![]() This quantitative information, assigned to each pixel of the generated image, supports a more objective, machine-based evaluation of the tissue that is less subject to observer bias. Importantly, the linearity of the mass spectrometry detection of the heavy metals conjugated to each antibody provides a quantitative assessment of the relative expression levels for the respective antigens, allowing researchers to detect changes not just in cell numbers and localization, but also in cell differentiation and signaling pathway activation. Analysis of functional markers generates information on disease states and can potentially be used to identify biomarkers. IMC allows immune cell markers to be investigated in both healthy and diseased tissues with their distribution pattern and proximity to tissue resident cells spatially mapped ( Giesen et al., 2014 Catena et al., 2020 Garcia-Melchor et al., 2021 Patel et al., 2021). The technique can therefore be employed to identify cellular markers that provide fundamental tissue architectural layout, as well as secondary cellular responses such as protein modifications, signaling pathway activation, cell injury states, and cell proliferation. cytoplasm), while the large number of antibodies that can be simultaneously analyzed supports both cell identification and cell activation state determination. ![]() The 1 μm 2 resolution provides the ability to identify basic subcellular localization of antigens (nucleus vs. IMC has also been applied in infectious and autoimmune diseases research and in drug profiling. IMC has been used to characterize malignancies, yielding information about the identity, number of, and spatial relationships between immune and resident cells ( Giesen et al., 2014 Bodenmiller, 2016 Bertocchi et al., 2021 Li et al., 2021). The absence of endogenous signal for the heavy metals results in a very low background and improved signal-to-noise ratio, making IMC particularly appealing as compared to fluorescence-based antibody imaging ( Zhang et al., 2018). ![]() Currently IMC can be employed for imaging up to 42 markers on a single section of tissue. The quantity of each metal present at each 1 μm 2 coordinate is used to reconstruct an artificial multilayer image of the initial tissue. Pulsed ablation of 1 μm 2 spots with a UV laser is performed, with the vaporized tissue analyzed by a mass spectrometer to identify the combination of heavy metals present in that 1 μm 2 region. In IMC, a cocktail of validated antibodies against defined protein epitopes, each of which is covalently bound to a unique rare-earth metal, is hybridized on a tissue section. Imaging mass cytometry (IMC) is a powerful analytical platform in which a high-resolution laser is combined with a mass cytometer that permits mass spectrometry-based, spatially preserved high-dimensional analysis of intact FFPE and frozen tissues at a resolution of 1 μm 2 per pixel ( Giesen et al., 2014 Bodenmiller, 2016 Guo et al., 2020). For a detailed comparison of the strengths and weaknesses of these technologies, please see the comprehensive review by Baharlou et al. To provide that spatial information on such a large number of cells, several platforms for multiplexed imaging have recently been developed, including serial immunofluorescence staining, staining with DNA-barcoded antibodies (CODEX, CO-Detection by indEXing), and staining with metal-tagged antibodies. The large numbers of distinct cell populations and complex cellular arrangement of the human kidney make it particularly difficult to adequately analyze without high-resolution spatial information. ![]() Single-cell RNA sequencing and single nucleus RNA sequencing have markedly increased the depth of information gained from a single biopsy, but lack the spatial information needed to determine cell proximity and cell–cell interactions ( Rost et al., 2017 Cippà et al., 2018 Wu et al., 2018, 2019 Lake et al., 2019 Deleersnijder et al., 2021). Thus, most analyses are descriptive, with few efforts to provide quantitative information about the tubular, stromal, and nonresident cell populations in disease states ( Kretzler et al., 2002 Zhang and Parikh, 2019). The limited amount of tissue obtained from renal biopsy confines the type of analysis performed, preventing extensive analysis at the molecular and cellular level. The predominant methods for cell identification during the pathological analysis of formalin-fixed paraffin-embedded (FFPE) samples from human renal core biopsy tissues include cell morphology and immunohistochemistry or immunofluorescence (IF). ![]()
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |