Cryo-Scanning Electron Microscope with Energy Dispersive Spectroscopy
Lead Research Organisation:
University of Warwick
Department Name: School of Life Sciences
Abstract
Electron microscopes use an electron beam to make images of samples - such as biological cells and tissues - at a very high level of detail. In a scanning electron microscope (SEM), the interaction of the beam with the sample is measured by looking at electrons that bounce off the sample (backscattered electrons) and electrons that are released by the sample when the beam hits it (secondary electrons). The electrons are also scattered by air molecules, so the inside of the microscope is held under vacuum.
The vacuum is a major problem for biomedical samples, because they contain up to 90% water, which will evaporate in the vacuum. This would distort the sample, se we would not get a good image of what it really looked like. To make the samples stable in the vacuum, we can dehydrate them, or replace this water with plastic. By cutting the plastic-embedded sample in thin slices and imaging each slice, we can make a 3D model of the original sample; this is called array tomography. This gives a lot of information about the spatial organisation within cells and tissues.
Alternatively, in cryo-SEM the sample is frozen to below -160 degrees C using liquid nitrogen, and kept cold in the vacuum of the microscope. This allows us to image the samples in an undisturbed (near-native) state without dehydration or embedding. This is particularly useful for samples that contain very large amounts of water, such as biofilms - these are slimy layers formed by bacteria sticking together. Biofilms are particularly important because they are found in many infections and protect bacteria from antibiotic treatments. Studying how the biofilms form and how they respond to drugs will help scientists develop better treatments for infections with a biofilm component, and surfaces that resist biofilm formation. Our research groups have life-like models for clinically relevant biofilms, such as those found in the lungs, wounds, or gut, and on urinary catheter tubing and ventilators, and they actively develop new ways of combating biofilms. The cryo-SEM will make our research work more precise and accurate than it is using a 'traditional' SEM. For instance, if we want to develop a new drug that penetrates biofilm slime better, then the disruption and distortion caused by dehydration of the biofilm sample makes it harder to see exactly where the drug goes in the biofilm.
Using light microscopy with fluorescent markers and comparing those images with the information from the SEM, we can localise molecules of interest (with fluorescent labels) and see their cellular context, as well as look for rare events that would be harder to find without the fluorescent marker. We will use this method to study membrane traffic, cell division, and the effect of environmental enrichment on mouse brain tissue.
When the electron beam in a SEM hits the sample, X-rays are also released. Using a technique called the energy dispersive spectroscopy (EDS), the X-ray signal can tell us about which elements are present. These elemental signatures allow us to determine where particular molecules are within the sample. Mapping areas with high concentrations of certain elements can also show where certain molecules occur.
These new and exciting techniques will enable research that has not previously been possible at the University of Warwick.
The vacuum is a major problem for biomedical samples, because they contain up to 90% water, which will evaporate in the vacuum. This would distort the sample, se we would not get a good image of what it really looked like. To make the samples stable in the vacuum, we can dehydrate them, or replace this water with plastic. By cutting the plastic-embedded sample in thin slices and imaging each slice, we can make a 3D model of the original sample; this is called array tomography. This gives a lot of information about the spatial organisation within cells and tissues.
Alternatively, in cryo-SEM the sample is frozen to below -160 degrees C using liquid nitrogen, and kept cold in the vacuum of the microscope. This allows us to image the samples in an undisturbed (near-native) state without dehydration or embedding. This is particularly useful for samples that contain very large amounts of water, such as biofilms - these are slimy layers formed by bacteria sticking together. Biofilms are particularly important because they are found in many infections and protect bacteria from antibiotic treatments. Studying how the biofilms form and how they respond to drugs will help scientists develop better treatments for infections with a biofilm component, and surfaces that resist biofilm formation. Our research groups have life-like models for clinically relevant biofilms, such as those found in the lungs, wounds, or gut, and on urinary catheter tubing and ventilators, and they actively develop new ways of combating biofilms. The cryo-SEM will make our research work more precise and accurate than it is using a 'traditional' SEM. For instance, if we want to develop a new drug that penetrates biofilm slime better, then the disruption and distortion caused by dehydration of the biofilm sample makes it harder to see exactly where the drug goes in the biofilm.
Using light microscopy with fluorescent markers and comparing those images with the information from the SEM, we can localise molecules of interest (with fluorescent labels) and see their cellular context, as well as look for rare events that would be harder to find without the fluorescent marker. We will use this method to study membrane traffic, cell division, and the effect of environmental enrichment on mouse brain tissue.
When the electron beam in a SEM hits the sample, X-rays are also released. Using a technique called the energy dispersive spectroscopy (EDS), the X-ray signal can tell us about which elements are present. These elemental signatures allow us to determine where particular molecules are within the sample. Mapping areas with high concentrations of certain elements can also show where certain molecules occur.
These new and exciting techniques will enable research that has not previously been possible at the University of Warwick.
Technical Summary
Scanning electron microscopy (SEM) is perfect for acquiring information about the topology and composition of larger samples to nanometer resolution. For conventional SEM, biomedical samples need to be processed to ensure vacuum compatibility. However, many biological samples depend on water for their structural integrity and dehydration changes their spatial organisation. Cryo-SEM allows biological specimens to be imaged without dehydration by freezing the specimen and keeping it cold during the analysis. Array tomography, the imaging of multiple sections arranged on a flat substrate by SEM, improves the throughput of imaging ultrathin sections compared to conventional transmission electron microscopy (TEM). Combined with in-resin fluorescence, array tomography bridges the gap between traditional high-resolution TEM imaging of embedded samples and fluorescence imaging, thus allowing for targeted imaging of rare events that is extremely time-consuming by conventional TEM. In addition, the composition of samples can be determined by elemental mapping through Energy Dispersion Spectroscopy (EDS) within the SEM.
The Advanced Bioimaging Research Technology Platform at the University of Warwick seeks to implement cryo-SEM and array tomography with EDS to enable cryo-SEM imaging of biological samples in near-native state, improve the throughput of resin section analysis and allow correlative light and electron microscopy. We will use these techniques to enhance research in MRC remit areas including Neurosciences and Mental Health, Molecular and Cellular Medicines, Infection and Immunity and Antimicrobial Resistance.
The Advanced Bioimaging Research Technology Platform at the University of Warwick seeks to implement cryo-SEM and array tomography with EDS to enable cryo-SEM imaging of biological samples in near-native state, improve the throughput of resin section analysis and allow correlative light and electron microscopy. We will use these techniques to enhance research in MRC remit areas including Neurosciences and Mental Health, Molecular and Cellular Medicines, Infection and Immunity and Antimicrobial Resistance.
