In-Liquid Electron Microscopy Reveals the Nanoworld in Motion
For the first time, scientists are watching nanoparticles form and biological molecules interact in their native liquid environment, revolutionizing how we understand everything from drug delivery to battery operation.
Explore the TechnologyImagine watching a virus assemble itself or seeing the very first moments of a nanoparticle's formation. For decades, these processes remained hidden, occurring too rapidly at too small a scale to observe directly. In-liquid electron microscopy (Liquid-EM) has shattered this barrier by transforming transmission electron microscopes from instruments that capture static snapshots of dead, dried samples into powerful movie cameras that record dynamic processes in liquid environments 2 6 .
From watching cancer cells uptake potential medicines to visualizing the crystallization of organic molecules, Liquid-EM is providing a unique glimpse into the invisible dance of the nanoworld.
Observe processes in their natural liquid state
Watch dynamic processes as they happen
Reveal structures at the nanoscale
At its core, in-liquid electron microscopy is a clever solution to a fundamental problem. Traditional transmission electron microscopy (TEM) requires a high vacuum to operate, as gas molecules would scatter the electron beam. This obviously prevents the study of any liquid process.
The breakthrough came with the development of specialized liquid cells. These are essentially miniature, sealed containers with ultra-thin windows transparent to the electron beam. A typical liquid cell, as used in commercial systems like the Poseidon AX, consists of two silicon chips with silicon nitride (SiN) windows that are only 5-50 nanometers thick 8 . These windows trap a thin layer of liquid—often just 100-1000 atoms thick—allowing the electron beam to pass through while maintaining a native liquid environment for the sample 1 2 .
When the electron beam interacts with the sample in the liquid cell, several phenomena occur:
What makes the analysis of these liquid-phase experiments particularly powerful is specialized software like LiquidDiffract, an open-source tool designed specifically for processing X-ray and electron diffraction data from liquids and disordered solids 3 .
This software helps researchers extract crucial structural information such as:
Louis de Broglie proposed in his PhD thesis that electrons could behave as waves 1 .
The Davisson-Germer experiment and George Paget Thomson confirmed electron diffraction 1 .
Development of robust yet ultra-thin silicon nitride windows enabled liquid cell technology 2 6 .
Liquid-EM now enables observation of dynamic processes across multiple scientific domains.
The capabilities of Liquid-EM extend across multiple scientific domains, revolutionizing how we study nanoscale processes:
Observing nanoparticle growth and transformation in solution with sub-nanometer resolution 6 .
Imaging virus assemblies and protein structures in fluid that mimics their native environment 8 .
Tracking how cancer cells interact with drug-carrying nanoparticles for targeted therapies 2 .
Visualizing crystallization processes and chemical reactions as they happen .
A recent landmark study published in Communications Chemistry vividly demonstrates the power of Liquid-EM to capture previously invisible processes . Researchers set out to visualize antisolvent crystallization (ASC), a method widely used in the pharmaceutical industry to produce ultrafine drug particles, but whose nanoscale mechanisms had never been directly observed.
The research team investigated the ASC of an organic molecule called R-BINOL-CN, a chiral compound relevant to drug development. In the ASC process, a substance is dissolved in one solvent (chloroform, for R-BINOL-CN), and then a "antisolvent" (methanol) in which the substance has poor solubility is added. This rapidly triggers desolvation, leading to precipitation .
| Component | Specification/Role |
|---|---|
| Liquid Cell Holder | DENSsolutions Ocean holder |
| Window Material | Silicon Nitride (SiN), ~50 nm thick |
| Solvent | Chloroform (dissolves R-BINOL-CN) |
| Antisolvent | Methanol (triggers precipitation) |
| Organic Molecule | R-BINOL-CN (beam-sensitive) |
| Imaging Mode | Scanning Transmission Electron Microscopy (STEM) |
The experimental procedure was meticulously designed to overcome the challenges of working with volatile solvents:
R-BINOL-CN was first dissolved in chloroform at a specific concentration.
The liquid cell was prepared with the first solution placed between the silicon nitride windows.
Methanol was carefully introduced into the holder using a precise flow system.
The interaction was immediately observed using STEM with controlled electron dose.
Images were recorded in real-time, capturing nucleation and growth processes.
The real-time visualization revealed a fascinating crystallization mechanism that had previously only been inferred. When the methanol came into contact with the R-BINOL-CN solution in chloroform, the organic molecules almost instantly self-assembled into chain-like structures of spherical particles .
This was strikingly different from what occurred when R-BINOL-CN was simply dissolved in chloroform without methanol, where the molecules formed single large particles without the chain-like organization. The observation provided direct visual evidence of how antisolvents trigger specific molecular assembly pathways that determine the final crystal morphology—a crucial insight for pharmaceutical manufacturing where particle shape and size directly impact drug efficacy and processing .
| Condition | Particle Morphology | Observation Method | Key Finding |
|---|---|---|---|
| With Methanol (in situ) | Chain-like structures of spherical particles | Liquid-EM (STEM) | Instantaneous self-assembly upon solvent mixing |
| Without Methanol | Single large particles | Liquid-EM (STEM) | No chain formation; simple coalescence |
| Ex Situ (drop-cast) | Discrete microspheres (1-6 μm) | FESEM/TEM | Morphology changes with aging time |
| Ex Situ (2 days aged) | Micropods from fused spheres | FESEM/TEM | Progressive assembly over time |
Conducting successful in-liquid electron microscopy requires a sophisticated suite of tools and reagents. The ecosystem extends from the microscope itself to specialized holders, microchips, and analytical software.
| Item | Function/Role | Example Specifications |
|---|---|---|
| Silicon Nitride Microchips | Creates liquid confinement cell with electron-transparent windows | 5-50 nm thick windows; various well sizes 8 |
| Liquid Cell Holder | Holds microchips, enables liquid flow in microscope vacuum | Poseidon AX, DENSsolutions Ocean 9 |
| Syringe Pump System | Controls precise introduction of liquids and reagents | Flow rates from μL/min to nL/min |
| Direct Electron Detectors | Captures high-resolution images with minimal noise | 6-micron pixel spacing (DirectView) 8 |
| Analytical Software | Processes diffraction data and extracts structural information | LiquidDiffract (open-source) 3 |
| Plasma Cleaner | Prepares microchip surfaces for optimal sample adhesion | Standard conditions: 30W, 15mA, 45s 8 |
Commercial systems like the Poseidon AX offer integrated workflows that streamline the entire process from sample preparation to data publication. These systems provide hundreds of possible chip configurations, allowing researchers to tailor liquid layer thickness and other parameters to their specific experimental needs 9 .
In-liquid electron microscopy has opened a window into a world that was once largely theoretical. By enabling real-time observation of dynamic processes in liquids, this technology is transforming our understanding of everything from pharmaceutical crystallization to viral assembly.
As one researcher noted, "The ultimate method to be implemented for this purpose is lacking for electrons mainly due to the difficulties in sample preparation and probe source design" 2 . Yet the progress has been remarkable—from the first observations of nanoparticle growth to the sophisticated visualization of antisolvent crystallization processes in organic molecules 6 .
The continued refinement of liquid cells, the development of more sensitive detectors, and the creation of specialized analysis software like LiquidDiffract promise to further expand the frontiers of what we can observe 3 . As these tools become more accessible, we stand at the threshold of a new era in nanoscale science—one where we no longer have to imagine how molecular dramas unfold, but can watch them, in real time, as they happen.