Environmental Scanning Electron Microscopy Techniques

Environmental scanning electron microscopy (ESEM) extends conventional scanning electron microscopy by permitting investigation of specimens in gaseous or hydrated states, thus preserving near-native conditions. By introducing a controlled pressure of water vapour or inert gas into the specimen chamber, ESEM minimises dehydration artefacts and enables imaging of biological, chemical and soft materials without extensive sample preparation. The technique relies on differential pumping to maintain high vacuum in the electron optics while sustaining a gaseous environment around the sample. Gas molecules interact with secondary electrons, amplifying the signal via ionisation and enabling high-contrast imaging even at low accelerating voltages. Modern systems incorporate rapid pressure regulation, cryo-stages and advanced detectors for energy-dispersive X-ray analysis, facilitating in-situ studies of dynamic processes such as condensation, corrosion and biological reactions. Developments in beam control, chamber design and signal processing have broadened ESEM applications from life sciences to materials engineering, environmental science and nanotechnology. This versatility positions ESEM as a key tool for characterising delicate interfaces, monitoring phase transformations and guiding the design of functional surfaces under realistic conditions.

Research from Nature Portfolio

Recent studies have refined in-situ sample preparation to preserve true morphology and chemistry of hydrated specimens. A foundational method employs an extended low-temperature approach within the chamber, stabilising delicate biological and botanical tissues without chemical fixation. This technique allows repeated transfer between low-vacuum ESEM and high-vacuum SEM modes, enabling high-resolution topographical imaging and elemental microanalysis on the same sample. Demonstrations include detailed visualisation of plant wax structures and early embryonic tissues, as well as elemental mapping of root cells, all achieved through a rapid, reagent-free workflow compatible with commercial ESEM platforms equipped with cooling stages.

Research from all publishers

An innovative imaging framework introduces the concept of quasi-force reconstruction to overcome signal attenuation in high-pressure gaseous environments. By analysing temporal variations in secondary electron yield, this approach reconstructs sample morphology at pressures up to several kilopascals without hardware modification, enhancing contrast for weakly scattering nanostructures. Complementary work has combined experimental measurements with computational fluid dynamics to map slip-flow regimes in apertures connecting specimen and intermediate chambers. This study validated a mathematical model of pressure and temperature distributions at low pressures, informing nozzle geometry for optimal beam stability. More recent analyses have focused on the impact of nozzle shaping on supersonic gas expansion behind critical apertures. Through a combination of pressure-temperature sensing and theoretical flow analysis, researchers demonstrated that specific nozzle profiles minimise electron scattering, yielding sharper images and more reliable X-ray microanalysis in differentially pumped systems.

Environmental Scanning Electron Microscopy Techniques publication trend

The graph below shows the total number of articles in environmental scanning electron microscopy techniques across all publications each year (not limited to Nature Index journals).

Technical terms

Environmental scanning electron microscopy (ESEM): A microscopy mode allowing specimen imaging under controlled gas or vapour pressure to preserve native or hydrated states.

Secondary electron: Low-energy electrons emitted from the sample surface upon primary beam interaction, used to generate high-resolution topographical contrast.

Differential pumping: A vacuum architecture employing sequential pumping stages separated by apertures to maintain distinct pressure zones between the electron gun and specimen chamber.

Extended Low Temperature Method (ELTM): An in-situ sample preparation technique using a cooled stage to stabilise specimens in ESEM without chemical treatment, enabling repeated analysis.

Quasi-force: A reconstructed imaging parameter derived from time-domain secondary electron signals, enhancing morphology contrast under high-pressure conditions.

References

1. Quasi‐Newtonian Environmental Scanning Electron Microscopy (QN‐ESEM) for Monitoring Material Dynamics in High‐Pressure Gaseous Environments. Advanced Science (2020).
2. In-situ preparation of plant samples in ESEM for energy dispersive x-ray microanalysis and repetitive observation in SEM and ESEM. Scientific Reports (2019).
3. Slip Flow Analysis in an Experimental Chamber Simulating Differential Pumping in an Environmental Scanning Electron Microscope. Sensors (2022).
4. Mathematical Physics Analysis of Nozzle Shaping at the Gas Outlet from the Aperture to the Differentially Pumped Chamber in Environmental Scanning Electron Microscopy (ESEM). Sensors (2024).