ZEISS EVO provides an easy to use and fast method for sample imaging in histology and pathology applications
Environmental scanning electron microscopy (environmental SEM) offers an efficient imaging method for histology and pathology. In environmental SEM samples are not dehydrated or embedded and can be placed in the microscope after a very few and simple preparation steps. Backscattered electron contrast, used for imaging wet samples under water vapour conditions, reveals high contrast sample details.
Our application note details how the resulting images help to understand 3D architecture of different types of tissue without any serial sectioning and reconstruction. In pathology, environmental SEM imaging can be used to provide direct evidence of pathological tissue conditions as illustrated by an example for liver cirrhosis.
In electron microscopy, the electron optics and sample chamber are usually kept under vacuum (1 mPa) to avoid any interference of gas molecules with the electron beam in order to maintain the beam resolution at the sample surface. However, there are applications where imaging benefits from a gaseous atmosphere within the sample chamber. Variable pressure scanning electron microscopy (VP SEM) is a versatile method for imaging of non-conductive samples. Charging of the sample surface can be avoided by leaking gas (typically air, N2 or H2O) into the sample chamber. Gas molecules positively ionized by the secondary electrons (SE) compensate negative charge build up on the sample surface. VP SEM is mainly used for SE (secondary electron) and BSE (backscattered electron) imaging of non-conductive samples. Typical chamber pressures for VP imaging ranges between 10 – 100 Pa. Even higher gas pressure is used if the sample is hydrated, which usually applies to biological samples. Water vapour is purged into the chamber and reduces the rate of water evaporation from the sample, which in turn stabilizes the sample and slows down the rate of dehydration. Under these conditions images of various biological samples can be acquired before drying artefacts occur.
As a consequence of the interaction of the electron beam with water molecules, the focused electron beam exhibits a skirting effect that degrades image resolution. Nevertheless this is acceptable, because this effect is only noticeable at magnifications above 5000x. On the other hand a wide range of samples can be imaged directly without cumbersome sample preparation procedures. Historically SE contrast is used for visualization of the sample surface in the majority of cases where SEM imaging is performed in a water vapour atmosphere. Typical samples for this imaging regime are plants, fungi and small insects. Recent developments in detector technology have enhanced the sensitivity of variable pressure secondary electron and backscattered electron detectors leading to an improved overall performance of environmental SEM imaging.
In this article we emphasize the application of environmental pressure SEM for another group of samples. In histology text books the microanatomy of different types of tissue is commonly illustrated utilizing images of paraffin sections prepared according to histology protocols. The preparation of these sections, including fixation, staining, embedding and sectioning, is a standardized but nevertheless extensive process. In certain cases, high resolution TEM images of ultrathin sections are added to understand the corresponding ultrastructural details. Quite rarely the text books feature a render perspective visualizing the 3D organization. Even if such illustrations are most helpful to understand how the structure of a tissue is linked to its function, such sketches are difficult to generate, since the artist needs to understand the 3D organization by viewing many single sections first.
We propose the idea to apply environmental SEM imaging as an easy method to visualize histological samples. Utilizing BSE contrast for image generation results in topography-like contrast which allows an immediate understanding of the 3D organization of the imaged tissue. Compared to conventional high vacuum SE contrast imaging, sample preparation for environmental SEM is much easier and less time consuming. Furthermore samples can be imaged in wet state and are therefore kept closer to their physiological morphology. Moreover, environmental SEM can also offer benefits for fast clinical diagnosis when an immediate imaging of samples is requested.
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ZEISS wishes to acknowledge Prof. Matthias Klinger (Institute for Anatomy, University of Luebeck) for his samples and scientific support.