Biology
has long sought a window into the human mind and with new technologies, today,
it is possible to literally see through the brain!
In a
recent report in the journal Nature, the authors have developed a new
technology, called CLARITY, that render big chunks of tissue optically clear
and permissive to immunolabeling.
Understanding
the functioning of the brain and its transformation into the mind is one of the
single biggest challenges facing biology today. And although work over the past
few decades has given us a fairly good understanding of the brain at a
microscopic level; we have lost out on the bigger picture that emerges from the
interactions among the different microscopic areas – largely due to
technological limitations (and the diffraction limit of light).
Our
current view of the brain is almost like a map of the world before the
satellite technology came in – remember the pre-google map/ pre-GPS era? Our
reconstructions of the brain either involve microscopic examination of many
smaller pieces aimed at re-creating the bigger picture or a gross overview of
the bigger areas without the finer details. Not only does this piece-meal
effort yield a partial and incomplete picture, it is labor intensive, expensive
and time consuming. Also most of these techniques are often incompatible with
molecular phenotyping of the intact tissue such that we most often get a crude map
without any labels or orientation. This makes it virtually impossible to
understand the landscape of the human brain. And mapping a place is truly
critical to visiting and understanding it!
One
possible solution to these technical challenges would be to render the intact
tissue transparent to light and permeable to our molecular tags in their native
location. Now, this sounds like the perfect dream but Prof. Karl Deisseroth and
his team at the Stanford have achieved exactly this with their technology
called CLARITY.
They
figured that lipid bilayers or the cell membranes are the single biggest
obstacle in making a tissue inaccessible to light (and hence imaging) and to
our molecular probes; and if they could be selectively removed without
destroying the structure and localization of the tissue, then we could get an
optically clear 3-dimensional tissues that can be imaged with ease. But these
cell membranes are what provide structure and integrity to the cells; without
them we would be staring at a broth of many proteins and nucleic acids with no
structure or localization. Thus, even as the lipid bilayers are removed, one
needs to replace that framework with an alternative scaffold that can
physically support the tissue and this is what they have achieved with their
technology.
Briefly,
they first immobilize the proteins and nucleic acids in the brain tissue by
fixing them and crosslinking them with formaldehye and a hydrogel (acrylamide and
bisacrylamide) that is liquid at low temperatures (and hence perfuses all
through the tissue), but solidifies at higher temperatures. Formaldehyde is a
strong, pungent smelling chemical that uses the amine groups (NH2 or nitrogen
groups) of proteins and nucleic acids and crosslinks them to each other. This
locks the cells and tissues in a state of complete inaction enabling additional
manipulation (sectioning, staining, storing). While formaldehyde is routinely
used in the hospitals and laboratories to store and “fix” tissue samples, it
would not preserve structural integrity in the absence of lipid bilayers. Once
the tissue is embalmed with this mix of a hydrogel and a fixative, the authors
can now move the tissue to higher temperatures (37C) where the hydrogel will
solidify and form a firm scaffold with the proteins and nucleic acids anchored
on it.
At
this point, the tissue is ready to be cleared off the lipids. And although
using organic solvents like alcohol, benzene etc. would probably be an easier
route to remove the lipid bilayers, this treatment usually quenches any
fluorescence that the tissue may carry due to the expression of genetically
encoded fluorescent proteins (like GFP, YFP, RFP etc.). To circumvent this
problem, the authors used ionic detergents to remove the lipid layers, much
like what your soap does. However, instead of relying on a slow (month-long)
passive diffusion process, the authors applied an electric charge on the tissue
to strip off the lipids along with the detergent. Since the proteins and the
nucleic acids (also charged) were already cross-linked to the hydrogel, a small
electric field should selectively strip out the lipids thus leaving behind an
optically clear scaffolding of the brain with the proteins and nucleic acids anchored
in place.
Using
CLARITY, or Clear Lipid-exchanged Acrylamide-Hybridized Rigid
Imaging/immunostaining/in situ hybridization compatible- Tissue hYdrogel, the
authors managed to transform a mouse brain in 8 days into an optically clear,
lipid-free, structurally stable hydrogel tissue hybrid. This tissue hybrid (map)
could then be labeled for specific areas by the use of specific molecular
probes (nucleic acid probes in FISH or antibodies against proteins) and imaged
as a whole with standard microscopy that is routinely used.
Intact adult mouse brain Imaging before and after CLARITY.
CLARITY: Tissue is crosslinked with formaldehyde (red) in the presence of hydrogel monomers (blue), covalently linking tissue elements to monomers that are then polymerized into a hydrogel mess (followed by a 4 day wash step). Electric fields applied across the sample in ionic detergent actively transport micelles into, and lipids out of, the tissue, leaving fine-structured and cross-linked biomolecules in places. The ETC chamber for the electrophoresis is depicted in the boxed region.
Imaging was
performed from an intact adult mouse brain using CLARITY. This is a
non-sectioned mouse bran showing the cortex, hippocampus and thalamus (X10
objective; stack size 3400 uM; step size, 2uM).
Clearing
tissues has been attempted for a long time now and for many end purposes like
imaging, generating tissue scaffolds, developing tissue niches and synthetic
organs. However, these approaches have relied on harsher methods that stripped
the tissues off all biomolecules – lipids, nucleic acids and proteins – thus
making the cleared tissue stripped of biological landmarks/ markers. Previous
methods used detergents like SDS, urea, Triton X 100 and chemicals like Scale to strip the lipids from the tissue
matrix and most of these methods also caused massive protein loss ranging
anywhere between 65% to 25%.
With
their improvements and modifications, CLARITY has enabled optical clearing of
bigger and denser tissues with only 8% loss of the tissue protein thus allowing
more landmarks in our map. The authors also found that replacing the impervious
lipid bilayers with the porous hydrogel enabled the diffusion of the molecular
probes deeper into the tissue – enabling greater access and better coverage.
Remarkably,
these hybrid tissue scaffolds were also compatible to banked specimens that
were fixed years ago with only formaldehyde. This further broadens the scope
and utility of the technique as it makes available for study tissue samples
banked and collected years before.
Although
further improvements in imaging and computational techniques would enable a
more refined, high throughput analysis, the use of CLARITY has certainly
enabled integrative visualization of large scale biological systems. And within
a few years, we might be able to have an accurate and labeled (satellite level)
map of the landscape of a mammalian brain.
References:
2)
http://www.nature.com/news/see-through-brains-clarify-connections-1.12768
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