Can your 3D microscopy system automatically produce perfectly aligned images from large volumes at EM resolution?

Joel Mancuso, Gatan, Inc.

Morphometry is an important and growing discipline within neuroscience.  Theoretical models of neuronal circuits require 3D information over extensive volumes.  Current models are based on real data obtained from serial sectioning brain tissue and subsequent reconstruction to show components present, for example synapses, axons, dendrites.  Realistic and meaningful analysis requires morphometric analysis at the ultrastructural level over large sample volumes. Large volumes are required in order to be statistically relevant and usable for model building.

Electron microscopy is key to providing information at the ultrastructural level.

Until now, the classical method to obtain such data was serial sections collected on grids and observed in the TEM.  This is a long and difficult process requiring much skill.  Sections are obtained as ribbons using an ultra-microtome.  Ribbons must be divided manually.  Multiple sections are then collected on grids using the surface tension of water near the diamond knife.  Sections on grids are processed in various chemicals.  All these steps are risky as the grids or sections can be broken.  Only one broken grid compromises the entire series of sections because it introduces an important gap in the series.

Based on groundbreaking work performed by Denk and Horstmann at MPI Heidelberg a revolutionary solution called 3View^® is now available from Gatan, using serial block face scanning electron microscopy (SBFSEM). This new method automates the cutting and imaging of the specimen.  Grids are no longer used because sections are not cut for collection.  It is the block of tissue itself that is directly introduced inside the scanning electron microscope and its surface is repeatedly shaved and scanned in order to obtain a stack of aligned images.  The raw images obtained can be directly exported to software for reconstruction and quantification.  Image stack alignment is due to the inherent design and stability of the system and typically does not require software post-processing.

Case Study #1: Mosquito fish spinal cord circuit reconstruction, a wide field of view application

In this example it was necessary to exploit the wide field of view of 3View to follow a specific circuit of interest.  Unlike traditional biological TEM, the blockface can be scanned without the grid bars interfering with the image acquisition.  3View is a technique compatible with immunohistochemistry.  However, only pre-embedding techniques are suitable because SBFSEM is not based on the observations of sections but on the imaging of the block face.  3,3'-Diaminobenzidine (DAB) was use as a pre-labeling agent in this study.  Once the animal is fixed and dissected open, a retrograde dye is applied using filter paper soaked with the dye.  The specimen is stained with DAB, and tissue is processed with a classical embedding protocol.  If the aim is to follow a labeled structure, one must check that the labeling is not restricted to the surfaces of the section but has penetrated through all the section.

This specimen preparation along with the wide field of view of 3View allowed targeting of the circuit of interest in over 6,000 serial images, roughly 300µm of tissue in the z orientation.  The image acquisition process was completely automated, collecting 50GB of data, and took under two weeks.



Figure 1. Example of pre-embedding labeling visualized with 3View.  The tissue has been processed for immunochemistry with DAB staining. This image shows a section from a mosquito fish spinal cord motor neuron.  The original image is 2048x2048 pixels. Image courtesy of Dr. Eduardo Rosa-Molinar, Biological Imaging Group, University of Puerto Rico-Rio Piedras.




Figure 2. A high resolution image showing the detail of the DAB immunochemically stained neuron. Image courtesy of Dr. Eduardo Rosa-Molinar, Biological Imaging Group, University of Puerto Rico-Rio Piedras. 

Case Study #2: New Frontiers in High Resolution

Not only can 3View image large fields of view, but it can also approach TEM resolution.  New staining technology developed by Tom Deerinck and Dr. Mark Ellisman, of the National Center for Microscopy and Imaging Research, University of California San Diego has extended the resolution limits of SBF-SEM.  It was important to resolve synaptic vesicles for the study.  An image size of 4096 x 4096 was chosen to maximize the field of view and still resolve synaptic vesicles.  Gatan’s 3View^® was installed on a FEI Quanta™ 200 FEG SEM for this study.



Figure 3. A low magnification overview from a 15,625µm³ volumetric data set of mouse brain (Case Study #2). The original data set was 4096x4096 pixels, and contained 500 serial images. Image courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center for Microscopy and Imaging Research, University of California, San Diego.
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Figure 4. High resolution extract from the data set in figure 3. Image courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center for Microscopy and Imaging Research, University of California, San Diego.

Figure 5. A high magnification movie of 100 x 50nm slices from the region indicated in Figure 3. 3View^® has perfect alignment as demonstrated in the slice movie, made with slice player in DigitalMicrograph™. This data set was not post processed and no alignment was necessary. Data courtesy of Tom Deerinck and Dr. Mark Ellisman, National Center for Microscopy and Imaging Research, University of California, San Diego.

Tissue processing

Like any microscopy technique, optimum results, especially spatial resolution in 3D, are often achieved by following certain protocols.  In the case of 3View specimen preparation, two key considerations are the level of contrast shown by the specimen,  and the resilience of the embedding resin to the electron beam radiation.  For a given specimen, higher image contrast can be achieved by increasing experimental variables such as the beam energy and the number of injected electrons per pixel point.  However, such higher injection conditions have other detrimental effects on the specimen and imaging quality, for example the cutting quality, additional scattering associated with the gas required to compensate the injected charge, as well as the depth resolution of the 3D data.  For this reason, a specimen optimized to give the highest possible 3D resolution data whilst minimizing the acquisition timeframe, is one with high contrast in the tissue, and embedded in a medium relatively resistant to electron beam damage.  In case study #2, the tissue has been postfixed in osmium tetroxide with ferrocyanide.  In a further step, uranyl acetate has been used to enhance the contrast of some organelles.

Setting of the microscope

Once the sample is ready to be cut, it is placed on the 3View ultramicrotome.  The 3View ultramicrotome is already attached to the door of the SEM and can be operated out of the chamber.  In this open configuration of the microscope, a binocular optical microscope can be adjusted on the door and the sample set relative to the diamond knife.  This is made convenient via a camera fixed on the optical microscope.  When the sample has been adjusted with respect to the diamond knife, the door is closed and the SEM evacuated.  The face of the block is now positioned just below the pole piece of the SEM.

Cutting and image acquisition

All the cutting and acquisition parameters are entered into Gatan’s DigitalMicrograph software.  Settings are chosen in order to fit the magnification and speed of scanning with the field of view and the resolution required.  The focus is adjusted at the same time and is then valid for the entire acquisition session.

Detection is via a backscattered electron detector (BSED), designed especially for low kV work on biological specimens.  The cutting and imaging process is based on the repetition of the following steps:

- The specimen is raised
- The diamond knife shaves the surface and then moves away
- Imaging the freshly cut block face

The advantages are the following:

- The block is stable so it is always the same field of view that is scanned and in focus. Hence all images are aligned at the time of acquisition.
- The upwards movement of the block sets the depth of shaving of the surface.  It was set to 50 nm, but this can be adjusted according to requirements.
- The cutting and imaging processes are automatic and do not require attendance near the microscope during the session. The system can typically work overnight.
- A file of images (image ‘stack’) is obtained that is directly exportable to software for analysis and reconstruction.

Three dimensional (3D) visualization and rendering

Alignment allows projection in all axes and visualization in all desired planes.  Gatan can provide an additional tool called the DigitalMicrograph^® 3D Visualization Tool, allowing 3D visualization as well as 3D automatic rendering from any image stack.  With this tool, the stacks can be visualized as volumes and can be rotated, projected on all the different axes and sliced through.  Images can also be exported and saved in TIFF format in an ascending numerical order, to be imported by a 3rd party software package.



Figure 6 Example of reconstructed motor neuron using serial images acquired using 3View (Case Study #1). Image courtesy of Dr. Eduardo Rosa-Molinar, Biological Imaging Group, University of Puerto Rico-Rio Piedras.