Peering directly into the nanoscale world of molecules in their native liquid environment—in real time
Imagine trying to observe a swimming fish while the water around it has been frozen solid. Your view would be static, unnatural, and missing the entire essence of its movement. For decades, this has been the fundamental challenge for scientists trying to observe the molecular machinery of life and materials in action.
Traditional electron microscopy requires samples to be placed in a high vacuum, forcing researchers to freeze-dry or crystallize their specimens.
In-liquid electron microscopy and diffraction allow us to peer directly into the nanoscale world of molecules dancing in their native liquid environment.
This isn't just an incremental improvement; it's a paradigm shift that is transforming how we study everything from protein interactions to battery materials, opening windows into processes we could previously only infer 1 .
Electron microscopes use beams of electrons instead of light to achieve magnifications thousands of times greater than possible with ordinary light microscopes. These electrons have extremely short wavelengths (around 2 picometers) compared to visible light, allowing them to resolve individual atoms .
The fundamental problem with studying liquid samples has been straightforward: you can't put a puddle of water in a high vacuum chamber and expect it to stay there.
The breakthrough came with the development of specialized nanofluidic cells that act as miniature aquariums for the electron microscope 6 .
Electron Wavelength
Beam Path Tunability 8
Resolution Level
Sample Environment
Simple design with stable environment for studying fundamental biological processes like DNA hybridization 1 .
Enable reagent mixing and chemical changes during observation, allowing study of dynamic processes 3 .
Thinnest windows with radical scavenging properties for high-resolution studies of radiation-sensitive samples 2 .
Graphene is not only the thinnest possible membrane (about 0.35 nanometers thick) but also exceptionally strong and conductive. Perhaps most importantly, graphene acts as a radical scavenger, protecting samples against radiation damage caused by the electron beam 2 . This protection significantly increases the maximum electron dose that samples can sustain before losing their structural integrity.
In 2023, researchers demonstrated a landmark achievement: high-resolution electron diffraction of hydrated protein crystals preserved in their natural environment at room temperature 2 . This experiment addressed a critical limitation in structural biology—the fragility of protein crystals and their deviation from natural functioning conditions when frozen or dried.
Resolution Achieved
Sufficient to resolve individual atoms within protein molecules 2
Lysozyme was crystallized as nanometer-sized bars using an acetate crystallization buffer, generating crystals suitable for electron diffraction while easily identifiable in TEM images.
TEM grids were covered with 3-5 monolayers of graphene (1-1.7 nanometers thick). This thickness provided robust sealing while minimizing background scattering.
The lysozyme crystals in their crystallization solution were applied to the graphene-coated grids. The flexibility of graphene membranes allowed them to adapt to different crystal morphologies.
The samples were hermetically sealed within liquid pockets formed between graphene layers. The quality of sealing was crucial—any cracks would lead to buffer evaporation and protein denaturation.
A low-dose data collection strategy was employed in combination with a hybrid-pixel direct electron detector to minimize radiation damage while maximizing signal detection.
Diffraction patterns were collected and indexed using a template-matching algorithm to identify reflection patterns indicative of the crystal structure.
| Parameter | Specification | Significance |
|---|---|---|
| Sample | Lysozyme nanocrystals in mother liquor | Benchmark protein maintained in native environment |
| Sealing Material | 3-5 layer graphene (1-1.7 nm) | Optimal balance of seal integrity and electron transparency |
| Window Material | Graphene | Thinnest possible membrane with radical scavenging properties |
| Detector Type | Hybrid-pixel direct electron detector | High sensitivity with low electron dose requirements |
| Achieved Resolution | 3 Å | Sufficient to resolve individual atoms |
| Data Collection Strategy | Low-dose | Minimized radiation damage to delicate protein crystals |
The advancement of in-liquid electron microscopy has relied on both novel materials and clever adaptations of existing technologies.
Hermetically seal liquid samples while allowing electron transmission.
Application: Preserving hydrated protein crystals in native state 2
Create viewing windows in nanofluidic cells.
Application: Standard material for MEMS liquid cells 8
High-contrast labels for biological tracking.
Application: Visualizing EGF receptors on fibroblast cells 5
Potential radioprotective solvent.
Application: Crystallization medium for reduced radiation damage 2
Control liquid flow and reagent mixing.
Application: Studying nanoparticle growth with changing chemical environments 3
Detect electrons with high efficiency and low noise.
Application: Low-dose data collection for radiation-sensitive samples 2
Researchers are using these techniques to observe battery materials during charging and discharging cycles, watching how the electrode materials change structure in real-time—knowledge crucial for developing longer-lasting batteries 6 .
The technology has revealed the growth trajectories of individual colloidal platinum nanocrystals, providing insights that could improve catalytic converters and fuel cells 6 .
Scientists have achieved nanometer resolution in whole cells immersed in liquid. In one striking example, researchers imaged single gold-tagged epidermal growth factor molecules bound to cellular receptors on fibroblast cells, achieving a spatial resolution of 4 nm 5 .
This technique has been used to observe the internalization of EGF receptors, revealing circular clusters consistent with the known behavior of receptor-activated endosomes 5 .
| Technique | Key Features | Best Suited Applications |
|---|---|---|
| Static Liquid Cell EM | Simple design, stable environment | DNA hybridization studies, cellular morphology 1 |
| Flow Liquid Cell EM | Enables reagent mixing, chemical changes | Nanoparticle growth, electrochemical reactions 3 |
| Graphene Liquid Cell EM | Thinnest windows, radical scavenging | High-resolution protein diffraction, radiation-sensitive samples 2 |
| Liquid-Phase STEM | Z-contrast imaging, thick liquid layers | Imaging labeled molecules in whole cells 5 |
| Environmental SEM | Open system, vapor pressure control | Low vapor pressure liquids, ionic liquids 6 |
Making molecular movies of chemical reactions and biological processes
Combining electron microscopy with light microscopy or spectroscopy
In-liquid electron microscopy and diffraction have fundamentally transformed our relationship with the nanoscale world. What was once a static gallery of frozen specimens has become a dynamic theater where we can watch the intricate dance of molecules in their native environment. This technology has not only broken the vacuum barrier that long constrained electron microscopy but has opened entirely new avenues for exploration across biology, materials science, and chemistry.
As we continue to refine these techniques and push the boundaries of resolution, speed, and sensitivity, we move closer to the ultimate goal of science: to observe nature as it truly is, not as we're forced to prepare it. The ability to witness molecular processes in action, from proteins folding to crystals growing, provides not just beautiful images but profound insights that will drive innovation for decades to come. The age of watching molecules dance has just begun, and the performance promises to be spectacular.