Integrated Workflow for Chromatin Architecture Analysis in Rare Thymic Cells Using Focused Ion Beam Scanning Electron Microscopy v1
Year: 2025
Type: preprint
Abstract: The thymus, a primary lymphoid organ, plays a critical role in T-cell development and selection including the establishment of central tolerance. To investigate chromatin organisation in cells that regulate thymocyte differentiation, we developed an optimised workflow to quantify heterochromatin-to-euchromatin ratios among different thymic cell types. Traditional electron microscopy (EM) sample preparation methods often require substantial cell quantities, posing challenges for studying rare or low-abundance cells. Our workflow addresses this limitation by enriching cells using fluorescence-activated cell sorting (FACS), followed by chemical fixation and embedding in alginate hydrogel. Samples were then prepared for electron microscopy using a reduced osmium-thiocarbohydrazide-osmium (rOTO) staining protocol, and lead citrate (Walton’s Lead) post-staining provided exceptional chromatin contrast. High-resolution 3D imaging using focused ion beam scanning electron microscopy (FIB-SEM) allowed detailed analysis of nuclear architecture. This approach revealed chromatin remodelling dynamics in thymic cells regulating T-cell development and offered a versatile tool for structural studies of rare cell populations. Graphical Abstract
Authors: Zulfeqhar A. Syed, Ye Sun, Mami Matsuda-Lennikov, Yousuke Takahama, Christopher K. E. Bleck
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4411363673