Plasma Focused Ion Beam Scanning Electron Microscopy

Why use PFIB SEM for volume electron microscopy? 

A plasma focused ion beam scanning electron microscope (plasma-FIB SEM) combines improved sputtering efficiency with nanometer imaging resolution at either room temperature or under cryogenic conditions. This provides a fast way to capture large specimen volumes. Multiple ion species (Xe, O, Ar, and N) are versatile options for site-specific, large-volume material removal.

 

Unlike Ga-based FIBs, curtain-free surfaces are easily generated for a wide range of materials such as LR-White, HM20, and EPON resins. Superior resin- and sample-processing compatibility allows for targeted FIB-SEM acquisition with direct, in-resin correlative imaging of the regions of interest (ROIs) and automated management of the data from each slice and region.

Mouse neuron visualized with PFIB SEM volume electron microscopy 3D reconstruction of mouse neuron prepared in epoxy resin and imaged with the Hydra Bio Plasma-FIB. Segmentation shows synaptic vesicles and mitochondria (red).

How does PFIB SEM work for volume electron microscopy?

The Thermo Scientific Hydra Bio Plasma-FIB is a versatile tool for high-throughput volumetric imaging - compatible with all commonly used sample-embedding media and preparation protocols. Four ion species (Xe, O, Ar, and N) can be used independently for site-specific, large-volume material removal for top-down and cross-section analysis in 2D and 3D. Selecting the ion beam that matches the requirements of each individual sample delivers excellent surface texture for complex samples, including sample-substrate interfaces and dental (mineralized tissue) materials.

 

A plasma FIB-SEM enables efficient, large-volume serial sectioning thanks to a broader, collimated beam delivering currents up to 2.5 μA higher than a Ga-FIB. Improved sputtering efficiency enhances performance, generating smoother cut faces and reducing curtaining artifacts, further improving throughput and providing fast access to ROIs. The combination of higher currents, higher sputter rates, and reduced damage makes it possible to access volumes hundreds of micrometers in size while still observing nanoscale features.

Depiction of the PFIB SEM volume electron microscopy technique.

PFIB Spin Mill Bio Method for large planar milling and imaging of resin-embedded samples

The optional Spin Mill Bio Method on the Hydra Bio Plasma-FIB provides large-area planar milling up to 1 mm in diameter and the ability to image large areas in a horizontal plane for 3D characterization. The Spin Mill Bio Method is fully automated and easy to set up using Thermo Scientific Auto Slice & View Software. You can select multiple areas for image acquisition within one Spin Mill Bio experiment and image each region of interest at different imaging settings. 

 

With the Spin Mill Bio Method, samples are milled at a near-glancing angle with the PFIB. Typical sample preparation for slice-and-view analysis (e.g. protective capping, trenching, or the use of fiducial markers) is not required. With such a large milled area, numerous regions can be selected. Sparse features are easily identified, and statistically relevant, multisite 3D data can be collected.

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Spin Mill Bio Method

The Spin Mill Bio Method on the Thermo Scientific Hydra Bio Plasma-FIB.

Adaptive Scanning for AI-driven high-resolution acquisition

Using Adaptive Scanning, the Hydra Bio Plasma-FIB uses AI to recognize features of interest in your specimen. Features of interest are annotated on the initial image prior to starting the volume acquisition. The features are scanned in high resolution while the “background” is scanned at lower resolution. This method significantly reduces the dose and scanning time while increasing throughput for volume EM of resin-embedded samples.

Representative data showing iFLM correlative microscopy system combing fluorescent imaging with PFIB SEM

Cryo-volume EM with PFIB SEM in the study of cells and tissues

Cryo-fixation allows specimens to be imaged in a hydrated state and avoids artifacts commonly associated with classic volume EM sample preparation steps like heavy metal staining, dehydration, and resin embedding. The Hydra Bio PFIB makes it possible to view 3D volumes at cryogenic temperatures to reveal the cellular complexity in near-native state using Auto Slice and View. 

 

For cryo-volume acquisitions, the AI-guided dynamic auto-functions significantly enhance consistency and reliability of results. The AI-powered analysis adjusts the defined auto-function position to effectively mitigate the impact of sample variability and the challenges associated with cryo-prepared specimens. It provides valuable assistance in achieving successful and unattended cryo-volume acquisitions.  

3D visualization of unstained Chlamydomonas reinhardtii imaged in cryo conditions on a Hydra Bio Plasma-FIB

3D visualization of unstained Chlamydomonas reinhardtii imaged in cryo conditions on a Hydra Bio Plasma-FIB and segmented with Amira software

Correlative microscopy combines PFIB SEM with fluorescence light microscopy

The Thermo Scientific iFLM Correlative System is an integrated wide-field light microscope for cryo-correlative imaging inside the Hydra Bio Plasma-FIB. It allows you to combine fluorescence imaging and ion milling within a single microscope for correlative light and electron microscopy (CLEM) at room temperature or under cryogenic conditions.

Representative data showing iFLM correlative microscopy system combing fluorescent imaging with PFIB SEM

PFIB SEM 3D example data

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3D imaging of unicellular alga, unstained

Contextual volume imaging in cryo conditions delivered this high contrast dataset of Chlamydomonas reinhardtii, demonstrating the benefit of milling with alternative plasma species to obtain curtain free images without the need for staining. Imaged on a Hydra Bio Plasma-FIB with cryo-stage. Sample prepared by plunge freezing with no added stains. Slice thickness is 20 nm.
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3D imaging of CHO cell in native state

Reach inside the cell: a CHO cell imaged in 3D with a Hydra Bio Plasma-FIB and automated with Auto Slice & View Software. The cell was plunge-frozen without any staining. 3D imaging revealed the detailed internal arrangement of cell organelles which are segmented and false-colored with Amira Software.
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3D reconstruction of mouse brain tissue

3D reconstruction of mouse brain tissue embedded in LR White (acrylic-based) resin acquired with an O+ ion beam to mitigate charging and curtaining on Hydra Bio Plasma-FIB with Auto Slice & View software for automated data collection.
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3D Reconstruction of Caenorhabditis Elegans L1

3D reconstruction of Caenorhabditis elegans L1 imaged with Hydra Bio Plasma-FIB using O+ focused ion beam. Sample prepared by high-pressure freezing (HPF) followed by freeze substitution (FS) and HM20 embedding.

Volume reconstruction of a Nicotiana benthamiana (tobacco) epidermal cell

The dataset was acquired using the Adaptive Scanning application in Auto Slice & View Software. Areas marked with light blue were scanned at low-resolution while regions of interest were acquired at high-resolution. Stack alignment, post-processing, and segmentation illustrating detail in the selected sub-volume were performed with Amira Software. Sample courtesy of Tessa Burch-Smith, Kirk Czymmek and Lolita Rotkina, Donald Danforth Plant Science Center.

Volume reconstruction of a Nicotiana benthamiana (tobacco) epidermal cell

Mouse hippocampus visualized with the Spin Mill Method

Spin Mill Method allows milling of large areas to reveal high-resolution sample details which can be further analyzed to focus in on regions of interest. Mouse hippocampal organotypic slice in EPON resin. Courtesy of S. Watanabe, John Hopkins University and J. Wang, Thermo Fisher Scientific.

Mouse hippocampus visualized using PFIB SEM Spin Mill method for large-area planar milling

Meet the Scientists using PFIB SEM for volume electron microscopy

Dr. Kirk Czymmek, Principal Investigator from Donald Danforth Plant Science Center.

“We are increasingly being challenged to understand how to improve crop resilience to environmental stress and disease.  The remarkable capabilities of our new Hydra vEM will allow us the unprecedented ability to reconstruct entire plant cells and tissue with exquisite detail. This technology will allow us to “freeze” organisms in time and space and build intricate 3D models that will help us solve our critical food security challenges.”

 

Kirk J. Czymmek, Principal Investigator, Director Advanced Bioimaging Laboratory, Donald Danforth Plant Science Center

PFIB SEM resources

Thermo Scientific Hydra Bio Plasma-FIB is a versatile PFIB SEM with multiple ion species for volume electron microscopy analysis.

Thermo Scientific Hydra Bio Plasma focused ion beam scanning electron microscope

When preparing biological specimens for analysis, the sample preparation method and resin type determine how well subcellular structures and fluorescent signals are preserved.

Application note on PFIB SEM sample compatibility

In this application note, we describe the Spin Mill method, for large-area planar milling with oxygen PFIB for accessing and investigating large areas (up to 1 mm).

PFIB SEM Spin Mill method for large-area planar milling of cells application note

For Research Use Only. Not for use in diagnostic procedures.