LVSED bee tongue
Secondary electron (SE) imaging reveals the topographic surface of the sample in high detail and is a valuable technique across scientific fields. However, the majority of secondary electron detectors are not able to function under low vacuum conditions and various backscattered electron (BSE) detector setups are used to investigate sample topography instead. Low vacuum analysis allows an amount of gas into the specimen chamber which can help reduce charging among other things, and is ideal for analysis of biological and/or uncoated samples.聽
The arrival of PEMC's new instrumentation in 2023 has expanded our imaging capabilities during low vacuum analysis with the introduction of the low vacuum secondary electron detector (LVSED) installed on our JEOL IT510 LV SEM, letting us image at low vacuum in higher detail than previously possible without additional time commitments.
Beam interaction
Origin locations of various analytical electrons and X-rays

Preparation of Whole Insects for EM Analysis

This case study features an intact bee that was found deceased prior to being brought into the lab. The bee was mounted onto a brass disc using silver paint to give provide both good conduction and adhesion, before being left in a plate degasser for a few days. This helps to ensure that the bee will be stable under vacuum and is appropriately dry for analysis, and is an easy way to prepare whole insect samples for analysis. The bee was then gold-coated during sample preparation for high vacuum imaging, but this does not mean that low vacuum imaging can't also be carried out.

Setting up the Low Vacuum Experiment

For this comparative study using the JEOL IT510 LV SEM, we made sure that the conditions used for all imaging modes were kept consistent. This means that that the accelerating voltage, spot size/probe current, working distance, magnification, stage rotation, field of view, and image capture settings did not change. Once an area of interest was identified, it was imaged using a typical low vacuum BSE mode, LVSED mode, and then the specimen chamber was brought down to high vacuum mode to engage standard SE imaging. The images below show the differences between these imaging modes.

Uses for LVSED Imaging

LVSED imaging provides significantly more topographic detail than BSE imaging under the same conditions. Whilst operating conditions can be optimised for low vacuum BSE, LVSED allows for faster analysis, greater ease of use, as well as improved resolution. The JEOL IT-510 LV SEM also allows for a good deal of control over the specimen chamber pressure up to 650 Pa, and can be adjusted during analysis to find the best balance between pressure and detector signal for the sample in question.聽
As such, LVSED imaging is a powerful tool for researchers and clients looking to investigate their samples without coating, or for delicate samples that do not hold up well under the electron beam, and can be combined with energy dispersive spectroscopy (EDS), allowing for qualitative compositional analysis as well as imaging.

Limitations to Consider

As the LVSED the detector is in a fixed position so setting up the analysis may require some time to find the best stage and specimen geometry for a given sample which should be factored in to experiment times. Additionally - although a significant improvement on BSE imaging - LVSED imaging cannot produce the same resolution as high vacuum SE imaging. We would recommend that samples that are able to be coated are done so in order to generate the best images possible.
Guidance from the team at Plymouth Electron Microscopy Centre (PEMC) regarding sample preparation and analysis is available to all our users.