High-efficiency detector of secondary and backscattered electrons for low-dose imaging in the ESEM.

A new Combined System for high-efficiency detection of Secondary and Backscattered Electrons (CSSBE) in the ESEM consists of three detectors: an ionisation SE detector, an improved scintillation BSE detector, and a new Ionisation Secondary Electron Detector with an electrostatic Separator (ISEDS). The ISEDS optimizes conditions for electron-gas ionisation phenomena in the ESEM to achieve a strongly amplified signal from the secondary electrons with a minimal contribution from backscattered and beam electrons. For this purpose, it is originally equipped with an electrostatic separator, which focuses signal electrons towards a detection electrode and controls the concentration of positive ions above the sample. The working principle of the ISEDS is explained by simulations of signal electron trajectories in gas using the EOD program with our Monte Carlo module. The ability to detect the signal electrons in a selected range of energies is described with Geant4 Monte Carlo simulations of electron-solid interactions and proven by experimental results. High-efficiency detection of the ISEDS is demonstrated by imaging a low atomic number sample under a reduced beam energy of 5 keV, very low beam currents of up to 0.2 pA, and gas pressure of hundreds of Pa.

Imaging of Norway spruce early somatic embryos with the ESEM, Cryo-SEM and laser scanning microscope.

This article describes the surface structure of Norway spruce early somatic embryos (ESEs) as a typical culture with asynchronous development. The microstructure of extracellular matrix covering ESEs were observed using the environmental scanning electron microscope as a primary tool and using the scanning electron microscope with cryo attachment and laser electron microscope as a complementary tool allowing our results to be proven independently. The fresh samples were observed in conditions of the air environment of the environmental scanning electron microscope (ESEM) with the pressure from 550Pa to 690Pa and the low temperature of the sample from -18°C to -22°C. The samples were studied using two different types of detector to allow studying either the thin surface structure or material composition. The scanning electron microscope with cryo attachment was used for imaging frozen extracellular matrix microstructure with higher resolution. The combination of both electron microscopy methods was suitable for observation of "native" plant samples, allowing correct evaluation of our results, free of error and artifacts.