Worth the Wait: EM Scope update

EM-3D Color tracking

(For the initial story of the scope's arrival, see the News Article from September 2017)

“This is only one of about 20 in the US and it can do images that no other in the state can do, so we’re very lucky.”

The frustration of last year’s doomed arrival is all in the past:  the Serial Block Face Scanning Electron Microscope (SBFSEM) is off and running into the third dimension.

After the original instrument was delivered in February 2017, totally destroyed by the shippers, a new one had to be built from scratch, which took another half year.  Then it had to be shipped from Germany and installed at Duke, where engineers performed magic, and trainers taught the EM staff to operate it.

“We became operational in mid December, and in the last couple of months, have performed over 25 different 3D “runs” or sessions, looking at different types of tissues.   Seventeen different laboratory groups are users on our grant; thus, we anticipate many more projects to be initiated in the next few weeks,” says Sara Miller, PhD, Director of the Research Electron Microscopy Service.  “3D EM is used to examine cells that communicate with each other, like neurons.   The lab is now imaging connections between cells in brain, heart, gut, and tissue cultures, and from different species such as zebrafish, mice, and C. elegans (a non-parasitic soil nematode).  For instance, finding the point of contact between a neuroendocrine cell and a neuron is of interest to elucidate how the brain senses satiety, and virologists are looking at how infected cells talk to each other.  This is all in addition to conventional scanning electron microscopy examinations that provide surface topography of specimens.”

To produce the 3D images, a micrograph is taken at high magnification (nanometer resolution) using electrons backscattered from the face of a small resin block containing a carefully selected, stained, and embedded specimen.  An ultramicrotome inside the EM shaves off a very thin (~50 nm) section using a diamond knife, and the block face is imaged again and recorded.  This process is repeated many hundreds of times, depending on the desired thickness.  After the image stack is recomposed by a computer into a virtual block of tissue, visualizing a slice in any dimension is possible.  This is similar in concept to the mechanism by which a confocal microscope produces serial “optical sections” of a specimen and reconstructs them into a 3D image that can be viewed at any angle.  However, the confocal scope produces its image layers optically rather than physically, and at lower resolution, while the SBFSEM allows high resolution imaging at a sub-cellular level.  Mitochondria, Golgi bodies, nuclei, and attachment sites, such as synapses, are all visible in great detail.  An exciting feature of the software allows for coloring a neuron or other structure to follow the path it takes through the tissue. (photo at top)  Another powerful computer tool allows the slices to play in video motion while moving up and down through the block.

Prior to the availability of the SBFSEM and a few other recently developed techniques, ultrastructural 3D imaging generally required identification of an area of interest in a resin-embedded tissue block, painstaking collection of serial ultrathin sections (similar to those used by our diagnostic EM laboratory), imaging of each section in a transmission electron microscope (without losing any), and somehow aligning all of the images (often manually).  Analysis of a single small block could easily take a year or more.  This new scope can automatically image a block, unattended, in a matter of hours.

What does Dr. Miller anticipate will be future subject matter for the scope?   “The field is completely open as far as what we look at, and researchers at Duke are a varied lot, examining cells from many different organisms.  We look forward to working with the community to discover new cellular processes and interconnections.”

You can contact the Research EM Service at 919-684-9141 (Dr. Miller) or 919 684-3452 (Dr. Vancini)

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First, the tissue block surface is imaged, and then that layer is sliced off and thrown out to reveal the next layer.  A single SBF picture of the block face looks like a traditional transmission electron micrograph (see gray surface).  (Shown here: zebrafish heart.)

NewsEM-Image-Of-Aligned-D313A_1a_2nd-stack_BSED_slice_3D.jpg

The imaging and slicing is repeated many times, and the recomposed stacked layers allow 3D viewing from any plane throughout the tissue block.  (Shown here: a 3D stack of sections of zebrafish heart.)

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The layers are recomposed to create a video showing movement through the tissue

EM-3d colortrack

The path of a structure can be followed through the tissue and colored to identify it. (shown here: Neurons in mouse brain)

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Conventional SEM showing pollen in amazing detail

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More pollen, showing the vast variety in design of the particles

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Dr. Miller with lab manager Dr. Ricardo Vancini and the working microscope              
(Photo: DUMC PhotoPath/Susan Reeves)