence microscope. This extension allows single charged nano- and microparticles to levitate being isolated in space. The proposed electrodynamic manipulator is based on the linear quadrupole Paul trap. We measured the luminescence spectrum of a single microparticle filled with semiconductor quantum dots to demonstrate the operation process of the electrodynamic manipulator. This paper reveals the manipulator design features and restrictions are imposed by microscopic equipment; the issues of optical spectra recording are also discussed. The electrodynamic manipulator is a cheap and robust tool that can be compatible with any commercial microscopes, and can be easily adapted and modified for various investigation needs.

EMinsight: a tool to capture cryoEM microscope configuration and experimental outcomes for analysis and deposition.

The widespread adoption of cryoEM technologies for structural biology has pushed the discipline to new frontiers. A significant worldwide effort has refined the single-particle analysis (SPA) workflow into a reasonably standardized procedure. Significant investments of development time have been made, particularly in sample preparation, microscope data-collection efficiency, pipeline analyses and data archiving. The widespread adoption of specific commercial microscopes, software for controlling them and best practices developed at facilities worldwide has also begun to establish a degree of standardization to data structures coming from the SPA workflow. There is opportunity to capitalize on this moment in the maturation of the field, to capture metadata from SPA experiments and correlate the metadata with experimental outcomes, which is presented here in a set of programs called EMinsight. This tool aims to prototype the framework and types of analyses that could lead to new insights into optimal microscope configurations as well as to define methods for metadata capture to assist with the archiving of cryoEM SPA data. It is also envisaged that this tool will be useful to microscope operators and facilities looking to rapidly generate reports on SPA data-collection and screening sessions.

A 3D tailored monolithic glass chip for stimulating and recording zebrafish neuronal activity with a commercial light sheet microscope

Microfluidic technology is unrivaled in its ability to apply soluble chemical stimuli with high spatiotemporal precision. Analogous, light–sheet microscopy is unmatched in its ability of low phototoxic but fast volumetric in vivo imaging with single cell resolution. Due to their optical translucency during the larval stages, zebrafish (Danio rerio) are an ideal model to combine both techniques; yet, thus far this required light–sheet microscopes, which were in most cases custom–built and adapted to the available softlithographic chip technology. Our aim was to use a commercial light–sheet microscope to illuminate a microfluidic chip from two opposite lateral directions and to record images with the detection objective placed orthogonally above the chip. Deep tissue penetration can be achieved by superimposing beams from opposite directions to form a single light sheet. But a microfluidic chip that allows a) targeted stimulus application in a closed microenvironment, b) interference–free incoupling of excitation light from two directions and c) outcoupling of fluorescence in the perpendicular direction through an optically perfect cover glass was not known until now. Here, we present a monolithic glass chip with the required plane-parallel sidewalls and cover slide closure at the top, constructed by advanced femtosecond laser ablation, thermal bonding and surface smoothing processes. In addition, the 3D shape of a fish fixator unit was tailored to match the body shape of a zebrafish larva to ensure stable positioning during whole–brain recording. With hydrodynamic focusing a targeted partial exposure of the larva’s head to chemical stimuli and fast position switching (in less than 10 s) was possible. With the capabilities of this unique monolithic glass chip and its up–scalable wafer–level fabrication process, the new NeuroExaminer is prone to become an excellent addition to neurobiology laboratories already equipped with high–quality commercial light sheet microscopes.

Achieving sub-0.5-angstrom-resolution ptychography in an uncorrected electron microscope.

Subangstrom resolution has long been limited to aberration-corrected electron microscopy, where it is a powerful tool for understanding the atomic structure and properties of matter. Here, we demonstrate electron ptychography in an uncorrected scanning transmission electron microscope (STEM) with deep subangstrom spatial resolution down to 0.44 angstroms, exceeding the conventional resolution of aberration-corrected tools and rivaling their highest ptychographic resolutions​. Our approach, which we demonstrate on twisted two-dimensional materials in a widely available commercial microscope, far surpasses prior ptychographic resolutions (1 to 5 angstroms) of uncorrected STEMs. We further show how geometric aberrations can create optimized, structured beams for dose-efficient electron ptychography. Our results demonstrate that expensive aberration correctors are no longer required for deep subangstrom resolution.

Spectral phasor analysis using 4 detectors of a commercial microscope for fast spectral imaging

Spectral phasor analysis using 4 detectors of a commercial microscope for fast spectral imaging

Segmented FCS on a commercial microscope to study dynamics and interactions in live cells

Segmented FCS on a commercial microscope to study dynamics and interactions in live cells