Standard Glass Slide Research Articles

Abstract 3645: Unraveling spatial complexity of the tumor microenvironment: A whole transcriptomic perspective with Visium HD

Abstract In recent years, advances in spatial transcriptomics have revolutionized our understanding of the tumor microenvironment, providing crucial insights into the complex interplay of different cell types within cancer tissues. In this study, we employed the new, high definition Visium spatial transcriptomics assay (Visium HD) to investigate the intricate molecular landscape of prostate cancer at single-cell scale resolution and across the whole transcriptome. Our research focused on deciphering the spatial heterogeneity of gene expression patterns within the tumor microenvironment, shedding light on the interactions between cancer cells, stromal cells, and vasculature. The Visium HD assay enables an unbiased exploration of the transcriptome on FFPE tissue sections mounted on standard glass slides. Combining gene expression data with H&E images from the same section, it allows precise characterization of local molecular features and disease states at single-cell scale. We analyzed a set of human prostate adenocarcinoma samples with the Visium HD assay, and identified spatially regulated gene signatures associated with specific cell types and functional pathways. Our findings have recapitulated previously identified molecular markers, including VEGFA and MYC which have been implicated in tumor progression and angiogenesis. The spatial gene expression patterns had good correlation with histological annotations of cancerous and normal tissue within the H&E image. However, Visium HD allowed us to further observe molecular processes and pathways specific to this adenocarcinoma sample. This integrative approach offers valuable implications for personalized cancer therapy and the development of targeted interventions tailored to the spatial context of individual tumors. Our study underscores the significance of understanding the spatial organization of the whole transcriptome in cancer tissues, and highlights the potential of the Visium HD platform as a powerful tool for unraveling the complexities of the tumor microenvironment. These insights pave the way for the development of innovative therapeutic strategies and precision medicine approaches, ultimately contributing to improved outcomes for cancer patients. Citation Format: Yifeng Yin, Jerald Sapida, David Sukovich, David Patterson, Augusto Tentori. Unraveling spatial complexity of the tumor microenvironment: A whole transcriptomic perspective with Visium HD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3645.

Towards the epitaxial growth of Au thin films on MgO substrates for plasmonic applications

Surface Plasmon Polaritons (SPPs) in Au thin films are nowadays intensively exploited for sensing applications that leverage the strong optical field confinement at the metal/dielectric interface and the easy functionalization of the Au surface. Moreover, Au thin films represent one of the common starting points for the top-down nanofabrication of plasmonic nanostructures supporting localized resonances. In this framework, strategies for the growth of high-quality Au films on transparent substrates are crucial and not yet fully established. In this study, we exploit MgO(001) substrates for the growth of thin (about 45 nm) Au films, also including an additional buffer layer of Fe. We successfully demonstrate Au samples with reduced roughness and presenting Low-Energy Electron Diffraction (LEED) features, indicating a high degree of crystalline ordering. This is supported by the experimental evidence of an increased (by almost a factor of 3) propagation length compared to a reference Au sample grown on standard glass slides, which is however still significantly lower than the one expected from first principles.

Comparison of Devices That Measure Sickle Red Cell Deformability

Background With the emergence of novel pharmacologic and gene-based therapies, identifying the rheological and biophysical RBC abnormalities of sickle cell disease (SCD) not captured by clinical laboratory techniques is crucial. Ektacytometry (LORRCA), the current standard to assess RBC function, does not reflect mechanical stress that RBCs experience in capillary microvasculature in the body. The device is costly, requires a nitrogen gas tank, and its use is difficult to translate outside of large-scale laboratory settings. In comparison, the Microfluidic Impedance Red Cell Assay (MIRCA) is a low-cost, portable device that mimics capillary microvasculature with micropillar arrays spaced 3-12 µm apart. Thus, MIRCA measures mechanical deformability, which is more physiologic, mimicking RBCs squeezing through microvasculature. An impedance analyzer calculates the MIRCA Occlusion Index (OI), representing the % occlusion of the chip. Here we compare the MIRCA to the LORRCA, assessing the correlations of OI and Elongation Index maximum (EI) to conventional laboratory tests and SCD related complications. Methods Peripheral blood from 53 adult (n = 28) and pediatric (n = 25) individuals (HbSS = 35, HbSB0 = 6, HbSC = 5, HbSB0 = 1, and HbAA controls = 6) were obtained under an Emory University IRB approved protocol. Patients transfused < 90 days prior were excluded. Hospitalizations / emergency department (ED) visits for pain within 12 months of sample collection were determined by chart review. Samples were collected in EDTA, stored at 4°C up to 48 hours, centrifuged at 500g, washed, and resuspended to a 20% hematocrit to run on MIRCA, an oxygen gradient ektacytometer (LORRCA), and an ADVIA hematology analyzer. Polydimethylsiloxane (PDMS) fabricated MIRCA chips were bonded to a standard glass slide with pairs of gold-sputtered electrodes adjacent to each array. Chips were prepared by perfusing ethanol, 1X phosphate-buffered saline (PBS), and 3% bovine-serum albumin (BSA) in 1X PBS via syringe pump and were incubated overnight at 4°C prior to use. Baseline impedance was taken for a 2-minute perfusion of 1X PBS, then OIs were calculated from impedance values 10 minutes after sample introduction normalized to baseline. The data was analyzed using the Mann-Whitney U test, Spearman correlation, and linear regression. OriginPro (Northampton, MA, USA) and Stata 18 (College Station, TX, USA) were used for the analyses and a p < 0.05 was considered significant. Results The correlation coefficients for OI and EI with laboratory tests (Table 1) and to each other (Figure 1) were comparable (p < 0.05). HbSS/SB0 had higher OIs than HbSC/SB+ (median = 13.7% vs 6.8%, p < 0.01) and HbAA (median = 4.6%, p < 0.01). Patients with ≥ 1 vaso-occlusive events (VOE) in the past year had higher OIs and lower EIs compared to those without a VOE (median = 15.3% vs 9.6%, p <0.01; median = 0.4 vs 0.5, p = 0.02, respectively). Patients that received acute medical care (admission or ED visit) had higher OIs and lower EIs compared to those that did not (median = 15.7% vs 9.8%, p < 0.01; median = 0.4 vs 0.5, p = 0.02, respectively). In multiple linear regression, HbSS/SB0 (p < 0.01), adults (p = 0.01), and DRBC (p < 0.01) were associated with OI while HbSS/SB0 (p = 0.01), DRBC (p < 0.01), hemoglobin % (p = 0.04), absolute neutrophil count (p = 0.02), and VOE+ (p = 0.02) were associated with EI. The models explained 55.2% and 66.8% variability of OI and EI, respectively. Lower Akaike's information criterion (AIC, 186 vs -80) and Bayesian information criterion (BIC, 192 vs -71) of the EI model suggest a higher correlation to conventional laboratory values and clinical features compared to OI; OI may possibly be a unique biomarker that both laboratory tests and LORRCA fail to fully characterize. Conclusion MIRCA OI and LORRCA EI normoxic deformability are correlated to each other, and both show statistically comparable correlations to many parameters associated with SCD severity. Both OI and EI are significantly associated with SCD outcomes like VOE and acute care. Due to lower device size, cost, and relative ease-of-use, MIRCA may be more convenient for routine clinical use and for use in low resource settings where LORRCA costs and requirements hamper implementation. As the addition of chemical hypoxia to MIRCA is now underway, future work will examine OI under hypoxia and its associations with SCD-relevant outcomes like pain events, stroke and acute chest syndrome.

Digital morphology and complete remote reporting capabilities by scientists and haematologists – a review

Aim: To establish a workflow for full remote reporting of blood films by laboratory haematologists. Methods: A review of a fully digital morphology workflow by a scientist and haematologist was undertaken as an audit to determine the ability of digital morphology to be utilised as a complete workflow process without the aid of a glass slide to assist with authorising referred cases requiring haematologists review. Over 100 slides were first reviewed by a haematology scientist and referred for haematologist examination on the basis of established laboratory referral criteria. These referred films were first analysed by a haematologist via the digital morphology platform. All cases were then analysed by traditional microscopy from the glass slide. Cases were assessed for reliability and concordance of the digital morphology platform to standard glass slide analysis. Cases not concordant between the digital platform and glass slide review were reassessed by an extended scan function of the digital morphology platform and concordance and reliability were again assessed and recorded. Results: One hundred and nineteen cases were assessed by both methods. There was concordance for 102 cases. The discrepant cases were discordant due to missing platelet clumping in two cases, one case missed low number myelocytes on film and the remainder of the cases were discordant due to discrepancies in assessing red cell morphology. Following the review of the extended scan file for each discordant case all 17 discordant cases were able to be assigned as concordant. Conclusion: A digital morphology workflow is a feasible alternative to standard glass slide review for FBC film cases requiring a haematologist review. The addition of the extended scan function allowed all cases to be assigned as concordant between digital morphology platform and glass slide review. This is a promising technology which will allow quicker remote authorisation of cases requiring haematologist intervention for blood film review. This approach provides flexibility in work arrangements and also offers increased access for rural and remote laboratories to centralised specialist expertise in a timely manner.

Polystyrene microsphere monolayer assembled on glass slide for label-free OIRD immunoassay with enhanced sensitivity

Oblique-incidence reflectivity difference (OIRD) is a newly established method to monitor various surface processes and shows great prospect for real-time and label-free immunoassay, yet its sensitivity needs essential improvement to meet the requirement of practical applications. In this work, a label-free OIRD immunoassay biochip was developed with improved sensitivity by using standard glass slide coated with polystyrene (PS) microsphere monolayer as the substrate. The immunoassay sensitivity of this biochip was found to be greatly improved compared with the conventional biochip built on glass slide. Further on-chip ELISA and optical calculation suggests that the improvement is not only associated with high capture efficiency of this biochip towards its target, but is also benefited from the porous PS monolayer enhanced intrinsic OIRD sensitivity via optical interference enhancement and effective dielectric constant effect. This work may provide valuable insight into the development of high performance label-free OIRD bio-detection devices.

Deep Learning Aided Small-Sized Portable Fluorescence Biochip Reader

We present here the development of a novel fluorescence-based portable biochip reader system, having a novel machine learning method for fluorescence measurement of multianalytes. It is suitable for reading DNA/protein biochips in basic research and clinical diagnosis. We aim to develop a point-of-care diagnostic device in the healthcare sector to accelerate biomedical research at an affordable cost. Innovation is involved in the design of each module of the system that is compact, detachable, and reconfigurable. The system can be used with multiple fluorescent dyes depending on specific requirements. It provides high-sensitivity and high-resolution fluorescent images with lesser scan time compared to currently available state-of-the-art systems. The system is autoaligned/autofocus with a standard microscopic glass slide and custom-developed membrane-based biochip. A new deep learning network Bio-U-Net has been developed for real-time detection and quantification of microarrays. On the real test images, it achieved the precision of 0.9928 and the sensitivity of 0.9698 along with <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$F1$ </tex-math></inline-formula> -score of 0.9812. The system and deep learning network have been extensively tested and these together provide a low-cost option while maintaining the required accuracy.

Flexible, Biocompatible PET Sheets: A Platform for Attachment, Proliferation and Differentiation of Eukaryotic Cells

Transparent, flexible, biaxially oriented polyethylene terephthalate (PET) sheets were modified by bioactive polymer-fibronectin top layers for the attachment of cells and growth of muscle fibers. Towards this end, PET sheets were grafted with 4-(dimethylamino)phenyl (DMA) groups from the in situ generated diazonium cation precursor. The arylated sheets served as macro-hydrogen donors for benzophenone and the growth of poly(2-hydroxy ethyl methacrylate) (PHEMA) top layer by surface-confined free radical photopolymerization. The PET-PHEMA sheets were further grafted with fibronectin (FBN) through the 1,1-carbonyldiimidazole coupling procedure. The bioactive PET-PHEMA-I-FBN was then employed as a platform for the attachment, proliferation and differentiation of eukaryotic cells which after a few days gave remarkable muscle fibers, of ~120 µm length and ~45 µm thickness. We demonstrate that PET-PHEMA yields a fast growth of cells followed by muscle fibers of excellent levels of differentiation compared to pristine PET or standard microscope glass slides. The positive effect is exacerbated by crosslinking PHEMA chains with ethylene glycol dimethacrylate at initial HEMA/EGDA concentration ratio = 9/1. This works conclusively shows that in situ generated diazonium salts provide aryl layers for the efficient UV-induced grafting of biocompatible coating that beneficially serve as platform for cell attachment and growth of muscle fibers.

Optical Photothermal Infrared Microspectroscopy Discriminates for the First Time Different Types of Lung Cells on Histopathology Glass Slides

The debate of whether a glass substrate can be used in Fourier transform infrared spectroscopy is strongly linked to its potential clinical application. Histopathology glass slides of 1 mm thickness absorb the mid-IR spectrum in the rich fingerprint spectral region. Thus, it is important to assess whether emerging IR techniques can be employed to study biological samples placed on glass substrates. For this purpose, we used optical photothermal infrared (O-PTIR) spectroscopy to study for the first time malignant and non-malignant lung cells with the purpose of identifying IR spectral differences between these cells placed on standard pathology glass slides. The data in this feasibility study showed that O-PTIR can be used to obtain good-quality IR spectra from cells from both the lipid region (3000-2700 cm-1) and the fingerprint region between 1770 and 950 cm-1 but with glass contributions from 1350 to 950 cm-1. A new single-unit dual-range (C-H/FP) quantum cascade laser (QCL) IR pump source was applied for the first time, delivering a clear synergistic benefit to the classification results. Furthermore, O-PTIR is able to distinguish between lung cancer cells and non-malignant lung cells both in the lipid and fingerprint regions. However, when these two spectral ranges are combined, classification accuracies are enhanced with Random Forest modeling classification accuracy results ranging from 96 to 99% across all three studied cell lines. The methodology described here for the first time with a single-unit dual-range QCL for O-PTIR on glass is another step toward its clinical application in pathology.

Dichromatic properties of a magnetic fluid thin layer

In the paper, we investigate spectral dependence of transmittance of a thin layer of a magnetic fluid with Fe2O3 nanoparticles and transformer oil ITO 100 as base fluid, as a function of its thickness. Thin layer samples of the magnetic fluid with various thicknesses were prepared using standard microscope glass slides and spacers with defined thicknesses ranging from 25 µm to 530 µm. The stable spatial localization of the thin layer was ensured due to the existence of a surface tension emerging at the interface between the magnetic fluid surface and surfaces of the microscope glass slides. The transmittance as a function of the layer thickness was investigated in visible (VIS) and near-infrared (NIR) spectral ranges and spectral dependence of the coefficient of absorption of the magnetic fluid was determined, too. At the same time, the change of color of the layers depending on their thicknesses was evidenced. The results show the transmittance dependence on the thin layer thickness can be used to design magnetic fluid-based passive optical attenuators and filters.

MO319BEDSIDE URINE SEDIMENT EXAMINATION IN IMMUNOGLOBULIN A NEPHROPATHY PATIENTS PERFORMED BY NEPHROLOGISTS

Abstract Background and Aims Urine sediment microscopy is mostly abandoned by nephrologists nowadays, however it is an important diagnostic tool in kidney and urinary tract diseases. The aim of this study is to emphasize the benefits of urine microscopy performed by a nephrologist. Method A prospective cohort study at Pauls Stradins Clinical University Hospital Nephrology center included patients with histologically confirmed diagnosis of IgA nephropathy from 1st January 2020 till December 2020. Appropriately collected urine samples were examined using manual microscopy within an hour after sample collection and by automated urinalysis. Samples were centrifuged at 4000 rpm for 4 minutes, the supernatant urine was carefully decanted, 1 - 1,5 ml of the left urine was mixed by gentle agitation and placed on a standard glass slide with a cover slip. Sample examination was performed using low (magnification x10) and high power (magnification x40) using brightfield microscopy with a minimum of 10 fields. Results A total of 37 patients (24 men, mean age 42.7 ± 10.9 years) were included in the study. 59.5 % of patients (n = 22) had hematuria based on automated urine sediment analysis and 62.2 % (n = 23) of patients had hematuria based on manual urine microscopy. 45.9 % of patients (n=17) had dysmorphic erythrocytes, 13.5 % of patients (n = 5) had isomorphic red blood cells (RBC) and 40.5 % of patients (n = 15) did not have RBC in urine samples by manual urine microscopy. 54.2 % (13/24) of men and 30.8 % (4/13) of women had dysmorphic RBC in urine. Conclusion Manual urine sediment examination was more sensitive than automated analysis. Majority of IgA nephropathy patients have active urine sediment with hematuria and dysmorphic RBC. Manual microscopy remains an effective and reliable method that can be easily and quickly performed by nephrologists.

Laser-induced scanning transfer deposition of silver electrodes on glass surfaces: A green and scalable technology

A pulsed laser ablation backwriting technique with high repetitive rates is implemented for the fabrication of silver coatings on glass surfaces. This method enables geometrical constraint-free deposition of metallic coatings. These exhibit sufficiently low electrical resistance to be used as electrodes in dielectric barrier discharge (DBD) plasma elements.Ambient air deposition of metallic silver electrodes on standard glass slides is explored using a sub-ns UV laser source, combined with hybrid beam scanning methods. The green nature of the overall deposition process includes a preliminary irradiation scan to homogenise the target surface before the subsequent backwriting step. Metal transfer is achieved by combining two phenomena within a simple beam scanning process: LIRT (laser-induced reverse transfer) of silver from the target to the glass, with a partial and secondary LIFT (Laser-Induced Forward Transfer) of silver from the glass to the target.Appropriate selection of pulse energy and pulse repetition rates, beam scanning velocities and target motion enable the growth of sufficiently thick Ag deposits on glass with the required low electrical resistivity and nearly 2D constraint-free geometry. This method avoids the use of vacuum and liquids, resulting in a cheap, facile and green methodology for the deposition of silver electrodes onto transparent substrate surfaces.

Fluorescence Enhancement on Silver-Plated Plasma Micro-Nanostructured 3D Polymeric Microarray Substrates for Multiplex Mycotoxin Detection

Oxygen plasma micro-nanostructured poly(methyl methacrylate) (PMMA) slides were modified through silver microparticle deposition to create microarray substrates that enhance the emitted fluorescence intensity. Silver deposition relied on a commercially available reagent and was completed in two 30-min incubation cycles of the substrate with the reagent. The fluorescence enhancement achieved using these substrates over flat PMMA slides was determined through the development of a microarray for the multiplexed detection of four mycotoxins, aflatoxin B1, ochratoxin A, fumonisin B1, and deoxynivalenol. It was shown that the implementation of silver-plated oxygen plasma micro-nanotextured PMMA substrates increased the signals obtained for aflatoxin B1 and ochratoxin A by approximately 2.8 times, 5.6 times for deoxynivalenol, and 16-times for fumonisin B1, compared to flat PMMA substrates. Most notably, this signal increase was not accompanied by a significant increase in the non-specific signal. In addition, the spot repeatability both across a single slide as well as between different slides was high, with coefficients of variation lower than 12%. The slides were also stable for at least three months, thus offering a microarray substrate with improved properties compared to standard glass slides, regarding both the absolute spot fluorescence intensity and between spots repeatability.

Flexural wave-based soft attractor walls for trapping microparticles and cells.

Acoustic manipulation of microparticles and cells, called acoustophoresis, inside microfluidic systems has significant potential in biomedical applications. In particular, using acoustic radiation force to push microscopic objects toward the wall surfaces has an important role in enhancing immunoassays, particle sensors, and recently microrobotics. In this paper, we report a flexural-wave based acoustofluidic system for trapping micron-sized particles and cells at the soft wall boundaries. By exciting a standard microscope glass slide (1 mm thick) at its resonance frequencies <200 kHz, we show the wall-trapping action in sub-millimeter-size rectangular and circular cross-sectional channels. For such low-frequency excitation, the acoustic wavelength can range from 10–150 times the microchannel width, enabling a wide design space for choosing the channel width and position on the substrate. Using the system-level acousto-structural simulations, we confirm the acoustophoretic motion of particles near the walls, which is governed by the competing acoustic radiation and streaming forces. Finally, we investigate the performance of the wall-trapping acoustofluidic setup in attracting the motile cells, such as Chlamydomonas reinhardtii microalgae, toward the soft boundaries. Furthermore, the rotation of microalgae at the sidewalls and trap-escape events under pulsed ultrasound are demonstrated. The flexural-wave driven acoustofluidic system described here provides a biocompatible, versatile, and label-free approach to attract particles and cells toward the soft walls.

Digital pathology for primary diagnosis of screen-detected breast lesions-experimental data, validation and experience from four centres.

The rate of deployment of digital pathology (DP) systems for primary diagnosis in the UK is accelerating. The flexibility and resilience of digital versus standard glass slides could be of great benefit in the NHS breast screening programme (NHSBSP). This study aims to document the safety and benefits of DP for preoperative tissue diagnosis of screen-detected breast lesions. Concordance data for glass and digital slides of the same cases from four sites were subjected to detailed concordance-discordance analysis. A literature review of DP in the primary diagnosis of breast lesions is presented, making this the most comprehensive synthesis of digital breast cancer histopathological diagnostic data to date. Detailed concordance analysis of experimental data from two histopathology departments reveals clinical concordance rates for breast biopsies of 96% (216 of 225) and 99.6% (249 of 250). Data from direct comparison validation studies in two histopathology departments, utilising the protocol recommended by the Royal College of Pathologists, found concordance rates for breast histology cases of 99.4% (180 of 181) and 99.0% (887 of 896). An intraobserver variation study for glass versus digital slides for difficult cases from the NHSBSP yielded a kappa statistic of 0.80, indicating excellent agreement. Discordances encountered in the studies most frequently concerned discrepancies in grading attributable to mitotic count-scoring and identification of weddelite. The experience of four histopathology laboratories and our review of pre-existing literature suggests that DP is safe for the primary diagnosis of NHSBSP breast histology specimens, and does not increase the risk of misclassification.

Miniaturized high-throughput synthesis and screening of responsive hydrogels using nanoliter compartments.

The traditional pipeline of hydrogel development includes individual one-by-one synthesis and characterization of hydrogels. This approach is associated with the disadvantages of low-throughput and high cost. As an alternative approach to classical one-by-one synthesis, high-throughput development of hydrogels is still tremendously under-represented in the field of responsive material development, despite the urgent requirement for such techniques. Here, we report a platform that combines highly miniaturized hydrogel synthesis with screening for responsive properties in a high-throughput manner. The platform comprises a standard glass slide patterned with 1 ​× ​1 ​mm hydrophilic regions separated by superhydrophobic liquid-impermeable barriers, thus allowing deposition of various precursor solutions onto the hydrophilic spots without cross-contamination. The confinement of these solutions provided by the hydrophilic/superhydrophobic pattern allows encapsulation of cells within the hydrogel, and enables variation in hydrogel height and width. We have also proved the proper mixing of chemicals within the nanoliter-sized droplets. We have successfully implemented this platform for the synthesis of hydrogels, constructing 53 unique hydrogels, to demonstrate the versatility and utility of the platform. Photodegradation studies were performed on 20 hydrogels, revealing structure/function relationships between the hydrogel composition and photodegradability, and covering the range of degradability from non-degradable to rapidly degradable materials.

Digital Whole Slide Imaging Compared With Light Microscopy for Primary Diagnosis in Surgical Pathology.

The adoption of digital capture of pathology slides as whole slide images (WSI) for educational and research applications has proven utility. To compare pathologists' primary diagnoses derived from WSI versus the standard microscope. Because WSIs differ in format and method of observation compared with the current standard glass slide microscopy, this study is critical to potential clinical adoption of digital pathology. The study enrolled a total of 2045 cases enriched for more difficult diagnostic categories and represented as 5849 slides were curated and provided for diagnosis by a team of 19 reading pathologists separately as WSI or as glass slides viewed by light microscope. Cases were reviewed by each pathologist in both modalities in randomized order with a minimum 31-day washout between modality reads for each case. Each diagnosis was compared with the original clinical reference diagnosis by an independent central adjudication review. The overall major discrepancy rates were 3.64% for WSI review and 3.20% for manual slide review diagnosis methods, a difference of 0.44% (95% CI, -0.15 to 1.03). The time to review a case averaged 5.20 minutes for WSI and 4.95 minutes for glass slides. There was no specific subset of diagnostic category that showed higher rates of modality-specific discrepancy, though some categories showed greater discrepancy than others in both modalities. WSIs are noninferior to traditional glass slides for primary diagnosis in anatomic pathology.

Unobstructed Multiscale Imaging of Tissue Sections for Ultrastructural Pathology Analysis by Backscattered Electron Scanning Microscopy.

Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workflow takes advantage of the very large scan fields possible with modern FE-SEMs that allow for the acquisition of wide-field overview images which can be explored at the ultrastructural level by digitally zooming into the images. Examples from applications in pulmonary research and neuropathology demonstrate the versatility and efficiency of this method. This BSE-SEM-based multiscale imaging procedure promises to substantially simplify and accelerate ultrastructural tissue analysis in pathology.

Wire bonding on glass substrates via vapour deposition of Ag-Ti film

Circuits on glass technology have recently developed in applications such as interposers, fibre optics, and displays. We report an automatable method towards interconnecting to glass circuits via wire bonding with maintained transparency. The method is based on wire bonding Au-Ag, using 25 μm diameter 99.99% pure Au wire. A 5 nm Ti and 500 nm Ag base film are initially deposited via e-beam evaporation onto a standard glass slide, where after wire bonding is preformed. The metal film is then etched from the substrate via ion-milling; remaining intact only in areas shielded from the beam. Cross-sections of the bonded balls before and after metallization removal show high quality continuous interfaces with no intermetallic or micro void formations. Good reliability was indicated by shear testing, remaining above 100 MPa for at least 8 days of ageing at 200 °C. Strong wire bonds were thus obtained on a glass substrate by deposition and selective removal of a Ag-Ti film.

Vibrating Sharp-edge Spray Ionization (VSSI) for voltage-free direct analysis of samples using mass spectrometry.

The development of miniaturized and field portable mass spectrometers could not succeed without a simple, compact, and robust ionization source. Here we present a voltage-free ionization method, Vibrating Sharp-edge Spray Ionization (VSSI), which can generate a spray of liquid samples using only one standard microscope glass slide to which a piezoelectric transducer is attached. Compared with existing ambient ionization methods, VSSI eliminates the need for a high electric field (~5000 V·cm-1 ) for spray generation, while sharing a similar level of simplicity and flexibility with the simplest direct ionization techniques currently available such as paper spray ionization (PSI) and other solid substrate-based electrospray ionization methods. The VSSI device was fabricated by attaching a piezoelectric transducer onto a standard glass microscope slide using epoxy glue. Liquid sample was aerosolized by either placing a droplet onto the vibrating edge of the glass slide or touching a wet surface with the glass edge. Mass spectrometric detection was achieved by placing the VSSI device 0.5-1 cm from the inlet of the mass spectrometer (Q-Exactive, ThermoScientific). VSSI is demonstrated to ionize a diverse array of chemical species, including small organic molecules, carbohydrates, peptides, proteins, and nucleic acids. Preliminary sensitivity experiments show that high-quality mass spectra of acetaminophen can be obtained by consuming 100 femtomoles of the target. The dual spray of VSSI was also demonstrated by performing in-droplet denaturation of ubiquitin. Finally, due to the voltage-free nature and the direct-contact working mode of VSSI, it has been successfully applied for the detection of chemicals directly from human fingertips. Overall, we report a compact ionization method based on vibrating sharp-edges. The simplicity and voltage-free nature of VSSI make it an attractive option for field portable applications or analyzing biological samples that are sensitive to high voltage or difficult to access by conventional ionization methods.

Abstract 2104: Near-infrared fluorescent tumor histology for multiplexed phenotypic identification of immunosuppressive cells

Abstract The purpose of our study was to develop near-infrared fluorophore-labeled antibodies to overcome autofluorescence interference in the visible spectrum, thereby enabling enhanced multiplexed identification of immune cells in human tumor tissue sections. Towards multiplexing, we conjugated each monoclonal primary antibody to a unique hapten tag. Detection of hapten-tagged primary antibodies was accomplished using high-affinity anti-hapten rabbit monoclonal secondary antibodies, which were each conjugated to a near-infrared fluorophore with Emax = 594, 650, 731, or 800 nm. For identification of immunosuppressive cells in tumor tissues, we used formalin-fixed, paraffin-embedded human tissue microarrays comprising malignant breast or prostate tumor core sections. Microarrays contained 12-60 patient biopsy cores with duplicate sections per case mounted on standard glass slides. Two antibody panels to detect immunosuppressive cell phenotypes were used in our study. The T regulatory panel targeted the antigens CD4, CD8, FOXP3, and LAG-3, allowing for phenotypic identification of CD4+ T helper cells, CD8+ cytotoxic T cells, CD4+ FOXP3+ Tregs, and highly immunosuppressive CD4+ FOXP3+ LAG-3+ Tregs. The macrophage panel targeted the antigens CD3, CD20, CD68, and CD163 for identification of CD3+ T cells, CD20+ B cells, CD68+ M1 macrophages, and highly immunosuppressive CD163+ M2 macrophages. Widefield fluorescence microscopy was used to image the stained tumor microarrays, utilizing a high-radiance mercury arc lamp, high-sensitivity digital CMOS camera, and single-band optical filter sets to detect near-infrared fluorescence. Triplicate images were taken per tissue core. Prior to image analysis, a spectral unmixing algorithm was created using a reference set of single-stained tissue slides in order to deconvolute minor spectral overlap of near-infrared fluorophores. The algorithm was then applied to the multiplexed digital raw images, and immunophenotyping analysis was conducted to enumerate cells of interest. Image analysis results indicated unique patterns of distribution of immune cells in tumor tissues correlated with tumor grade and stage. In breast cancer tissues, higher levels of LAG-3+ immunosuppressive cells were found in tumors of the aggressive, treatment-resistant "triple-negative" tumor subtype. In prostate cancer tissues, an increase in CD68+ macrophages and loss of immunostimulatory CD3+ T cells and CD20+ B cells were found to be associated with advanced tumor grade and Gleason score. The use of near-infrared fluorophores and hapten-tagged detection antibodies facilitated enhanced, rapid multiplexed phenotyping of immunosuppressive cells in tumor tissue. These findings demonstrate our approach to be a potentially paradigm-shifting technology for detection of immune infiltrates in patient samples. Citation Format: Amy Flor, Annalise Vaccarello, Matt Levin, Helen Snyder, David Schwartz, Stephen Kron. Near-infrared fluorescent tumor histology for multiplexed phenotypic identification of immunosuppressive cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2104.