Seq-Scope-eXpanded: spatial omics beyond optical resolution.

Spatial omics integrates molecular profiling with spatial tissue context, enabling high-resolution analysis of gene expression, protein interactions, and epigenetic modifications. This approach provides critical insights into disease mechanisms and therapeutic responses, with applications in cancer, neurology, and immunology. Spatial omics technologies, including spatial transcriptomics, proteomics, and epigenomics, facilitate the study of cellular heterogeneity, tissue organization, and cell-cell interactions within their native environments. Despite challenges in data complexity and integration, advancements in multi-omics pipelines and computational tools are enhancing data accuracy and biological interpretation. This review provides a comprehensive overview of key spatial omics technologies, their analytical methods, validation strategies, and clinical applications. By integrating spatially resolved molecular data with traditional omics, spatial omics is transforming precision medicine, biomarker discovery, and personalized therapy. Future research should focus on improving standardization, reproducibility, and multimodal data integration to fully realize the potential of spatial omics in clinical and translational research.

SAT0275 PERFORMANCE OF THE ANTISYNTHETASE ANTIBODIES AND THEIR INDIRECT IMMUNOFLUORESCENCE PATTERNS IN THE ANTISYNTHETASE SYNDROME DIAGNOSIS

Spatially mapping the transcriptome and proteome in the same tissue section can profoundly advance our understanding of cellular heterogeneity and function. Here we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multimodal spatial omics approach combining sequencing-based spatial transcriptomics and multiplexed protein imaging on the same section, enabling both single-cell-resolution cell typing and transcriptome-wide interrogation of biological pathways. DBiTplus utilizes spatial barcoding and RNase H-mediated cDNA retrieval, preserving tissue architecture for multiplexed protein imaging. We developed computational pipelines to integrate these modalities, allowing imaging-guided deconvolution to generate single-cell-resolved spatial transcriptome atlases. We demonstrate DBiTplus across diverse samples including frozen mouse embryos, and formalin-fixed paraffin-embedded human lymph nodes and lymphoma tissues, highlighting its compatibility with challenging clinical specimens. DBiTplus uncovered mechanisms of lymphomagenesis, progression and transformation in human lymphomas. Thus, DBiTplus is a unified workflow for spatially resolved single-cell atlasing and unbiased exploration of biological mechanisms in a cell-by-cell manner at transcriptome scale.

Convergence: Multi-omics and AI are reshaping the landscape biomedical research.

Automated classification of immunostaining patterns in breast tissue from the human protein atlas

Pompe disease (PD) is caused by deficiency of the enzyme acid α-glucosidase resulting in glycogen accumulation in lysosomes. Clinical symptoms include skeletal myopathy, respiratory failure, and cardiac hypertrophy. We studied plasma proteomic and lipidomic profiles in 12 PD patients compared to age-matched controls. The proteomic profiles were analyzed by nLC-MS/MS SWATH method. Wide-targeted lipidomic analysis was performed by LC-IMS/MS, allowing to quantify >1100 lipid species, spanning 13 classes. Significant differences were found for 16 proteins, with four showing the most relevant changes (GPLD1, PON1, LDHB, PKM). Lipidomic analysis showed elevated levels of three phosphatidylcholines and of the free fatty acid 22:4, and reduced levels of six lysophosphatidylcholines. Up-regulated glycolytic enzymes (LDHB and PKM) are involved in autophagy and glycogen metabolism, while down-regulated PON1 and GPLD1 combined with lipidomic data indicate an abnormal phospholipid metabolism. Reduced GPLD1 and dysregulation of lipids with acyl-chains characteristic of GPI-anchor structure suggest the potential involvement of GPI-anchor system in PD. Results of proteomic analysis displayed the involvement of multiple cellular functions affecting inflammatory, immune and antioxidant responses, autophagy, Ca2+ -homeostasis, and cell adhesion. The combined multi-omic approach revealed new biosignatures in PD, providing novel insights in disease pathophysiology with potential future clinical application.

T cells mediate antigen-specific immune responses to disease through the specificity and diversity of their T-cell receptors (TCRs). Although determining the spatial distributions of T cell clonotypes in tissues is essential to understanding T cell maturation and behavior, spatial sequencing methods remain unable to profile the TCR repertoire. We have developed Slide-TCR-seq, a 10-μm-resolution method to sequence whole transcriptomes and TCRs within the spatial context of intact tissues. We first confirmed the ability of Slide-TCR-seq to map the characteristic architecture of T cells and their receptors in mouse spleen. In human reactive lymph node and tonsil, we identified spatially distinct TCR repertoires both between germinal center (GC) and non-GC regions as well as between GCs. We then spatially profiled T cell clonotypes and their infiltration in renal cell carcinoma specimens before and following immune checkpoint blockade, which identified uniquely distributed T cell clonotypes and transcriptional states, highlighting the infiltration and expression heterogeneity both between clonotypes and within a T cell clonotype. Our method is anticipated to facilitate dissection of the immune microenvironment, yielding insights into the complex spatial relationships between T cell clonotypes, neighboring cell types, and gene expression that drive T cell development and response to disease. This work was supported in part by the NIH U19AI082630, the Dana-Farber/Harvard Cancer Center Kidney Cancer SPORE (NCI P50CA101942), and the NCI IMAT grant.

Introduction: Low-cost optical microscopes are under development by several groups to bring point-of-care malaria diagnosis services to under-served communities in low-resource settings. We describe a Fourier ptychography microscope (FPM) system based on an open-source design and a method of measuring spatial resolution in terms of the optical modulation transfer function (MTF) using images of the 1951 USAF resolution test device. <br/> <br clear="all"/> Methods: The FPM system uses a Raspberry Pi computer and 196-LED matrix light source. FPM images were reconstructed using in-house Python code. The MTF was determined from bar-patterns using a least-squares analysis to fit a square wave, including odd-harmonic terms, to image profiles through the bars. These were normalized to larger uniform regions of the pattern and combined to generate the MTF. <br/> <br clear="all"/> Results: Total component cost of the FPM was less than $200. The theoretical diffraction limit imposed by the pupil function of this system was 280 cycles/mm, slightly less than the measured MTF 10% frequency of 300 cycles/mm. The 10% frequency in FPM images was 550 cycles/mm. <br/> <br clear="all"/> Conclusions: The USAF test pattern provides a practical method for assessing FPM performance in terms of the achieved MTF of the $200 Raspberry Pi based Fourier ptychography microscope. The limiting frequency in FPM images, 550 cycles/mm, was slightly less than the Nyquist sampling cut-off frequency of 670 cycles/mm imposed by pixel spacing.

A pump-probe variant of the photothermal microscope is used to enhance the resolution of the scanning optical microscope. The improved optical resolution is demonstrated on etched structures in Si. Special attention is paid to non-linear effects that can both improve and degrade the resolution. It is shown that the deterioration of the resolution in Si is due to saturation of the photoreflectance signal.

SUMMARYThe resolution of modern transmission electron microscopes reaches the physical limits imposed by lens aberrations and energy width. One of the many conditions to be fulfilled, the alignment of illuminating and imaging beam onto the coma‐free objective axis, is particularly discussed here since axial coma cannot be detected by the usual resolution‐checking methods. Space consumption of specimen stages prevents the full utilization of the magnetic saturation limit only in the 100 keV range. With higher energies, this handicap is obviated, and some additional advantages can be gained which promote material investigations at atomic resolution, and which are presently utilized in instrumental research projects. High resolution with biological specimens has up to now been unsuccessful because of radiation damage. Optimum utilization of all electrons scattered at the specimen must thus be given priority over optical resolution. Important instrumental requirements are minimum exposure beam control, imaging modes with high collection efficiency, and recording devices with high detection quantum efficiency connected on‐line to image processors. A remarkable decrease in beam sensitivity of organic crystals, by more than one order, has been found by cooling the specimen down to 4 K which, by the use of superconducting lenses, can be combined with both ultra high vacuum and the stability requirements for high resolution. Yet up to now, such protection has not been achieved with He cryostates in conventional lenses, perhaps because a temperature increase even of only a few degree K is harmful.Purely magnetic imaging energy filters are about to be developed to a high optical quality but have been employed so far in only a few high resolution instruments. Such filters allow removal of the inelastic background and thus improvement of contrast of images of low‐Z specimens, particularly in the dark field mode.Finally, some ‘non‐conventional’ projects have made progress. Correction of spherical and chromatic aberration by multipole lenses offers a chance to improve remarkably the resolution in the 100 keV range, to extend the bandwidth of phase contrast transfer and to obtain highly resolved information about inelastic images when an energy filter is also applied. Electron holography provides possibly useful large area phase contrast, particularly if the electron energy is decreased, which may be of great benefit in investigations of unstained specimens.

Targeting rheumatoid arthritis biomarkers: High-abundance protein depletion by silicomolybdic acid-functionalized metal-organic frameworks.

Proteomic biomarkers in rotator cuff disease: Current evidence and translational implications from a systematic review and network analysis.

Clusterin is an enigmatic glycoprotein with a nearly ubiquitous tissue distribution. It plays important roles in various pathophysiological processes, including tissue remodeling, reproduction, lipid transport, complement regulation, and apoptosis. Clusterin appears to have two main isoforms that result from alternative splicing. The secreted and nuclear forms of clusterin have been reported to play different roles in human malignancies. The purpose of this study was to examine clusterin immunoexpression and its clinical significance in a group of Chinese patients with non-small cell lung cancer (NSCLC). Tissue samples from the primary tumors of 121 patients with completely resected NSCLC were obtained. Clusterin protein expression was evaluated by immunohistochemical staining with an antibody against all clusterin isoforms. Staining patterns were observed and graded based on intensity and density and were correlated with clinical and pathological data. Both cytoplasmic and nuclear clusterin immunostaining patterns were observed. Clusterin staining was observed only in the cytoplasm in 70 patients (57.9%), only in the nucleus in 27 patients (22.3%), and in both the cytoplasm and nucleus in 16 patients (13.2%). A significant association was observed between positive cytoplasmic clusterin expression and histologic type as indicated by adenocarcinomas that were more likely to have clusterin staining only in the cytoplasm. Clusterin immunostaining was neither associated with recurrence-free survival (RFS) nor overall survival of patients by univariate or multivariate analysis. For patients undergoing chemotherapy, those with only cytoplasmic clusterin staining had worse survival than other patients. In conclusion, both cytoplasmic and nuclear immunostaining patterns of clusterin were detected in the tumors of patients with NSCLC. Adenocarcinomas were more likely to have only cytoplasmic staining. The immunoexpression of clusterin was not associated with prognosis, and cytoplasm-only immunostaining of clusterin was inversely correlated with chemosensitivity in this group of patients.

BackgroundCorynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage.ResultsThe experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen.ConclusionsThis study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America