Upright Microscope Research Articles

Mechanically sheared axially swept light-sheet microscopy.

We present a mechanically sheared image acquisition format for upright and open-top light-sheet microscopes that automatically places data in its proper spatial context. This approach, which reduces computational post-processing and eliminates unnecessary interpolation or duplication of the data, is demonstrated on an upright variant of axially swept light-sheet microscopy (ASLM) that achieves a field of view, measuring 774 × 435 microns, that is 3.2-fold larger than previous models and a raw and isotropic resolution of ∼460 nm. Combined, we demonstrate the power of this approach by imaging sub-diffraction beads, cleared biological tissues, and expanded specimens.

Bringing cell biology into classroom: tips to culture and observe skeletal muscle cells in high school and college

Watching living cells through a microscope is much more exciting than seeing pictures of cells in high school and college textbooks. However, bringing cell cultures into the classroom is challenging for biology teachers since culturing cells requires sophisticated and expensive instruments such as a CO2 incubator and an inverted phase-contrast microscope. Here, we describe easy and affordable methods to culture and observe skeletal muscle cells using the L-15 culture medium, tissue culture flask, standard dry incubator, standard upright microscope, and modified Smartphone microscope. Watching natural living cells in a “Do-It-Yourself (DIY)” way may inspire more students’ interest in cell biology.

PSIV-A-7 Effects of late gestational nutrient restriction of beef heifers on small intestinal crypt depth in neonatal calves

Abstract We hypothesized that late gestational nutrient restriction would impair small intestinal development in neonatal calves, including crypt cell depth and proliferation. Fall-calving crossbred beef heifers [body weight (BW) = 466 ± 30 (SD) kg; BCS: 5.4 ± 0.5] bred to a single sire were individually-fed 100% (control; n = 10) or 70% (nutrient restricted; n = 12) of NASEM metabolizable energy and metabolizable protein requirements for maintenance, pregnancy, and growth from d 160 of gestation to calving. Calves were removed from their dams immediately post-calving and fed artificial colostrum and milk replacer relative to body weight until euthanasia at 48.7 ± 0.4 h of age. The gastrointestinal tract was removed, and proximal jejunum samples were dissected from each calf and immersion-fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin blocks and cut into 5-µm sections that were mounted on glass slides. Slides were incubated with Ki-67 (marker of nuclear proliferation) and counterstained with Hematoxylin using an autostainer. Images were taken at 5× magnification using an upright microscope. QuPath software was used to measure crypt depth, starting at the base of the crypt region and ending at the furthest cluster of Ki-67 positive cells, perpendicular to the base of the mucosa. For each calf, 10 crypt depth measurements were taken from 4 images, resulting in 40 crypt depth measurements that were averaged. Nutritional plane was a fixed effect in the model, and maternal sire, date of study initiation, and calf sex (when P < 0.25) were considered covariates. We previously reported that calves born to nutrient restricted dams weighed less (P ≤ 0.05) at birth and 48 h than calves born to control dams. Small intestinal mass tended to be less (P = 0.11) in offspring of nutrient restricted dams but was not affected (P = 0.58) when expressed relative to body weight. Calves born to nutrient restricted dams also tended to have less (P = 0.11) small intestinal mass relative to brain weight. Maternal nutrient restriction did not affect (P = 0.95) jejunal crypt depth. Additionally, crypt depth relative to body weight was not affected (P = 0.20) by nutrient restriction. Although crypt depth was not affected, further analysis of proliferation and morphology will help to understand the relationship between maternal nutrient restriction and small intestine development in neonatal calves.

Visualization-enhanced under-oil open microfluidic system for in situ characterization of multi-phase chemical reactions

Under-oil open microfluidic system, utilizing liquid-liquid boundaries for confinements, offers inherent advantages including clogging-free flow channels, flexible access to samples, and adjustable gas permeation, making it well-suited for studying multi-phase chemical reactions that are challenging for closed microfluidics. However, reports on the novel system have primarily focused on device fabrication and functionality demonstrations within biology, leaving their application in broader chemical analysis underexplored. Here, we present a visualization-enhanced under-oil open microfluidic system for in situ characterization of multi-phase chemical reactions with Raman spectroscopy. The enhanced system utilizes a semi-transparent silicon (Si) nanolayer over the substrate to enhance visualization in both inverted and upright microscope setups while reducing Raman noise from the substrate. We validated the system’s chemical stability and capability to monitor gas evolution and gas-liquid reactions in situ. The enhanced under-oil open microfluidic system, integrating Raman spectroscopy, offers a robust open-microfluidic platform for label-free molecular sensing and real-time chemical/biochemical process monitoring in multi-phase systems.

Polarized hyperspectral microscopic imaging system for enhancing the visualization of collagen fibers and head and neck squamous cell carcinoma.

Polarized hyperspectral microscopes with the capability of full Stokes vector imaging have potential for many biological and medical applications. The aim of this study is to investigate polarized hyperspectral imaging (PHSI) for improving the visualization of collagen fibers, which is an important biomarker related to tumor development, and improving the differentiation of normal and tumor cells on pathologic slides. We customized a polarized hyperspectral microscopic imaging system comprising an upright microscope with a motorized stage, two linear polarizers, two liquid crystal variable retarders (LCVRs), and a compact SnapScan hyperspectral camera. The polarizers and LCVRs worked in tandem with the hyperspectral camera to acquire polarized hyperspectral images, which were further used to calculate four Stokes vectors: , , , and . Synthetic RGB images of the Stokes vectors were generated for the visualization of cellular components in PHSI images. Regions of interest of collagen, normal cells, and tumor cells in the synthetic RGB images were selected, and spectral signatures of the selected components were extracted for comparison. Specifically, we qualitatively and quantitatively investigated the enhanced visualization and spectral characteristics of dense fibers and sparse fibers in normal stroma tissue, fibers accumulated within tumors, and fibers accumulated around tumors. By employing our customized polarized hyperspectral microscope, we extract the spectral signatures of Stokes vector parameters of collagen as well as of tumor and normal cells. The measurement of Stokes vector parameters increased the image contrast of collagen fibers and cells in the slides. With the spatial and spectral information from the Stokes vector data cubes (, , , and ), our PHSI microscope system enhances the visualization of tumor cells and tumor microenvironment components, thus being beneficial for pathology and oncology.

3D printed chamber for live cell imaging on an upright epifluorescence microscope

ABSTRACT Live cell imaging is a standard technique in experimental biology that enables the observation of isolated cells and tissue slices in real time and the testing of cellular responses to changes in buffer composition. However, most live cell imaging devices require the use of dedicated microscopes and/or specialised stage adaptors, and come at a reasonably high cost. We employed 3D printing technology to create a low-cost imaging chamber with side ports to exchange fluids, to be used on upright microscopes. The chamber increased the functionality of a standard upright epifluorescent microscope to allow dynamic, real-time calcium imaging of cultured hypothalamic astrocytes from mice, and to test the effects of ATP stimulation upon calcium signalling. It was also used on slices obtained from the mouse brain using a brain matrix slicer. The advantages of this chamber include a very simple design that can be used with upright epifluorescence microscopes, does not require any special stage adaptor, and includes ports to permit fluid exchange during imaging. This chamber is ideal for educational settings with undergraduate laboratories that do not have access to dedicated inverted fluorescent microscopes for tissue culture experiments.

High-Sensitivity Intrinsic Optical Signal Imaging Through Flexible, Low-Cost Adaptations of an Upright Microscope.

Intrinsic optical signal imaging (IOSI) is a staple technique in modern neuroscience. Pioneered >30 years ago, IOSI allows macroscopic mapping of neuronal activity throughout the cortex. The technique has been used to study sensory processing and experience-dependent plasticity, and is often used as an adjunctive procedure to localize cortical areas for subsequent targeting by other imaging or physiology techniques. Despite the ubiquity of IOSI in neuroscience, there are few commercially available turn-key IOSI systems. As a result, investigators have typically resorted to building their own imaging systems. Over the years, simplified systems built either as dedicated rigs or incorporated into existing microscope platforms have been developed. Here we present a straightforward set of adaptations that can be applied to any standard upright microscope, using readily available, inexpensive, commercial parts for illumination, optics, and signal detection, that enables high-sensitivity IOSI. Using these adaptations, we are able to readily map sensory-evoked signals across the somatosensory and visual cortex, including single-whisker barrel cortical activity maps in mice. We show that these IOSI maps are highly reproducible across animals and can be used to study plasticity mechanisms in the somatosensory cortex. We also provide open-source applications to control illumination and analyze raw data to generate activity maps. We anticipate that these resources will be useful for neuroscience investigators looking to add IOSI capabilities to an existing microscope in the laboratory on a budget.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

Single-shot wavelength-multiplexed phase microscopy under Gabor regime in a regular microscope embodiment

Phase imaging microscopy under Gabor regime has been recently reported as an extremely simple, low cost and compact way to update a standard bright-field microscope with coherent sensing capabilities. By inserting coherent illumination in the microscope embodiment and producing a small defocus distance of the sample at the input plane, the digital sensor records an in-line Gabor hologram of the target sample, which is then numerically post-processed to finally achieve the sample’s quantitative phase information. However, the retrieved phase distribution is affected by the two well-known drawbacks when dealing with Gabor’s regime, that is, coherent noise and twin image disturbances. Here, we present a single-shot technique based on wavelength multiplexing for mitigating these two effects. A multi-illumination laser source (including 3 diode lasers) illuminates the sample and a color digital sensor (conventional RGB color camera) is used to record the wavelength-multiplexed Gabor hologram in a single exposure. The technique is completed by presenting a novel algorithm based on a modified Gerchberg–Saxton kernel to finally retrieve an enhanced quantitative phase image of the sample, enhanced in terms of coherent noise removal and twin image minimization. Experimental validations are performed in a regular Olympus BX-60 upright microscope using a 20X 0.46NA objective lens and considering static (resolution test targets) and dynamic (living spermatozoa) phase samples.

NIR Light-Mediated Photocuring of Adhesive Hydrogels for Noninvasive Tissue Repair via Upconversion Optogenesis.

The surgical treatments of injured soft tissues lead to further injury due to the use of sutures or the surgical routes, which need to be large enough to insert biomaterials for repair. In contrast, the use of low viscosity photopolymerizable hydrogels that can be inserted with thin needles represents a less traumatic treatment and would therefore reduce the severity of iatrogenic injury. However, the delivery of light to solidify the inserted hydrogel precursor requires a direct access to it, which is mostly invasive. To circumvent this limitation, we investigate the approach of curing the hydrogel located behind biological tissues by sending near-infrared (NIR) light through the latter, as this spectral region has the largest transmittance in biological tissues. Upconverting nanoparticles (UCNPs) are incorporated in the hydrogel precursor to convert NIR transmitted through the tissues into blue light to trigger the photopolymerization. We investigated the photopolymerization process of an adhesive hydrogel placed behind a soft tissue. Bulk polymerization was achieved with local radiation of the adhesive hydrogel through a focused light system. Thus, unlike the common methods for uniform illumination, adhesion formation was achieved with local micrometer-sized radiation of the bulky hydrogel through a gradient photopolymerization phenomenon. Nanoindentation and upright microscope analysis confirmed that the proposed approach for indirect curing of hydrogels below the tissue is a gradient photopolymerization phenomenon. Moreover, we found that the hydrogel mechanical and adhesive properties can be modulated by playing with different parameters of the system such as the NIR light power and the UCNP concentration. The proposed photopolymerization of adhesive hydrogels below the tissue opens the prospect of a minimally invasive surgical treatment of injured soft tissues.

Identification of prominent airborne pollen in a city situated in foot-hills of Himalayas, Chandigarh, India.

Pollen allergy is considered one of the important critical thrust areas, as 20-30% of the world population suffers from allergic rhinitis. The increase in global surface temperature directly affects pollen physiological (e.g., pollen production) and morphological parameters and indirectly affects the distribution pattern, the allergenic potential of pollen, and plant species. Therefore, periodic sampling and pollen studies of a region have become necessary to assess the status of change in species and its morphological characteristics of different taxa. The current study is conducted to identify the airborne pollen based on studying their unique and distinctive morphological characters to serve as a reference pollen guide for future research. The airborne pollens were trapped using the Burkard volumetric sampler at three different locations in Chandigarh from 2018 to 2020 and analyzed under Leica DM5500B-Automated Upright Microscope System. The study investigated various pollen features such as shape, size, aperture type, and exine/surface pattern for taxonomic classification of plant groups. The majority of LM-analyzed pollen grains were prolate-spheroidal or oblate, whereas the aperture types were 3-zonocolporate, 3-colpate, and 3-zonocolporate. Exine patterns were predominantly psilate, reticulate, and straite and were easily discernible. Nonetheless, the vast majority of airborne pollen belonging to both arboreal and non-arboreal was quite small and fall into small pollen size classes, i.e., 10-24μm. The exine pattern was readily apparent and were predominantly psilate, reticulate, and straight. The current study improved the knowledge on airborne pollen biodiversity, which will help to understand the regional distribution, long-range transport, and construct the current status of morphological features of species/taxa.

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

Poster 206: TIPE2 Mitigates the Histopathology of Osteoarthritis in An Accelerate Aging Mouse Model

Poster 206: TIPE2 Mitigates the Histopathology of Osteoarthritis in An Accelerate Aging Mouse Model

Nanoporous Vesicular Membranes of Amphiphilic Polymers Containing Trans / Cis Isomers

Nanoporous Vesicular Membranes of Amphiphilic Polymers Containing <i>Trans</i> / <i>Cis</i> Isomers

499 Seizures in a Dish: Demonstrating and Analyzing Coordinated Network Activity in Human and Mouse Brain Slice with Calcium Imaging

INTRODUCTION: Childhood epilepsy is a common and often devastating disease, and chronic seizures lead to a significant increase in premature mortality and developmental delay. Focal cortical dysplasia (FCD), a malformation of cortical development, is the primary cause of pharmacoresistant epilepsy in children undergoing resective surgery. There are no targeted medical or surgical therapies for FCD patients who have failed surgery or who are not surgical candidates. METHODS: Human tissue from patients with MCDs was resected during epilepsy surgery. Comparison tissue from CD1 mouse brains was also used. 400-µm brain slices were made with a vibratome. The slices were then incubated the slices in Fluo-8L, a low-affinity calcium-sensitive indicator for 40-180 minutes. Slices were imaged using an sCMOS imager at 15 fps on an upright microscope and standard patch-clamp rig with a custom-built temperature-controlled chamber with high aCSF flow rate (5-15mL per minute) to optimize tissue oxygenation. Network bursts are induced with a pro-ictal ACSF containing high potassium (8 mM) and 4-aminopyridine (100 micromolar). RESULTS: We developed a workflow for calcium imaging in human tissue as follows. Preliminary semi-manual analysis on each image stack was performed using ImageJ. Image stacks were motion corrected using the NoRMCorre algorithm and then processed using the calcium imaging analysis workflow (CaImAn) appropriate for 1-photon imaging. Hundreds of cells can be analyzed simultaneously. Ictal network activity may be induced with the pro-ictal ACSF, or with gabazine (1 mM, a GABA-A receptor antagonist, data not shown). CONCLUSION: We have demonstrated the feasibility of performing ex vivo calcium imaging to directly study the aberrant epileptic networks in human MCD tissue resected from children with intractable epilepsy. In future work, we will use this novel technique to uncover network anomalies underlying the epileptic networks in this disorder.

Commercially derived versatile optical architecture for two-photon STED, wavelength mixing and label-free microscopy.

Multimodal microscopy combines multiple non-linear techniques that take advantage of different optical processes to generate contrast and increase the amount of information that can be obtained from biological samples. However, the most advanced optical architectures are typically custom-made and often require on-site adjustment of optical components performed by trained personnel for optimal performance. Here, we describe a hybrid system we built based on a commercial upright microscope. We show that our multimodal imaging platform can be used to seamlessly perform two-photon STED, wavelength mixing and label-free microscopy in both ex vivo and in vivo turbid samples. The system is stable and endowed with remote alignment hardware that ensures long-term operability also for non-expert users, using the alignment protocol described in this article and in the related material. This optical architecture is an important step forward towards a wider practical applicability of non-linear optics to bioimaging.

Approach for patch‐clamping using an upright microscope with z‐axis movable stage

We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping. RESEARCH HIGHLIGHTS: Approach for efficient patch-clamping using an upright microscope with a z-axis movable stage. Routine study of live cell physiology. Custom microscope/patch-clamp configuration that is cost-effective and overcomes equipment limitations.

Particulate matter 2.5 induced hyperpigmentation in reconstructed human epidermis model (MelaKutis®).

Particulate matter 2.5 induced hyperpigmentation in reconstructed human epidermis model (MelaKutis®).

Phase imaging microscopy under the Gabor regime in a minimally modified regular bright-field microscope

Quantitative phase imaging (QPI) is nowadays a powerful tool for visualization and analysis of biological processes. QPI is usually attained from specifically designed optical microscopes retrieving phase information in a quantitative way. In this paper we report on an extremely simple, low cost and compact way to update a standard bright-field microscope with coherent sensing capabilities. It is based on the in-line Gabor holography concept and only needs to replace the illumination broadband source of the regular microscope with a coherent one. The proposed methodology is completed by the recording of a digital in-line Gabor hologram instead of regular imaging conditions and by the numerical processing of the recorded hologram to finally achieve QPI. The selection of the defocus distance is critical to finally achieve high quality phase imaging, so calibration considering phase and amplitude test targets is presented for the proper definition of such defocus distance. In addition, the selected configuration is experimentally validated using different samples (microbeads, cheek cells and alive spermatozoa). All the experiments are implemented in a regular Olympus BX-60 upright microscope using a 20X 0.46NA objective lens. The proposed method shows how to convert a regular microscope into a holographic one with probably the minimum needed modifications and with the main limitation coming from the Gabor imaging conditions (weak diffractive samples and twin image presence).

Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells

Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging.