Tools for imaging intracellular lipid distributions with high-resolution secondary ion mass spectrometry

Lipid metabolism and intracellular transport regulate the relative abundances of lipids and cholesterol within organelles. Thus, disturbances in lipid metabolism and transport result in abnormal accumulations of lipids within intracellular membranes and are often a hallmark of lipid-mediated health defects. Cellular imaging with a Cameca NanoSIMS in depth profiling mode has enabled the visualization of the three-dimensional (3D) distributions of isotopically labeled sphingolipids and cholesterol with high spatial resolution. However, identifying organelles can be challenging because their shapes are distorted in the z-direction in depth profiling images, and they must contain distinct stable isotopes or nonnative elements to enable their detection with a NanoSIMS. Therefore, we have developed strategies that facilitate better visualization and identification of organelles. First, we created a cell line that expresses organelle-specific proteins fused to small enzymes that covalently attach to small-molecule ligands. Functionalizing these ligands with fluorophores and distinctive elements or isotopes enables detection of the organelle-specific protein-enzyme construct they attach to with both SIMS and fluorescence microscopy. Additionally, we designed new sample preparation procedures to enable more robust cell-to-substrate attachment and high pressure freezing (HPF) of cells in a near-native state. Cryopreservation with HPF better resolved intracellular features when imaged with TEM. 3D NanoSIMS depth profiling images of cells expressing the organelle-specific protein-enzyme construct labeled with our custom ligands and preserved using high pressure freezing will allow assessment of the distributions of cholesterol and sphingolipids in hard to resolve organelles. Second, we use the secondary electron images acquired during secondary ion mass spectrometry (SIMS) depth profiling to predict the cell morphology and reshape the 3D NanoSIMS depth profiling images. This revealed the distributions of the sphingolipids and cholesterol in organelles and other intracellular compartments. Following strategy development, improvements allow detecting nonconstant sputter rates and increasing accessibility through automation and a user interface. Reshaping 3D NanoSIMS depth profiles of organelles using the novel depth correction strategy will improve organelle morphology and allow spatial assessment of the lipid distribution in cells. This information may provide insight into lipid-related health problems.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Quant Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Gorman, Brittney L

Contributors dc:contributor

• Kraft, Mary L
• Brooke, Christopher B
• Das, Aditi
• Shen, Mei

• NanoSIMS
• secondary ion mass spectrometry
• MATLAB
• secondary ion mass spectrometry (SIMS)
• Topograpic correction
• Morphology
• Depth profiling
• Lipids
• Cholesterol
• Sphingolipids
• Organelle
• Endosomes
• Cellular membranes
• sputter rate
• reconstruction
• secondary electron
• Focused ion beam

Statement dc:rights

• Copyright 2022 Brittney L. Gorman

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/115869

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F115869