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[Methods in Molecular Biology] T-Cell Motility Volume 1930 (Methods and Protocols) || Live Imaging of Resident T-Cell Migration in Human Lymphoid Tissue Slices Using Confocal Microscopy
摘要： In order to mount a potent immune response, immune cells must move actively through tissues. As an example, T-cell need to migrate within lymph nodes in order to scan the surface of many dendritic cells and recognize rare expressed antigens. The recent development of improved imaging approaches, such as two-photon microscopy, and the use of powerful mouse models have shed light on some of the mechanisms that regulate the migration of immune cells in many organs. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tissue samples. However, these studies lack a temporal dimension since samples have been fixed. In order to overcome some of these limitations, we describe, in this methodology chapter, an experimental system of fresh human adenoid slices to monitor the dynamics of resident T-lymphocytes that have been stained with directly-coupled fluorescent antibodies. Combined with confocal fluorescent imaging, this preparation offers an effective approach to imaging immune cells in a three-dimensional (3D) human lymphoid tissue environment.
关键词： Immunostaining,Confocal microscopy,Motility,Vibratome,T-cells
更新于2025-09-23 15:22:29
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[Methods in Molecular Biology] T-Cell Motility Volume 1930 (Methods and Protocols) || Three-Dimensional Structured Illumination Microscopy (3D-SIM) to Dissect Signaling Cross-Talks in Motile T-Cells
摘要： Visualization of signal transduction events in T-cells has always been a challenge due to their miniscule size. Recent advancement in super-resolution microscopy techniques presents many new opportunities to navigate the spatial and temporal signaling cross-talks in motile T-cells. Here, we provide technical details, optimal conditions, and critical practical considerations that need to be taken into account during cell handling, sample preparation, and image acquisition of motile T-cells for performing three-dimensional structured illumination microscopy (3D-SIM).
关键词： 3D-SIM,Immunostaining,Super-resolution microscopy
更新于2025-09-23 15:22:29
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Nanoscale Imaging of Kidney Glomeruli Using Expansion Pathology
摘要： Kidney glomerular diseases, such as the minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and other nephrotic syndromes, are typically diagnosed or confirmed via electron microscopy. Although optical microscopy has been a vital tool to examine clinical specimens for diagnoses in pathology for decades, the optical resolution is constricted by the physical diffraction limit of the optical microscope, which prevents high-resolution investigation of subcellular anatomy, such as of the podocyte tertiary foot processes. Here, we describe a simple, fast, and inexpensive protocol for nanoscale optical imaging of kidney glomeruli. The protocol is based on Expansion Pathology (ExPath), a new principle of microscopy that overcomes optical diffraction limit by chemically embedding specimens into a swellable polymer and physically expanding it homogenously prior to imaging. Our method uses only commercially available reagents, a conventional fluorescence microscope and it can be applied to both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. It requires minimal operative experience in a wet lab, optical microscopy and imaging processing. Finally, we also discuss challenges, limitations and prospective applications for ExPath-based imaging of glomeruli.
关键词： imaging,kidney glomerulus,immunostaining,expansion microscopy,tissue expansion,nanoscopy
更新于2025-09-09 09:28:46