3D rendering of coronal section of a mouse brain imaged with STP tomography at 20x at a resolution of half a micron. GFP-expressing pyramidal neurons in hippocampus and cortex are targeted. |
A
new technology developed by neuroscientists at Cold Spring Harbor
Laboratory (CSHL) transforms the way highly detailed anatomical images
can be made of whole brains. Until now, means of obtaining such
images—used in cutting-edge projects to map the mammalian brain—have
been painstakingly slow and available only to a handful of highly
specialized research teams.
By
automating and standardizing the process in which brain samples are
divided into sections and then imaged sequentially at precise spatial
orientations in two-photon microscopes, the team, led by Assoc. Prof.
Pavel Osten and consisting of scientists from his CSHL lab and the
Massachusetts Institute of Technology, has opened the door to making
whole-brain mapping routine.
Specifically,
says Osten, “the new technology should greatly facilitate the
systematic study of neuroanatomy in mouse models of human brain
disorders such as schizophrenia and autism.”
The
new technology, developed in concert with TissueVision of Cambridge,
Mass. and reported on in a paper appearing online Jan. 15 in Nature Methods,
is called Serial Two-Photon Tomography, or STP tomography. Tomography
refers to any process (including the familiar CAT and PET scans used in
medical diagnostics) that images an object section by section, by
shooting penetrating waves through it. Computers powered by mathematical
formulae reassemble the results to produce a three-dimensional
rendering. Two-photon imaging is a type used in biology laboratories,
particularly in conjunction with fluorescent biomarkers, which can be
mobilized to illuminate specific cell types or other anatomical
features. The two-photon method allows deeper optical penetration into
the tissue being sampled than conventional confocal microscopy.
As
Osten explains, STP tomography achieves high-throughput fluorescence
imaging of whole mouse brains via robotic integration of the two
fundamental steps—tissue sectioning and fluorescence imaging. In their
paper, his team reports on the results of several mouse-brain imaging
experiments, which indicate the uses and sensitivity of the new tool.
They conclude that it is sufficiently mature to be used in whole-brain
mapping efforts such as the ongoing Allen Mouse Brain Atlas project.
One
set of experiments tested the technology at different levels of
resolution. At 10x magnification of brain tissue samples, they performed
fast imaging “at a resolution sufficient to visualize the distribution
and morphology of green-fluorescent protein-labeled neurons, including
their dendrites and axons,” Osten reports.
A
full set of data, including final images, could be obtained by the team
in 6.5 to 8.5 hours per brain, depending on the resolution. These sets
each were comprised of 260 top-to-bottom, or coronal, slices of mouse
brain tissue, which were assembled by computer into three-dimensional
renderings themselves capable of a wide range of “warping,” i.e.,
artificial manipulation, to reveal hidden structures and features.
“The
technology is a practical one that can be used for scanning at various
levels of resolution, ranging from 1 to 2 microns to less than a
micron,” Osten says. Scans at the highest resolution level take about
24 hours to collect. This makes possible an impressive saving of time,
Osten says, compared to methods that are now in use. Using these, it
would take an experienced technician about a week to collect a set of
whole-brain images at high resolution, he noted.
“What
is most exciting about this tool is its application in the study of
mouse models of human illness, which we are already doing in my lab,”
Osten says. “We are focusing on making comparisons between different
mouse models of schizophrenia and autism. Many susceptibility genes
have been identified in both disorders—one recent estimate by Dr. Mike
Wigler’s team here at CSHL put the figure at over 250 for autism
spectrum disorders, for instance. Dr. Alea Mills at CSHL has published a
mouse model of one genetic aberration in autism—a region on chromosome
16 – and soon we will have tens of models, each showing a different
aberration.
“We
will want to compare these mice, and that is essentially why we
designed STP tomography—to automate and standardize the process of
collecting whole-brain images in which different cell-types or circuit
tracings have been performed. This makes possible comparisons across
different mouse models in an unbiased fashion.”
This
research was supported by grants from: The Simons Foundation, The
McKnight Foundation, the Howard Hughes Medical Institute, and the
National Institutes of Health.
Serial two-photon tomography for automated ex vivo mouse brain imaging
