DOI: 10.6084/m9.figshare.26831113
title: A standardized protocol for sample preparation for scanning electron microscopy to visualize extrachromosomal DNA

description: Extrachromosomal DNA (ecDNA) are circular DNA structures associated with cancer and drug resistance. One specific type, double minute (DM) chromosomes, has been studied since the 1960s using imaging techniques like cytogenetics and fluorescence microscopy. Specialized techniques such as atomic force microscopy (AFM) and scanning electron microscopy (SEM) offer micro to nano-scale visualization, but current sample preparation methods may not optimally preserve ecDNA structure. Our study introduces a systematic protocol using SEM for high-resolution ecDNA visualization. We have optimized the end-to-end procedure, providing a standardized approach to explore the circular architecture of ecDNA and address the urgent need for better understanding in cancer research. Despite advances in extrachromosomal DNA (ecDNA) detection, current methods struggle to reveal ecDNA's architecture within cells. Specialized techniques like scanning electron microscopy (SEM) provide the needed resolution, but existing sample preparation may not preserve ecDNA well. Our study introduces a systematic method using SEM, optimizing procedures for preparing and visualizing metaphase spread samples. This offers a standardized approach to study ecDNA's circular architecture, addressing a pressing need in cancer research. Extrachromosomal DNA (ecDNA) are circular DNAs linked to cancer and drug resistance. Double minute (DM) chromosomes, a type of ecDNA, have been studied since the 1960s using imaging techniques. Although advanced sequencing helps detect ecDNA, it cannot fully reveal their details within cells. Specialized methods like atomic force microscopy (AFM) and scanning electron microscopy (SEM) offer the needed resolution but may not preserve ecDNA well during preparation. Our study introduces a systematic protocol using SEM to visualize ecDNA with high resolution. We have optimized the procedure, offering a standardized approach to study ecDNA's circular architecture, addressing an urgent need in cancer research. Extrachromosomal DNA (ecDNA) amplifies oncogenes, contributing to tumor heterogeneity and drug resistance. Circular ecDNA architecture is proposed to enhance chromatin accessibility and transcriptional capacity over chromosomes. Current methods lack standardized approaches for resolving ecDNA structure at high resolution, posing challenges in understanding its characteristics. Metaphase spread preparation: crucial for scanning electron microscopy (SEM) karyotyping, involving colcemid treatment, hypotonic swelling and fixation. SEM sample preparation: in-depth exploration of contrast enhancement using uranyl acetate, evaluation of dehydration methods and sputter coating optimization. SEM microscope settings: identification of optimal settings with the SE2 detector at 3 kV for obtaining high-quality images of chromosomes and ecDNA. Contrast enhancement findings:Uranyl acetate alone or in combination with osmium tetroxide provides optimal contrast enhancement.Osmium tetroxide use has additional handling concerns and increases processing time. Uranyl acetate alone or in combination with osmium tetroxide provides optimal contrast enhancement. Osmium tetroxide use has additional handling concerns and increases processing time. Dehydration and drying methods:Ethanol dehydration followed by air drying is identified as the most suitable method for preserving chromosome and ecDNA structure.Critical point drying and hexamethyldisilazane (HMDS) result in structural compromises, with HMDS causing chromosome expansion. Ethanol dehydration followed by air drying is identified as the most suitable method for preserving chromosome and ecDNA structure. Critical point drying and hexamethyldisilazane (HMDS) result in structural compromises, with HMDS causing chromosome expansion. SEM microscope settings and sample age effects:SE2 detector at 3 kV is identified as the best choice for obtaining high-quality images.Freshly prepared samples retain chromosome structures better and contain more ecDNA compared with samples stored for months. SE2 detector at 3 kV is identified as the best choice for obtaining high-quality images. Freshly prepared samples retain chromosome structures better and contain more ecDNA compared with samples stored for months. Optimized protocol importance: current SEM sample preparation practices may require optimization for studying ecDNA ultrastructure. Broad applicability: the optimized protocol offers inroads for studying ecDNA structure–function relationships in other cells with double minute chromosomes. Contribution to research community: valuable for microscopy cores and scientific labs interested in optimal imaging of ecDNA, contributing to cancer research and broader scientific understanding.