Hi, I am Kedar Narayan, a group leader at the Center for Molecular Microscopy, or CMM,
at the National Cancer Institute and Frederick National Laboratory for Cancer Research.
Imaging three dimensional objects in 2-D can be limiting, yielding an incomplete or even
a misleading picture. And so, at the CMM, we apply cutting edge electron microscopy,
or EM, technologies to image biological samples at the highest possible resolutions. My group
specializes in the imaging of cells and tissues, that is, larger volumes, in 3-D and at nanoscale
resolutions.
In this short video, I am going to give you a flavor of an imaging technique that we employ
in our lab: Focused Ion Beam Scanning Electron Microscopy, or FIB-SEM. FIB-SEM, as you will
see, is a powerful 3-D EM imaging approach, and in this specific example, we highlight
a correlative light microscopy and FIB-SEM, or CLEM/FIB-SEM, workflow. CLEM/FIB-SEM combines
the advantages of light and electron microscopy to generate image reconstructions of targeted
features of interest, in 3-D, at resolutions of tens of nanometers.
So here you have a group of cells, of which a subset is fluorescently labeled. These cells
are grown on a gridded cover slip, a glass substrate on which an alphanumeric code has
been etched.
You can now image these cells, either live or after fixation, using fluorescence microscopy,
and also record their XY coordinates using the gridded pattern.
Once this step is completed, the cells can be fixed, stained, dehydrated and resin-embedded
in situ. When the gridded cover slip is removed, the alphanumeric pattern is transferred to
the resin surface in relief, with the cells embedded just beneath the surface. In other
words, you now know exactly where to find your cells of interest.
At this point, the cell sample is transferred to the FIB-SEM instrument.
This martian looking surface is a close-up of the resin, where you expect to find your
targeted cell. The resin-embedded cell is protected by a patterned platinum and carbon
pad deposited by the focused ion beam or FIB, which appears as the rapidly moving blue beam
in this movie.
Remarkably, the same focused ion beam that deposits the platinum and carbon pad can now
be used to mill a trench in front of the targeted cell. The tightly controlled FIB inches toward
the cell, ablating away the resin and eventually reveals the cell itself in cross section.
The scanning electron imaging beam, shown here as a yellow band, now rasters over the
polished resin surface, and a detector records a high-resolution back-scatter electron signal.
This process is repeated over and over. As the FIB moves forward and mills away a few
more nanometers of the resin, the SEM images the newly exposed section of the cell to generate
the next image. And so on.
Typically, we generate SEM images at 3-5 nm pixel size in the imaging plane and 3-15 nm
FIB step size, meaning that this automated loop is often repeated several thousand times
to generate a highly information-rich ultrastructural image stack covering entire mammalian cells.
These images are then registered using the notch marks in the pad, and converted into
an isotropic image volume that allows you to visualize architectural features throughout
the bulk of the cell.
The final step is segmentation; here, we show the extraction and rendering of the plasma
membrane of the cell. FIB-SEM imaging reveals that membrane extensions in this cell that
looked like spaghetti in 2-D cross section are actually veils when visualized in three
dimensions.
FIB-SEM imaging can be applied to a wide variety of systems to confirm observations, generate
hypotheses, and most fun of all, make unexpected discoveries in cell biology.
If you'd like to know more, you can contact me, Kedar Narayan at the Center for Molecular
Microscopy at the National Cancer Institute and Frederick National Laboratory for Cancer
Research. You can also visit cmm.nci.nih.gov.