High Speed EELS and EFTEM Analysis across the Visual Cortex

Abstract

Unstained biological materials are traditionally difficult to analyze in the TEM as they show very little contrast and more importantly, they are quite susceptible to electron beam damage if extra care is not taken when performing the experiment. Biological materials are almost entirely composed of carbon but in some areas they show other elements in small amounts. Electron Energy Loss Spectroscopy (EELS) is well suited to studying such materials given EELS high sensitivity to light element and its high collection efficiency. The paper shows how quantitative information can be obtained from a region in a TEM sample across a blood vessel capillary region.Electron Energy Loss Spectroscopy (EELS) is the analysis of the energy distribution of the electrons that have passed through a thin sample and have interacted with it inelastically. EELS is a very powerful technique capable of providing chemical and electronic information from particular areas in the sample. Spatial information can be obtained using two approaches: the first method is to combine EELS with a scanning transmission electron microscope (STEM) where the electron probe is scanned across a selected area in the sample and an EELS spectrum is collected point by point across the scan giving a Spectrum Image (SI). The second method is to use Energy Filtering Transmission Electron Microscopy (EFTEM) approach. EFTEM utilizes a special type of EELS spectrometer which has the capability to create images using only electrons that have lost a narrow range of energies after interacting with the specimen. Concentrating on a particular ionization edge, it is possible to build up images which show a two dimensional distribution of a particular element. In this paper, both EFTEM and EELS SI approaches have been used to reveal the elemental distribution across a selected area in a sample obtained from human autopsy tissue The TEM sample, obtained from human autopsy, was fixed in formalin and glutaraldehyde, post fixed in osmium tetroxide and uranyl acetate, dehydrated in ethyl alcohols and propylene oxide embedded in epoxy resin. The TEM sample was also coated with 5Å carbon on both sides to prevent any charging effect due to the electron beam interacting with the sample. EFTEM and EELS STEM SI datasets were acquired using a JEOL 2010 equipped with a LaB 6 electron source and STEM capability. Attached to the bottom of the microscope was a Gatan GIF Quantum ER imaging filter and EELS spectrometer. Figure 1 shows an unfiltered conventional TEM image of the region in the sample across a capillary blood vessel. Even though the TEM sample was slightly stained, the contrast shown in the unfiltered TEM image is poor. This is due to the fact that the sample is mainly made of carbon and other light elements such as O. However by removing the contribution of inelastic scattering as shown in Figure 2, the contrast can be greatly improved and hidden details and features are now revealed. These features are typically related to the varying composition and presence of other element...

DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos

“…1c ). We next used energy-filtered transmission electron microscopy (EFTEM) for elemental mapping of nitrogen (indicative of proteins) and phosphate (indicative of nucleic acids) in 2-cell embryos 21 . Similar to what was observed with OsO 4 /uranyl acetate staining, phosphorus was enriched in a clear compartment within each vesicle of each DDX1 aggregate (Fig.…”