Optimal Diverse Biological Sample Preparation Methods for 2D and 3D Electron Microscopy Imaging
Year: 2023
Type: conference-abstract
Abstract: Modern electron microscopy permits scientists to study the fine details of cells and tissues, utilizing both two-dimensional and three-dimensional imaging modalities due to a resurgence of interest in the ultrastructural complexes of biological samples, and the development of novel instrumentation and advances in specimen preparation technologies [1, 2, 3, 4]. As a research driven microscopy core laboratory sited in a fast-growing medical school, we have opportunities working on a wide spectrum of biological samples. To achieve the best possible imaging results, it is important to design each individual project in details with proper experimental approach in order to obtain optimal preservation of ultrastructure for any given biological samples [5], and apply practical strategy to localize targeted proteins on subcellular level[6]. In vitro cultured cells serve as a model system is quite popular in biological research field to study ultrastructural changes under different experimental conditions. Morphological well-preserved mitochondria in cells or tissues are essential to the quantitative measurement of mitochondrial fusion or fragmented dynamics linked to cellular metabolism of cancer research. We have established successful protocols to visualize mitochondrial structure in different kinds of in vitro cultured cells, and figured out methodology to visualize mitochondria using limited amount of in vivo isolated immune cells such as T cells and B cells[5]. Microsporidia are a divergent group of single-celled eukaryotic parasites, which infect a wide range of hosts including nematodes, insects and vertebrates, and they form spores to survive outside of the host. Study the packaging of polar tube, the dynamics of polar tube extrusion, and investigate the mechanism of polar tube-based cell infection, is quite interesting. We compared different sample preparation methods, ranging from conventional chemical fixation using glutaraldehyde-osmium tetroxide (GA-OsO4), to reduced osmium-thiocarbohydrazide (TCH)-osmium (rOTO) and high pressure freezing-freeze substitution (HPF-FS), and imaged samples under SEM, TEM and serial block face-SEM (SBF-SEM). Our results indicated that traditional GA-OsO4 fixation can preserve the fine structure of bacteria spores with superior resolution comparing with the rOTO procedure. However, the rOTO procedure generates better imaging contrast by SBF-SEM, and the resolution of SBF-SEM images are sufficient for building three-dimensional model of microsporidian spores [6]. Recently, we developed appropriate sample preparation strategies for imaging whipworm eggs which related to malnutrition, stunted growth and dysentery in human. As current available therapies only target adult worms, a therapy that targets the vulnerable egg hatching stage might be an important new method to tackle this global health burden. We used experimental model of whipworm to study hatching induction of the egg, as a part of research to develope the drug treatments of these infections. Given the unique shape and size of those eggs, we established an experimental approach to prevent sample lost during SEM sample preparation. By comparing different sample preparation protocols, we concluded that microwave assisted sample preparation method is superior in preservation the ultrastructure of the eggs for serial block face-SEM imaging.
Source: Microscopy and Microanalysis
Author: Feng-Xia (Alice) Liang
Institution: New York University
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4385072370