Hydra Bio Plasma-FIB Applications

Hydra Bio Plasma-FIB applications for volume imaging

Explore across length scale from tissues to proteins with the Thermo Scientific Hydra Bio Plasma-FIB. Investigate subcellular structures within the contextual environment by volume imaging. Study resin-embedded specimens at room temperature and unstained biological samples in the near-native state at cryogenic temperatures. Bridge the gap in terms of specimen volume and resolution between light microscopy and cellular cryo-electron tomography.

Thermo Scientific Hydra Bio Plasma-FIB SEM compatible sample types and applications.

Hydra Bio Plasma-FIB applications in cryogenic volume electron microscopy

Chlamydomonas reinhardtii imaged in 3D

Contextual volume imaging in cryo conditions delivered this high contrast image 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. Sample prepared by plunge freezing with no added stains. Slice thickness is 20 nm. Visualized with Thermo Scientific Amira Software.

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 Thermo Scientific Auto Slice & View Software. The cell was plunge-frozen without any staining. 3D imaging revealed the detailed internal arrangement of cell organelles which have been segmented and false-colored with Thermo Scientific Amira Software.

Hydra Bio Plasma-FIB applications for volume electron microscopy at room temperature

3D reconstruction of mouse neuron

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

Mouse neuron visualized with PFIB SEM volume electron microscopy

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.

3D reconstruction of mouse brain embedded in epoxy resin

3D reconstruction of mouse brain tissue in EPON imaged with Hydra Bio Plasma-FIB with Spin Mill Bio Method. Field of view is 175 µm with 5 nm slice thickness.

Mouse hippocampus visualized with the Spin Mill Bio Method

Spin Mill Bio 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 Bio for millimeter-wide area planar milling and imaging

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.

3D reconstruction of a mouse brain

3D reconstruction of a mouse brain tissue acquired with a Hydra Bio Plasma-FIB using O+ focused ion beam. Sample preparation: Conventional chemical fixation. Epon resin. 721 slices (thickness ~ 4 nm). HFW 16.4 µm (xy resolution 5x4 nm). Acquisition time ~17 h.

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. Horizontal field of view = 61.25 μm. 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.

Cross section of a tobacco leaf

Cross section of a tobacco leaf, stained and embedded in Quetol resin. The large field of view is made possible with the Spin Mill Bio Method. Imaging with oxygen rather than gallium reduces charging and avoids curtaining artifacts that would otherwise be experienced with Quetol resin. Sample courtesy of Kirk Czymmek, Donald Danforth Plant Science Center, USA.

Cross section of tobacco leaf visualized with Hydra Bio PFIB

Cryo-tomography with the Hydra Bio Plasma-FIB

Cryo-electron tomography (cryo-ET) is a technique that allows the study of the 3D structure of cells and tissues at near-native conditions. Sample preparation is a critical step in cryo-ET, which can greatly affect the quality and resolution of the results. Cryo-ET has been applied to many different sample types, from single molecules to protein complexes, viruses, bacteria, cells, to tissue cells and large tissue samples. Samples are either vitrified through plunge-freezing or high pressure freezing (HPF). After the vitrification step, depending on the size of the sample, a thinning step is included in the sample preparation using (plasma) focused ion beam milling to produce cryo-lamellae that are thin enough to be penetrated by electron beams. The Hydra Bio Plasma-FIB supports automated preparation of lamellae with a thickness of 150 nm or less for the cryo-electron tomography workflow, while avoiding gallium-ion implantation. The guided workflow allows the selection of multiple points of interest and then automatically prepares several lamellae autonomously in unattended runs. The Hydra Bio Plasma-FIB prepares lamellae from within large (HPF) volumes with plasma FIB, CLEM localization through integrated light and electron imaging (iFLM Correlative System), and cryo-lift out with Thermo Scientific Easylift Cryo.

Sample types prepared with high pressure freezing, plunge freezing, and cryo-FIB milling.

Correlative light and electron microscopy (CLEM)

The Thermo Scientific Integrated Fluorescence Light Microscope (iFLM) Correlative System is an integrated wide-field light microscope for correlative imaging inside the Hydra Bio Plasma-FIB, enabling fluorescent imaging and ion milling within a single microscope at room temperature or under cryo conditions.

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

AI-assisted serial section electron microscopy with Auto Slice & View Software

Combined with Thermo Scientific Auto Slice & View Software, the Hydra Bio Plasma-FIB offers high throughput and fully automated 3D data collection, even at cryogenic temperatures. Benefitting from real-time AI analysis, dynamic auto functions effectively mitigate the impact of sample variability, which is an essential advantage in unattended, large-volume acquisition of cryo-prepared specimens.

Serial section electron microscopy with the Hydra Bio Plasma-FIB.

Lamella preparation with AutoTEM Cryo Software

Thermo Scientific AutoTEM Cryo Software offers the highest level of automation and an intuitive user interface with hints and instructive graphics. This simplifies and shortens the lamella preparation time for experts and enables beginners to routinely obtain the highest quality results. Create unattended batch milling even for overnight runs with configurable steps from milling to polishing and drift correction.

Lamella preparation on the Hydra Bio Plasma-FIB

Array tomography with the Hydra Bio Plasma-FIB

Array tomography can provide rapid localization of cells and their interaction partners in tissues. With the largest imaging field, array tomography is ideal for tissue histology. Ultrastructure analysis of whole animals or whole organs is not needed, so array tomography provides easy navigation for samples that can be stored and re-imaged.Like any Thermo Scientific SEM, the volume EM capability of the Hydra Bio Plasma-FIB can be extended with the addition of automated Maps for Array Tomography Software.

Maps Software for Array Tomography

Thermo Scientific Maps Software is an imaging and correlative workflow software suite compatible with Hydra Bio Plasma-FIB.

Automated array tomography of large microscopy images at high resolution

JOVE: Array Tomography Workflow

Learn more about preparation of serial sections and automated imaging procedures in a SEM.

Protocol: Targeted 3D EM and Correlative Microscopy Method using SEM Array Tomography

Optimized scanning electron microscopy workflow for volume array tomography for asymmetric samples and model organisms.

Array tomography of nonlinear sample C. elegans larva.

Header image courtesy of Tessa Burch-Smith, Kirk Czymmek and Lolita Rotkina, Donald Danforth Plant Science Center.

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

1x1 image pixel for data collection