High Magnification Objective Research Articles

A Simple and Fast Optical Clearing Method for Whole-Mount Fluorescence In Situ Hybridization (FISH)Imaging.

We report a single-step optical clearing method that is compatible with RNA fluorescence in situ hybridization (FISH) imaging. We previously demonstrated microscopy imaging with immunohistochemistry and genetic reporters using a technique called lipid-preserving refractive index matching for prolonged imaging depth (LIMPID). Our protocol reliably produces high-resolution three-dimensional (3D) images with minimal aberrations using high magnification objectives, captures large field-of-view images of whole-mount tissues, and supports co-labeling with antibody and FISH probes. We also custom-designed FISH probes for quail embryos, demonstrating the ease of fabricating probes for use with less common animal models. Furthermore, we show high-quality 3D images using a conventional fluorescence microscope, without using more advanced depth sectioning instruments such as confocal or light-sheet microscopy. For broader adoption, we simplified and optimized 3D-LIMPID-FISH to minimize the barrier to entry, and we provide a detailed protocol to aid users with navigating the thick and thin of 3D microscopy.

A novel 3D imaging approach for quantification of GLUT4 levels across the intact myocardium.

Cellular heterogeneity is a well-accepted feature of tissues, and both transcriptional and metabolic diversity have been revealed by numerous approaches, including optical imaging. However, the high magnification objective lenses needed for high-resolution imaging provides information from only small layers of tissue, which can result in poor cell statistics. There is therefore an unmet need for an imaging modality that can provide detailed molecular and cellular insight within intact tissue samples in 3D. Using GFP-tagged GLUT4 as proof of concept, we present here a novel optical mesoscopy approach that allows precise measurement of the spatial location of GLUT4 within specific anatomical structures across the myocardium in ultrathick sections (5 mm×5 mm×3 mm) of intact mouse heart. We reveal distinct GLUT4 distribution patterns across cardiac walls and highlight specific changes in GLUT4 expression levels in response to high fat diet-feeding, and we identify sex-dependent differences in expression patterns. This method is applicable to any target that can be labelled for light microscopy, and to other complex tissues when organ structure needs to be considered simultaneously with cellular detail.

Full-Automatic High-Efficiency Mueller Matrix Microscopy Imaging for Tissue Microarray Inspection.

This paper proposes a full-automatic high-efficiency Mueller matrix microscopic imaging (MMMI) system based on the tissue microarray (TMA) for cancer inspection for the first time. By performing a polar decomposition on the sample's Mueller matrix (MM) obtained by a transmissive MMMI system we established, the linear phase retardance equivalent waveplate fast-axis azimuth and the linear phase retardance are obtained for distinguishing the cancerous tissues from the normal ones based on the differences in their polarization characteristics, where three analyses methods including statistical analysis, the gray-level co-occurrence matrix analysis (GLCM) and the Tamura image processing method (TIPM) are used. Previous MMMI medical diagnostics typically utilized discrete slices for inspection under a high-magnification objective (20×-50×) with a small field of view, while we use the TMA under a low-magnification objective (5×) with a large field of view. Experimental results indicate that MMMI based on TMA can effectively analyze the pathological variations in biological tissues, inspect cancerous cervical tissues, and thus contribute to the diagnosis of postoperative cancer biopsies. Such an inspection method, using a large number of samples within a TMA, is beneficial for obtaining consistent findings and good reproducibility.

Optogenetic Interrogation of Electrophysiological Dendritic Properties and Their Effect on Pacemaking Neurons from Acute Rodent Brain Slices.

Understanding dendritic excitability is essential for a complete and precise characterization of neurons' input-output relationships. Theoretical and experimental work demonstrates that the electrotonic and nonlinear properties of dendrites can alter the amplitude (e.g., through amplification) and latency of synaptic inputs as viewed in the axosomatic region where spike timing is determined. The gold-standard technique to study dendritic excitability is using dual-patch recordings with a high-resistance electrode used to patch a piece of distal dendrite in addition to a somatic patch electrode. However, this approach is often impractical when distal dendrites are too fine to patch. Therefore, we developed a technique that utilizes the expression of Channelrhodopsin-2 (ChR2) to study dendritic excitability in acute brain slices through the combination of a somatic patch electrode and optogenetic activation. The protocol describes how to prepare acute slices from mice that express ChR2 in specific cell types, and how to use two modes of light stimulation: proximal (which activates the soma and proximal dendrites in a ~100 µm diameter surrounding the soma) with the use of a high-magnification objective and full-field stimulation through a low-magnification objective (which activates the entire somato-dendritic field of the neuron). We use this technique in conjunction with various stimulation protocols to estimate model-based spectral components of dendritic filtering and the impact of dendrites on phase response curves, peri-stimulus time histograms, and entrainment of pacemaking neurons. This technique provides a novel use of optogenetics to study intrinsic dendritic excitability through the use of standard patch-clamp slice physiology. Key features • A method for studying the effects of electrotonic and nonlinear dendritic properties on the sub- and suprathreshold responses of pacemaking neurons. • Combines somatic patch clamp or perforated patch recordings with optogenetic activation in acute brain slices to investigate dendritic linear transformation without patching the dendrite. • Oscillatory illumination at various frequencies estimates spectral properties of the dendrite using subthreshold voltage-clamp recordings and studies entrainment of pacemakers in current clamp recordings. • This protocol uses Poisson white noise illumination to estimate dendritic phase response curves and peri-stimulus time histograms.

Integration of wide-field imaging system with droplet microfluidics for monitoring living bacteria

Droplet microfluidics has been widely used to analyze chemicals and biological reactions at the single-molecule level. Microscopic systems are commonly used for imaging and analyzing droplets using high-magnification objective lenses. However, these systems are expensive, complex, and large, limiting the high-throughput characterization of droplets in reactions targeting disease-related biomarkers, including nucleic acids, proteins, and pathogens. Another concern is the time gap between droplet analysis within a microfluidic channel and imaging chamber, which can cause discrepancies in reaction time between droplets. To address this issue, we introduce the droplet analysis system for vast imaging and statistical tool (DropVIST)—a wide-field imaging system integrated with droplet microfluidics for rapid and accurate droplet analysis. DropVIST can detect eight differently colored droplets simultaneously using a wide-field imaging system and dFinder software, which can quantify the reacted droplets. The smallest droplet diameter for detection was 30 μm, and the maximum detection area was 201.84 cm2, suggesting that the system can theoretically analyze 3103,377 droplets in real-time. We validated the monitoring performance of DropVIST using clinical isolates of five types of living pathogenic bacteria and compared this with conventional approaches, including the microbroth dilution method and colony-forming unit assay. This platform provides a rapid, simple, and accurate tool for monitoring living bacteria at the single-cell level.

Low-cost 3D printed lenses for brightfield and fluorescence microscopy.

We present the fabrication and implementation of low-cost optical quality 3D printed lenses, and their application as microscope objectives with different prescriptions. The imaging performance of the 3D printed lenses was benchmarked against commercially available optics including a 20 mm focal length 12.7 mm diameter NBK-7 plano-convex lens used as a low magnification objective, and a separate high magnification objective featuring three 6 mm diameter NBK-7 lenses with different positive and negative focal lengths. We describe the design and manufacturing processes to produce high-quality 3D printed lenses. We tested their surface quality using a stylus profilometer, showing that they conform to that of commercial glass counterpart lenses. The 3D printed lenses were used as microscope objectives in both brightfield and epi-fluorescence imaging of specimens including onion, cyanobacteria, and variegated Hosta leaves, demonstrating a sub-cellular resolution performance obtained with low-cost 3D printed optical elements within brightfield and fluorescence microscopy.

Extended depth-of-field resolution enhancement microscopy imaging for neutralizing the impact of mineral inhomogeneous surface

<p>One of the most fundamental experimental methods in geoscience is to observe minerals under high magnification objectives. However, uneven microsurfaces in thin sections occur due to the irregular constituent distribution and varying hardness of minerals in natural rocks. Consequently, the conflict between large depth-of-field (DOF) and high-resolution in microscopy imaging leads to random out-of-focus issues when observing thin sections with high resolution microscopy. Although existing super-resolution algorithms promise to improve visual performance, reconstructing images with both large DOF and high-resolution simultaneously remains challenging. We address this problem by guiding the networks with optical information. Utilizing DOF information from low-resolution data, we propose an optically induced generative adversarial network (OIGAN) to neutralize the impact through computational imaging. In OIGAN, optical DOF information from low-resolution data facilitates to achieve spatial-adaptive extended-DOF resolution enhancement imaging, without incorporating extended DOF high-resolution data for supervision. The approach, trained and evaluated on the dataset with 233,156 images (115,346 pairs of low- and high-resolution data), outperforms four comparison methods on various minerals and optical conditions, leading to at least 1.54dB increase on peak signal-to-noise ratio (PSNR). Specifically, OIGAN significantly improves the accuracy of fluid inclusion ice-melting temperature measurement, reducing mean error by 65%, and enhances mineral classification accuracy with 1.5%~15% increase. OIGAN offers an insight of integrating physical knowledge into neural networks, facilitating self-identification of minerals, automatic microthermometry of fluid inclusions and other geoscience tasks via microscopy.</p>

A dynamic parallel image acquisition method for slide scanning process

Automated microscope systems have played an important role in the screening of numerous diseases. However, it is a very time-consuming process to continuously acquire images under the high magnification objective lens. This paper proposes a dynamic parallel image acquisition method, which can greatly improve image acquisition speed. Due to the relative motion between the x-y stage and the camera, some of the captured images have motion blur To this end, we also designed a motor variable speed motion curve to ensure the quality of the collected images. The experimental results show that the traditional image scanning mode needs 47.3 ms to obtain continuous microscopic images, while the dynamic parallel image acquisition method only needs 25.4 ms, which improves the acquisition speed without affecting the clarity of the acquired images.

3D dynamic observation of human sperm by parallel phase-shifting digital holographic microscopy based on pixelated polarization

Morphology and motility are essential criteria for assessing sperm viability. However, the human sperm head is small (∼3–4 μm) and requires a relatively high-magnification microscope objective, while the sperm flagella (∼45 μm) are poorly visible with complex 3D properties. Microscopic dynamic observation of intact sperm in 3D is challenging. Conventional inspection methods with a limited depth of field are inadequate for this issue. To provide a solution to this critical need, we develop pixelated polarization-based parallel phase-shifting digital holographic microscopy for the 3D dynamic observation of human sperm. Compared to conventional holographic imaging, this approach can effectively separate the object wavefront and avoid image quality degradation while fully exploiting the spatial bandwidth of the camera. We propose the use of the Stokes parameter reconstruction method to reconstruct the object wavefront and investigate the effect of the sampling interval on the system resolution by spectral analysis. The methodology achieves the retrieval of the 3D trajectory and motion parameters of sperm and reconstructs the sperm head orientation and the thin, highly-dynamic flagellum. The system allows for more comprehensive information on sperm motility and morphology, which is significant for male reproductive research. It also has significant potential for 3D dynamic observation of micro-organisms.

Multi-Resolution 3D Optical Mapping of Immune Cell Infiltrates in Mouse Asthmatic Lung.

Asthma is a chronic inflammatory airway disease driven by various infiltrating immune cell types into the lung. Optical microscopy has been used to study immune infiltrates in the asthmatic lungs. Confocal laser scanning microscopy (CLSM) identifies the phenotypes and locations of individual immune cells in lung tissue sections by employing high-magnification objectives and multiplex immunofluorescence staining. In contrast, light-sheet fluorescence microscopy (LSFM) can visualize the macroscopic and mesoscopic architecture of whole-mount lung tissues in three dimensions (3D) by adopting an optical tissue clearing method. Despite each microscopy method producing image data with unique resolution from a tissue sample, CLSM and LSFM have not been applied together due to the difference in the tissue preparation procedures. Here we introduce a new approach combining LSFM and CLSM into a sequential imaging pipeline. We built a new optical tissue clearing workflow in which the immersion clearing agent can be switched from an organic solvent to an aqueous sugar solution for sequential 3D LSFM and CLSM of mouse lungs. This sequential combination microscopy offered quantitative 3D spatial analyses of the distribution of immune infiltrates in the same mouse asthmatic lung tissue at the organ, tissue, and cell level. These results show that our method facilitates multi-resolution 3D fluorescence microscopy as a new imaging approach providing comprehensive spatial information for a better understanding of inflammatory lung diseases. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Sub-50 nm optical imaging in ambient air with 10× objective lens enabled by hyper-hemi-microsphere

Optical microsphere nanoscope has great potential in the inspection of integrated circuit chips for semiconductor industry and morphological characterization in biology due to its superior resolving power and label-free characteristics. However, its resolution in ambient air is restricted by the magnification and numerical aperture (NA) of microsphere. High magnification objective lens is required to be coupled with microsphere for nano-imaging beyond the diffraction limit. To overcome these challenges, in this work, high refractive index hyper-hemi-microspheres with tunable magnification up to 10× are proposed and realized by accurately tailoring their thickness with focused ion beam (FIB) milling. The effective refractive index is put forward to guide the design of hyper-hemi-microspheres. Experiments demonstrate that the imaging resolution and contrast of a hyper-hemi-microsphere with a higher magnification and larger NA excel those of a microsphere in air. Besides, the hyper-hemi-microsphere could resolve ~50 nm feature with higher image fidelity and contrast compared with liquid immersed high refractive index microspheres. With a hyper-hemi-microsphere composed microscale compound lens configuration, sub-50 nm optical imaging in ambient air is realized by only coupling with a 10× objective lens (NA = 0.3), which enhances a conventional microscope imaging power about an order of magnitude.

Synchronous measurement of surface deformation and gradient distribution in microstructures

In the traditional microfield speckle pattern interference measurement system, due to the use of high-magnification objective lens, the working distance is small and the shearing device cannot be introduced to implement shearography measurement. Thus the distribution of the deformation gradient on a microstructure surface cannot be measured. A system for the synchronous measurement of the surface deformation and gradient distribution of a microstructure was designed. Through the combination of focusing lens, convex lens, and low-magnification objective lens, the microstructure surface was clearly magnified and the internal distance of the system was enlarged. Thus the shearing device can be introduced to realize the shearography measurement under the microfield view. The synchronous full-field measurement of the structural surface deformation and its gradient distribution was further realized by the introduction of a reference light and designing of an optical path switch. In addition, the designed system had a zoom feature, which can facilitate the measurement in variable field of view in a certain range. The feasibility of the system was verified by theoretical analysis and experiment, and the practicability was validated by testing the chip and circuit board.

Content aware multi-focus image fusion for high-magnification blood film microscopy.

Automated digital high-magnification optical microscopy is key to accelerating biology research and improving pathology clinical pathways. High magnification objectives with large numerical apertures are usually preferred to resolve the fine structural details of biological samples, but they have a very limited depth-of-field. Depending on the thickness of the sample, analysis of specimens typically requires the acquisition of multiple images at different focal planes for each field-of-view, followed by the fusion of these planes into an extended depth-of-field image. This translates into low scanning speeds, increased storage space, and processing time not suitable for high-throughput clinical use. We introduce a novel content-aware multi-focus image fusion approach based on deep learning which extends the depth-of-field of high magnification objectives effectively. We demonstrate the method with three examples, showing that highly accurate, detailed, extended depth of field images can be obtained at a lower axial sampling rate, using 2-fold fewer focal planes than normally required.

Effect of inoculum size and antibiotics on bacterial traveling bands in a thin microchannel defined by optical adhesive

Phenotypic diversity in bacterial flagella-induced motility leads to complex collective swimming patterns, appearing as traveling bands with transient locally enhanced cell densities. Traveling bands are known to be a bacterial chemotactic response to self-generated nutrient gradients during growth in resource-limited microenvironments. In this work, we studied different parameters of Escherichia coli (E. coli) collective migration, in particular the quantity of bacteria introduced initially in a microfluidic chip (inoculum size) and their exposure to antibiotics (ampicillin, ciprofloxacin, and gentamicin). We developed a hybrid polymer-glass chip with an intermediate optical adhesive layer featuring the microfluidic channel, enabling high-content imaging of the migration dynamics in a single bacterial layer, i.e., bacteria are confined in a quasi-2D space that is fully observable with a high-magnification microscope objective. On-chip bacterial motility and traveling band analysis was performed based on individual bacterial trajectories by means of custom-developed algorithms. Quantifications of swimming speed, tumble bias and effective diffusion properties allowed the assessment of phenotypic heterogeneity, resulting in variations in transient cell density distributions and swimming performance. We found that incubation of isogeneic E. coli with different inoculum sizes eventually generated different swimming phenotype distributions. Interestingly, incubation with antimicrobials promoted bacterial chemotaxis in specific cases, despite growth inhibition. Moreover, E. coli filamentation in the presence of antibiotics was assessed, and the impact on motility was evaluated. We propose that the observation of traveling bands can be explored as an alternative for fast antimicrobial susceptibility testing.

Improved microscopy with ultraviolet surface excitation (MUSE) using high-index immersion illumination.

Microscopy with ultraviolet surface excitation (MUSE) typically has an optical sectioning thickness significantly larger than standard physical sectioning thickness, resulting in increased background fluorescence and higher feature density compared to formalin-fixed, paraffin-embedded physical sections. We demonstrate that high-index immersion with angled illumination significantly reduces optical sectioning thickness through increased angle of refraction of excitation light at the tissue interface. We present a novel objective dipping cap and waveguide-based MUSE illuminator design with high-index immersion and quantify the improvement in optical sectioning thickness, demonstrating an e-1 section thickness reduction to 6.67 µm in tissue. Simultaneously, the waveguide illuminator can be combined with high or low magnification objectives, and we demonstrate a 6 mm2 field of view, wider than a conventional 10x pathology objective. Finally, we show that resolution and contrast can be further improved using deconvolution and focal stacking, enabling imaging that is robust to irregular surface profiles on surgical specimens.

Photolithographic Patterning of FluorAcryl for Biphilic Microwell-Based Digital Bioassays and Selection of Bacteria.

FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 μm and various well depths ranging from 300 nm to 1 μm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 μm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.

Influence of laser shape on thermal increase during micro‐Raman spectroscopy analyses

AbstractMicro‐Raman spectrometers are used in various domains, from archaeology to space exploration. These systems use microscope objectives to focus the laser onto the sample to be analysed. Although this method drastically increases the spatial resolution down to a few hundreds of nanometres, it is also commonly admitted that this focused laser beam may heat and alter the studied material. The setting of the laser power is thus a universal problem for any micro‐Raman spectroscopy user who has to find a compromise between the Raman signal intensity and the risk of thermal alteration. This parameter is not easy to set based on bibliographic references because, depending on the architecture of the system used, on the laser wavelength and on the sample, damage may occur at very different laser power levels. Here, we describe the different parameters that influence thermal increase induced by the laser and propose easy to make experiments to measure them. A series of experiments is then carried out to study the thermal alteration of various materials when exposed to a laser beam focused with the different microscope objectives of a micro‐Raman system. It is shown that, except for very thin samples or powdered materials, the relevant parameters to consider are the laser power at the sample surface and the duration of exposure to the laser beam, whatever the objective used. We also demonstrate that the risk of sample alteration is almost non‐existent for thick transparent materials. Interestingly, it is shown that, for semi‐transparent materials, alteration may occur at lower laser power for low magnification objectives than for high magnification objectives. These results are explained by the high aperture angle of microscope objectives which induces high dispersion of the laser within the sample.

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging, a powerful analytical method to study single-cell behavior, such as gene expression and protein profiling, provides an essential basis for modern medical diagnosis. The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years. Simultaneously, chip-based living single-cell imaging is also limited by complicated trapping steps, low cell utilization, and difficult high-resolution imaging. To solve these problems, an ultra-thin temperature-controllable microwell array chip (UTCMA chip) was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells. The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope (or confocal microscope) with a high magnification objective (100 × oil lens), and the temperature of the chip can be controlled by combining it with a home-made temperature control device. High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall. The cell utilization rate, single-cell capture rate, and microwell occupancy rate are all close to 100% in the microwell array. This method will be useful in rare single-cell research, extending its application in the biological and medical-related fields, such as early diagnosis of disease, personalized therapy, and research-based on single-cell analysis.

Integration of polymeric membrane/dielectric sphere assemblies in microfluidic chips for enhanced-contrast imaging with low-magnification systems

Current microscopy systems for the imaging of microorganisms are expensive because of their optimized design toward resolution maximization and aberration correction. In situations where such an optimization is not needed, for instance to merely detect the presence of pathogens in liquids for on-site analyses, a potential approach is to use highly refractive spheres in combination with low-magnification objectives to increase the resolution and the sensitivity of the optical sensing system in a cost-effective fashion. Indeed, for point-of-need assays, integration of optical elements on a microfluidic device can bring several advantages, such as test parallelization/automation and low-volume consumption. We report a study on BaTiO3 spheres that are partially embedded in thin polymeric membranes of mismatched refractive index. We computed the transformation that the polymeric membrane/dielectric sphere assembly (PMDSA) mediates on the light originating from the sample toward the optical detector and shows its enhanced-detection potential for a low-magnification objective. We then propose a method to easily fabricate chips with custom designs and precise location of such dielectric spheres relative to the microfluidic structures for enhanced imaging of microorganisms. We applied this concept to the detection of living fluorescent bacteria, either flowing in aqueous medium or immobilized in hydrodynamic traps. We quantified the contrast gain provided by the PMDSA for short exposure when used with a low-magnification objective. By comparing with a high-magnification objective, we also show how longer-term imaging can be still reliably performed with a more cost-effective system. Since the present PMDSA concept combines the optical enhancement of low-magnification systems with the flexibility of microfluidic handling, it can be highly suitable for portable and cost-effective systems for on-site analysis, from flow cytometry to longer-term antibiotic testing.

Coaxial spectroscopic imaging ellipsometry for volumetric thickness measurement.

We propose spectroscopic imaging ellipsometry that can measure spectral ellipsometric signals in the entire field of view simultaneously without areal scanning or operation of polarization devices. The proposed imaging ellipsometry is configured in a coaxial optical structure so that the high magnification objective lens is applicable and the spatial resolution is highly increased. Without the operation of polarization components and to efficiently obtain the spectral data in the object plane, the ellipsometric parameters are encoded into the high frequency in the spectral domain and are measured by an imaging Michelson interferometer. The volumetric thickness measurement by the proposed method was verified by comparing the thickness results of the SiO2/Si sample that has four different thicknesses with commercial ellipsometer results.