Solve the multi-length scale challenge with automated correlative microscopy from ZEISS

The world’s only manufacturer of light, electron, X-ray and ion microscopes

Next Generation 3D Science

Three-dimensional (3D) characterization and modeling of microstructures at length scales from micrometers to nanometers is experiencing a surge in activity and interest across a variety of scientific fields. Advanced computing power with new characterization approaches enable new pathways to research. In this new era, automated and efficient workflows capture information from the same sample volume across length scales and modalities.

It is clear that one technique by itself cannot span all of the necessary length scales in materials characterization. Increasingly, several research fields, such as energy materials, electronics, neuroscience, environmental and metals research, are using an array of characterization techniques where the combination of several microscopes are required to obtain a fundamental understanding of the microstructure and performance.

ZEISS has created the infrastructure required to efficiently navigate modalities and length scales with a combination of software and underlying high performance imaging systems. These correlative workflows automate the tasks such as navigating to sub-surface features with FIB-SEM by using the information from an X-ray microscopy (XRM) dataset from the same sample. 

A correlative workflow. Stepping from XRM volumetric data and registration to SEM surface image allows for the targeting and automation of FIB-SEM analysis from identified buried features of interest.

Hierarchical materials such as porous membranes, rocks and biological materials naturally exhibit features from mm to nm. Understanding the property of these multi length scale materials requires characterization of a single volume in 3D with multiple imaging modalities across a spectrum of sample sizes (volumes), with corresponding imaging resolution spanning six orders of magnitude.

Images of hierarchical structure in bone were collected using multi-scale X-ray microscopes (ZEISS Xradia Versa and Ultra). 3D rendered datasets collected a range from a FOV of 50 mm (30 μm voxel) to 16 μm (16 nm voxel).

Correlative microscopy can increase throughput and improve validity in conventional microscopy. Localize features in 3D to target and find specific buried structures for physical extraction or cross-sectional imaging. This is important because examination of surface features may not yield results or may not represent “native” information, i.e. structures unmodified by sample preparation or environmental conditions.

Image A shows volume rendering for XRM interior tomography dataset; Image B shows import of XRM reconstructed 3D dataset into Atlas environment.

A                                                                          B

Light Microscopy

Light microscopy introduces several additional modalities and further overlap in scales. The Atlas framework has been used for several years to perform advanced SEM and FIB-SEM functions, alongside the ability to acquire, visualize, and navigate large (multiple gigapixel) SEM datasets and combine these with light microscopy data.

X-ray Microscopy

X-ray microscopy obtains tomographic structural information non-destructively under conditions and environments in the laboratory at scales that were previously only conceived of at synchrotron facilities. Modern laboratory XRM systems achieve spatial resolution ranging from tens of micrometers to tens of nanometers. They offer the flexibility to image the same volume non-destructively in 3D at multiple time steps throughout an evolution experiment, probing, for example, a specimen’s response to electrochemical, mechanical, corrosive, or thermal treatments. This type of study has been deemed “4D,” representing three spatial dimensions plus time, and is a key enabler in the pursuit of understanding microstructure evolution. Ideally, such experiments are conducted in situ, with specimens contained in environmental chambers that replicate real world conditions.

FIB-SEM Tomography

Scanning electron microscopy is an established and versatile surface imaging technique that spans the widest range of length scales from millimeter to sub-nanometer. It is widely used in materials and life sciences while encompassing a wide range of imaging methods and analytical tools. In combination with focused ion beam (FIB-SEM), its capabilities extend into the third dimension. FIB-SEM tomography enables the volumetric reconstruction of micrometer-sized volumes with nanometer resolution from SEM images of serial FIB cross-sections. The technique ideally complements XRM at higher spatial resolution using backscattered electron detection to generate complementary mass density contrast. In addition, it supplements XRM with a collection of analytical capabilities (e.g. EDS, EBSD, etc.).

By combining multiple microscopy techniques, it is possible to obtain complementary information by exploiting separate contrast mechanisms.

A modern correlative workspace: a new paradigm for microscopy

The implementation of an automated 3D correlative workflow solution is enabled by Zeiss Atlas, an integrated correlative software environment.

Atlas software extends capabilities to acquire, visualize, and navigate large (multiple gigapixel) datasets, combining with other data to enable an array of correlative workflows across several platforms into the 3rd dimension. For example, non-destructive XRM data acquisition can be used to inform and automate FIB-SEM tomography at multiple sites within the same sample volume. Atlas correlative software provides an environment that enables seamless integration of different data sources, facilitates navigation, and allows contextual visualization and correlation of results in 2D. 3D and now in 4D.

In combination, these microscopy techniques provide a unique opportunity to study materials evolution at multiple length scales in 3D/4D. The ability to understand data within its larger spatial context elevates the significance and efficiency of high resolution techniques.