Developing Electron Microscopy Tools for Profiling Plasma Lipoproteins Using Methyl Cellulose Embedment, Machine Learning and Immunodetection of Apolipoprotein B and Apolipoprotein(a)

Abstract

Plasma lipoproteins are important carriers of cholesterol and have been linked strongly to cardiovascular disease (CVD). Our study aimed to achieve fine-grained measurements of lipoprotein subpopulations such as low-density lipoprotein (LDL), lipoprotein(a) (Lp(a), or remnant lipoproteins (RLP) using electron microscopy combined with machine learning tools from microliter samples of human plasma. In the reported method, lipoproteins were absorbed onto electron microscopy (EM) support films from diluted plasma and embedded in thin films of methyl cellulose (MC) containing mixed metal stains, providing intense edge contrast. The results show that LPs have a continuous frequency distribution of sizes, extending from LDL (> 15 nm) to intermediate density lipoprotein (IDL) and very low-density lipoproteins (VLDL). Furthermore, mixed metal staining produces striking “positive” contrast of specific antibodies attached to lipoproteins providing quantitative data on apolipoprotein(a)-positive Lp(a) or apolipoprotein B (ApoB)-positive particles. To enable automatic particle characterization, we also demonstrated efficient segmentation of lipoprotein particles using deep learning software characterized by a Mask Region-based Convolutional Neural Networks (R-CNN) architecture with transfer learning. In future, EM and machine learning could be combined with microarray deposition and automated imaging for higher throughput quantitation of lipoproteins associated with CVD risk.

SARS-CoV-2 virus-like-particles via liposomal reconstitution of spike glycoproteins

“…To prevent artifacts from drying or vitrication of liposomes and VLPs for TEM, a methylcellulose matrix-assisted drying process was used as a structural support for the particles before negative staining with uranyl acetate. 80,81 Briey, 3 mm copper grids with carbon lm (CF200-CU-50 200 mesh) were glow discharged (PELCO easiGlow), then grids were inverted on drops of sample and incubated at room temperature for 60 minutes. The grids were washed with 1× PBS for 1 minute, xed for 5 minutes with 4% paraformaldehyde + 2.5% glutaraldehyde in 0.1 M sodium cacodylate, and then stained with 2% uranyl acetate for 5 minutes.…”

“…80,81 Briey, 3 mm copper grids with carbon lm (CF200-CU-50 200 mesh) were glow discharged (PELCO easiGlow), then grids were inverted on drops of sample and incubated at room temperature for 60 minutes. The grids were washed with 1× PBS for 1 minute, xed for 5 minutes with 4% paraformaldehyde + 2.5% glutaraldehyde in 0.1 M sodium cacodylate, and then stained with 2% uranyl acetate for 5 minutes.…”

Beyond coincidence in Hong-Ou-Mandel interferometry

“…EV were imaged in a JEOL 1200EX electron microscope equipped with a GATAN Orius 200 digital camera. EV were selected for quantification using unbiased counting rule applied to a scanning band [36] and calliper diameters measured either directly on screen or estimated using stereological techniques as previously described [33,36].…”