U.S. patent application number 15/431388 was filed with the patent office on 2017-08-03 for methods and compositions for preparing biological specimens for microscopic analysis.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Kwanghun Chung, Karl A. Deisseroth.
Application Number | 20170219465 15/431388 |
Document ID | / |
Family ID | 50068460 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170219465 |
Kind Code |
A1 |
Deisseroth; Karl A. ; et
al. |
August 3, 2017 |
Methods and Compositions for Preparing Biological Specimens for
Microscopic Analysis
Abstract
Methods and compositions are provided for preparing a biological
specimen for microscopic analysis. These methods find many uses,
for example in medicine and research, e.g., to diagnose or monitor
disease or graft transplantation, to study healthy or diseased
tissue, to screen candidate agents for toxicity and efficacy in
disease modification. Also provided are reagents, devices, kits and
systems thereof that find use in practicing the subject
methods.
Inventors: |
Deisseroth; Karl A.; (Palo
Alto, CA) ; Chung; Kwanghun; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Palo Alto |
CA |
US |
|
|
Family ID: |
50068460 |
Appl. No.: |
15/431388 |
Filed: |
February 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14403050 |
Nov 21, 2014 |
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PCT/US2013/031066 |
Mar 13, 2013 |
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15431388 |
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61681551 |
Aug 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G02B 21/34 20130101;
G01N 1/40 20130101; G01N 2001/4038 20130101; G01N 1/31 20130101;
G01N 33/4833 20130101; G01N 27/44747 20130101; G01N 1/30 20130101;
G01N 27/44743 20130101 |
International
Class: |
G01N 1/31 20060101
G01N001/31; G01N 1/40 20060101 G01N001/40; G01N 27/447 20060101
G01N027/447; G01N 33/483 20060101 G01N033/483 |
Claims
1.-53. (canceled)
54. A system for preparing a biological specimen for imaging, the
system comprising an electrophoretic tissue clearing device for
electrophoresing a three-dimensional hydrogel-embedded biological
specimen to substantially remove a plurality of cellular components
from the specimen before imaging of the hydrogel-embedded
biological specimen, wherein the device comprises: an
electrophoresis chamber for containing the hydrogel-embedded
biological specimen; a plurality of electrodes; a power supply; and
a temperature-controlled buffer circulator.
55. The system according to claim 54, wherein the device further
comprises a buffer filtering component.
56. The system according to claim 54, wherein the device further
comprises a plurality of fluid inlet and/or outlet ports.
57. The system according to claim 54, wherein the device further
comprises a component configured to support the hydrogel-embedded
biological specimen.
58. The system according to claim 57, wherein the component is
configured to support the hydrogel-embedded biological specimen in
a position that is substantially inside an electric field generated
between two or more of the electrodes.
59. The system according to claim 54, wherein one or more of the
electrodes comprises an expansion component for increasing the size
of an electric field generated by the electrodes.
60. The system according to claim 59, wherein the expansion
component comprises one or more S-shaped bends.
61. The system according to claim 54, wherein the length and the
width of the one or more electrodes are approximately equal.
62. The system according to claim 54, further comprising a lid that
forms a fluid-tight and/or air-tight seal with the electrophoresis
chamber.
63. The system according to claim 54, further comprising a buffer
solution that comprises an ionic surfactant.
64. The system according to claim 63, wherein the ionic surfactant
is sodium dodecyl sulfate.
65. The system according to claim 54, further comprising the
hydrogel-embedded biological specimen.
66. The system according to claim 65, wherein the hydrogel
comprises an acrylamide.
67. The system according to claim 65, wherein the biological
specimen is central nervous system tissue.
68. The system according to claim 65, wherein the biological
specimen is a biopsy specimen or an autopsy specimen.
69. The system according to claim 54, wherein the cellular
components comprise lipids.
70. The system according to claim 54, wherein the imaging is
performed using a microscope.
71. The system according to claim 70, wherein the microscope is an
optical microscope, a laser microscope, an electron microscope, or
a scanning probe microscope.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit to the filing date
of U.S. Provisional Patent Application Ser. No. 61/681,551, filed
on Aug. 9, 2012, the disclosure of which application is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention pertains to preparing biological specimens
for microscopic analysis.
BACKGROUND OF THE INVENTION
[0003] To study complex organs and tissues, such as brain and
tumor, it is necessary to understand its integrated 3-D structure
and fine molecular details throughout the whole tissue. Current
methods, exemplified by array tomography or serial block-face
scanning electron microscopy can provide sub-cellular fine details,
but involve prohibitively inefficient and damaging mechanical
sectioning and reconstruction. Optical sectioning techniques
combined with tissue clearing methods have been developed, in which
light-scattering is reduced to increase the depth at which tissue
can be imaged. While these methods can bypass laborious mechanical
sectioning and reconstruction processes, they are not compatible
with immunostaining/molecular phenotyping. What is needed is a
technology for the preparation of biological tissue for microscopic
analysis that maintains the 3-D integrity of the tissue and of the
sub-cellular structures therein, while also making biomolecules
within the tissue e.g., proteins, lipids, steroids, nucleic acids,
and small molecules, accessible for labeling with molecular probes
at deeper regions in the tissue. The present invention addresses
these and other issues.
SUMMARY OF THE INVENTION
[0004] Methods and compositions are provided for preparing a
biological specimen for microscopic analysis. These methods find
many uses, for example in medicine and research, e.g., to diagnose
or monitor disease or graft transplantation, to study healthy or
diseased tissue, and to screen candidate agents for toxicity and
efficacy in disease modification. Also provided are reagents,
devices, kits and systems thereof that find use in practicing the
subject methods.
[0005] In some embodiments, the present disclosure provides methods
of preparing a biological specimen for microscopic analysis, the
methods including fixing the specimen with a plurality of hydrogel
subunits, polymerizing the hydrogel subunits to form a
hydrogel-embedded specimen, and clearing the hydrogel-embedded
specimen. In some embodiments, clearing the hydrogel-embedded
specimen involves substantially removing a plurality of cellular
components from the specimen. In some embodiments, the cellular
components include lipids.
[0006] In some embodiments, clearing the hydrogel-embedded specimen
comprises electrophoresing the specimen. In some embodiments, the
specimen is electrophoresed using a buffer solution that comprises
an ionic surfactant. In some embodiments, the ionic surfactant is
sodium dodecyl sulfate (SDS). In some embodiments, the specimen is
electrophoresed using a voltage ranging from about 10 to about 60
volts. In some embodiments, the specimen is electrophoresed for a
period of time ranging from about 15 minutes up to about 10 days.
In some embodiments, the methods further involve incubating the
cleared specimen in a mounting medium that has a refractive index
that matches that of the cleared tissue. In some embodiments, the
mounting medium increases the optical clarity of the specimen. In
some embodiments, the mounting medium comprises glycerol.
[0007] In some embodiments, the microscopic analysis is optical
microscopy, laser microscopy, electron microscopy, and scanning
probe microscopy. In some embodiments, fixing the specimen involves
contacting the specimen with a paraformaldehyde. In some
embodiments, the hydrogel subunits include an acrylamide. In some
embodiments, polymerizing the specimen comprises thermal
crosslinking.
[0008] In some embodiments, the methods further involve contacting
the specimen with a polypeptide, nucleic acid, or small molecule.
In some embodiments, the contacting involves electrophoresis,
hydrodynamic pressure, ultrasonic vibration, solute contrasts,
microwave radiation, or vascular circulation. In some embodiments,
the polypeptide, nucleic acid, or small molecule includes a
component that can be rendered visible when the specimen is
microscopically analyzed. In some embodiments, the tissue is
central nervous system (CNS) tissue. In some embodiments, the CNS
tissue is a whole brain.
[0009] In some embodiments, the present disclosure provides methods
of imaging a biological specimen by microscopy, the methods
including preparing a biological specimen as described above and
imaging the biological specimen with a microscope. In some
embodiments, the microscope is an optical microscope, laser
microscope, electron microscope, or a scanning probe microscope. In
some embodiments, cellular or subcellular aspects of the specimen
are labeled with one or more small molecules, nucleic acids or
proteins transported into the prepared tissue. In some embodiments,
the methods further involve removing one or more small molecules,
nucleic acids, or proteins that were previously transported into
the prepared tissue.
[0010] In some embodiments, the present disclosure provides methods
of mapping the connectivity of nervous system tissue, the methods
including preparing a nervous system tissue specimen as described
above and imaging one or more neurons in the specimen with a
microscope. In some embodiments, the subject methods further
include labeling the one or more neurons in the specimen with a
component that can be rendered visible when the specimen is
microscopically analyzed. In some embodiments, the neurons are
labeled before fixing the tissue. In some embodiments, the neurons
are labeled after polymerizing the hydrogel.
[0011] In some embodiments, the present disclosure provides kits
for preparing a tissue for microscopic analysis, the kits including
a fixative and a plurality of hydrogel subunits. In some
embodiments, the kits further include an apparatus for
electrophoresing a three-dimensional hydrogel-embedded specimen to
substantially remove a plurality of cellular components from the
specimen. In some embodiments, the cellular components include
lipids.
[0012] In some embodiments, the present disclosure provides systems
for preparing a biological specimen for imaging, the systems
including an apparatus for electrophoresing a three-dimensional
hydrogel-embedded specimen to substantially remove a plurality of
cellular components from the specimen, a power supply and a
temperature-controlled buffer circulator. In some embodiments, the
cellular components include lipids.
[0013] In some embodiments, the present disclosure provides
electrophoretic tissue clearing devices, the devices including an
electrophoresis chamber for containing a three-dimensional
hydrogel-embedded specimen, a plurality of electrodes, a power
supply, and a temperature-controlled buffer circulator. In some
embodiments, the subject devices further include a buffer filtering
component. In some embodiments, the subject devices further include
a plurality of fluid inlet and/or outlet ports. In some
embodiments, the subject devices further include a component
configured to support the hydrogel-embedded specimen. In some
embodiments, the component is configured to support the
hydrogel-embedded specimen in a position that is substantially
inside an electric field generated between two or more of the
electrodes. In some embodiments, one or more of the electrodes
comprises an expansion component for increasing the size of an
electric field generated by the electrodes. In some embodiments,
the expansion component comprises one or more S-shaped bends. In
certain embodiments, the length and the width of the one or more
electrodes are approximately equal. In some embodiments, the
subject devices further include a lid that forms a fluid-tight
and/or air-tight seal with the electrophoresis chamber.
[0014] In some embodiments, the present disclosure provides methods
of preserving a biological specimen, the methods involving fixing
the specimen with a plurality of hydrogel subunits, polymerizing
the hydrogel subunits to form a hydrogel-embedded specimen, and
clearing the hydrogel-embedded specimen. In some embodiments, the
subject methods further involve storing the cleared
hydrogel-embedded specimen in a mounting medium. In some
embodiments, the subject methods further involve analyzing the
cleared hydrogel-embedded specimen for evaluation, diagnosis, or
prognosis of a pathological state. In some embodiments, the
specimen is a biopsy specimen or an autopsy specimen. In some
embodiments, the pathological state is cancer, immune system
dysfunction, neuropsychiatric disease, endocrine/reproductive
disease, cardiovascular/pulmonary disease, musculoskeletal disease,
or gastrointestinal disease.
[0015] In some embodiments, the specimen includes normal tissue,
and the method further involves analyzing the specimen to evaluate
cell, tissue, organ or system function and/or relationships between
cells and tissues, including during development. In some
embodiments, the subject methods further involve conducting a
genetic, transcriptomic, genomic, proteomic, metabolomic and/or
drug screening analysis on the specimen. In some embodiments, the
subject methods further involve storing the specimen for future
analysis, assessment, or functionalization.
[0016] In some embodiments, the present disclosure provides systems
for infusing hydrogel monomers into biological tissue and
subsequently triggering the monomers to form a polymer, gel, mesh,
or network with desired stiffness, transparency, pore size,
conductivity, or permeability properties, the system including a
biological specimen and a plurality of hydrogel subunits. In some
embodiments, the subject systems further include nanoscale hardware
devices, proteins, oligonucleotides, and/or fluorescent staining
reagents. In some embodiments, the components of the system are
activated or functionalized by energy or external signals such as
heat, light, chemical triggers, and/or accelerators.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The invention is best understood from the following detailed
description when read in conjunction with the accompanying
drawings. The patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee. It is
emphasized that, according to common practice, the various features
of the drawings are not to-scale. On the contrary, the dimensions
of the various features are arbitrarily expanded or reduced for
clarity. Included in the drawings are the following figures.
[0018] FIG. 1 is an illustration showing an overview of a process,
termed "CLARITY," that facilitates imaging of tissues without
tissue sectioning.
[0019] FIG. 2 is an illustration of an electrophoretic tissue
clearing (ETC) device and related instrumentation.
[0020] FIG. 3 is collection of images from intact adult mouse brain
samples that were imaged using the CLARITY process.
[0021] FIG. 4 is a collection of images and data showing molecular
phenotyping results in intact tissue volumes processed using
CLARITY.
[0022] FIG. 5 is a collection of images showing multi-round
molecular phenotyping of intact tissue using CLARITY.
[0023] FIG. 6 is a collection of images and data showing results of
human brain structural mapping and molecular phenotyping using
CLARITY.
[0024] FIG. 7 is a collection of images showing results from mouse
brain tissue that was imaged using CLARITY.
[0025] FIG. 8 is a collection of drawings showing an example of an
ETC chamber design. Indicated dimensions are in millimeters.
[0026] FIG. 9 is a collection of images showing optical tissue
clearing of intact adult mouse brain using FocusClear.TM. and
glycerol.
[0027] FIG. 10 is a collection of images showing results from
electron microscope (EM) imaging of CLARITY-processed mouse brain
tissue. The images demonstrate EM-compatibility of the CLARITY
process.
[0028] FIG. 11 is a collection of images showing results from whole
mouse brain molecular phenotyping.
[0029] FIG. 12 is a collection of images showing axonal fibers of
the TH-positive neurons in the prefrontal cortex of a mouse brain
imaged using the CLARITY process.
[0030] FIG. 13 is a collection of images showing axonal fibers of
the TH-positive neurons in the nucleus accumbens and striatum of a
mouse brain imaged using the CLARITY process.
[0031] FIG. 14 is a collection of images showing axonal fibers of
the TH-positive neurons in amygdala of mouse brain imaged using the
CLARITY process.
[0032] FIG. 15 is a series of graphs showing the average
immunofluorescence cross-section of PSD-95 puncta at different
depths (0-200 .mu.m, 20 .mu.m interval).
[0033] FIG. 16 is a series of images showing the average PSD-95
puncta at different depths (0-200 .mu.m, 20 .mu.m interval).
[0034] FIG. 17 is a collection of images showing
microtubule-associated protein 2 (MAP2) staining showing uniform
labeling of dense dendritic fibers and neuronal cell bodies
throughout a 1 mm-thick mouse brain tissue sample imaged using the
CLARITY process.
[0035] FIG. 18 is an image showing results from whole adult
zebrafish brain molecular phenotyping.
[0036] FIG. 19 is a collection of images showing results from the
traced PV-positive neurons in the neocortex of a brain tissue
sample from a subject with autism.
[0037] FIG. 20 is a collection of images showing results from the
traced PV-positive neurons in the neocortex of a brain tissue
sample from a normal control subject.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Methods and compositions are provided for preparing a
biological specimen for microscopic analysis. These methods find
many uses, for example in medicine and research, e.g., to diagnose
or monitor disease or graft transplantation, to study healthy or
diseased tissue, to screen candidate agents for toxicity and
efficacy in disease modification. Also provided are reagents,
devices, kits and systems thereof that find use in practicing the
subject methods. These and other objects, advantages, and features
of the invention will become apparent to those persons skilled in
the art upon reading the details of the compositions and methods as
more fully described below.
[0039] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to
particular method or composition described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0040] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0041] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supercedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0042] As will be apparent to these of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible. Any steps of a method may
be separated from another step of the method by an optional storage
step, i.e. storage at room temperature, at 16.degree. C., at
4.degree. C., at -12.degree. C., at -20.degree. C., at -70.degree.
C., or on -130.degree. C.
[0043] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the peptide" includes reference to one or more
peptides and equivalents thereof, e.g. polypeptides, known to those
skilled in the art, and so forth.
[0044] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Methods
[0045] In aspects of the invention, methods are provided for
preparing biological specimens for microscopic analysis. By
"microscopic analysis" is meant the analysis of a specimen using
techniques that provide for the visualization of aspects of a
specimen that cannot be seen with the unaided eye, i.e., that are
not within the resolution range of the normal eye. Such techniques
may include, without limitation, optical microscopy (e.g., bright
field, oblique illumination, dark field, phase contrast,
differential interference contrast, interference reflection,
epifluorescence, confocal, etc., microscopy), laser microscopy,
electron microscopy, and scanning probe microscopy. By "preparing a
biological specimen for microscopic analysis" is generally meant
rendering the specimen suitable for microscopic analysis at an
unlimited depth within the specimen.
[0046] In practicing the subject methods, a biological specimen is
fixed in the presence of hydrogel subunits. By "fixing" the
specimen it is meant exposing the specimen, i.e., cells of the
specimen, to a fixation agent such that the cellular components
become crosslinked to one another. By "hydrogel" or "hydrogel
network" is meant a network of polymer chains that are
water-insoluble, sometimes found as a colloidal gel in which water
is the dispersion medium. In other words, hydrogels are a class of
polymeric materials that can absorb large amounts of water without
dissolving. Hydrogels can contain over 99% water and may comprise
natural or synthetic polymers, or a combination thereof. Hydrogels
also possess a degree of flexibility very similar to natural
tissue, due to their significant water content. A detailed
description of suitable hydrogels may be found in published U.S.
patent application 20100055733, herein specifically incorporated by
reference. By "hydrogel subunits" or "hydrogel precursors" is meant
hydrophilic monomers, prepolymers, or polymers that can be
crosslinked, or "polymerized", to form a three-dimensional (30)
hydrogel network. Without being bound by scientific theory, it is
believed that this fixation of the biological specimen in the
presence of hydrogel subunits crosslinks the components of the
specimen to the hydrogel subunits, thereby securing molecular
components in place, preserving the tissue architecture and cell
morphology.
[0047] Any convenient fixation agent, or "fixative," may be used in
the fixative/hydrogel composition to fix the specimen in the
presence of hydrogel subunits, for example, formaldehyde,
paraformaldehyde, glutaraldehyde, acetone, ethanol, methanol, etc.
Typically, the fixative will be diluted in a buffer, e.g., saline,
phosphate buffer (PB), phosphate buffered saline (PBS), citric acid
buffer, potassium phosphate buffer, etc., usually at a
concentration of about 1-10%, e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
or 10%, for example, 4% paraformaldehyde/0.1M phosphate buffer; 2%
paraformaldehyde/0.2% picric acid/0.1M phosphate buffer; 4%
paraformaldehyde/0.2% periodate/1.2% lysine in 0.1 M phosphate
buffer; 4% paraformaldehyde/0.05% glutaraldehyde in phosphate
buffer; etc. The type of fixative used and the duration of exposure
to the fixative will depend on the sensitivity of the molecules of
interest in the specimen to denaturation by the fixative, and will
be known by the ordinarily skilled artisan or may be readily
determined using conventional histochemical or immunohistochemical
techniques, for example as described in Buchwalow and Bocker.
Immunohistochemistry: Basics and Methods. Springer-Verlag Berlin
Heidelberg 2010.
[0048] The fixative/hydrogel composition may comprise any
convenient hydrogel subunits, such as, but not limited to,
poly(ethylene glycol) and derivatives thereof (e.g. PEG-diacrylate
(PEG-DA), PEG-RGD), polyaliphatic polyurethanes, polyether
polyurethanes, polyester polyurethanes, polyethylene copolymers,
polyamides, polyvinyl alcohols, polypropylene glycol,
polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide,
poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate),
collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin,
alginate, protein polymers, methylcellulose and the like. In some
instances, the hydrogel subunits may be modified to add specific
properties to the hydrogel; for example, peptide sequences can be
incorporated to induce degradation (see, e.g., West and Hubbell,
1999, Macromolecules, 32:241) or to modify cell adhesion (see, e.g.
Hem and Hubbell, 1998, J. Biomed. Mater. Res., 39:266). Agents such
as hydrophilic nanoparticles, e.g., poly-lactic acid (PLA),
poly-glycolic acid (PLO), polylactic-co-glycolic acid) (PLGA),
polystyrene, poly(dimethylsiloxane) (PDMS), etc. may be used to
improve the permeability of the hydrogel while maintaining
patternability (see, e.g., U.S. patent application Ser. No.
13/065,030; Lee W. et al. 2010 Proc. Natl. Acad. Sci. 107,
20709-20714). Materials such as block copolymers of PEG, degradable
PEO, polylactic acid) (PLA), and other similar materials can be
used to add specific properties to the hydrogels (see, e.g., Huh
and Bae, 1999, Polymer, 40:6147). Crosslinkers (e.g.
bis-acrylamide, diazirine, etc.) and initiatiors (e.g.
azobisisobutyronitrile (AIBN), riboflavin, L-arginine, etc.) may be
included to promote covalent bonding between interacting
macromolecules in later polymerization steps.
[0049] Typically, the concentration and molecular weight of the
hydrogel subunit(s) and modifying agents will depend on the
selected polymer and the desired characteristics, e.g., pore size,
swelling properties, conductivity, elasticity/stiffness (Young's
modulus), biodegradability index, etc., of the hydrogel network
into which they will be polymerized. For example, it may be
desirable for the hydrogel to comprise pores of sufficient size to
allow the passage of macromolecules, proteins, nucleic acids, or
small molecules as described in greater detail below, into the
specimen. The ordinarily skilled artisan will be aware that pore
size generally decreases with increasing concentration of hydrogel
subunits and generally increases with an increasing ratio of
hydrogel subunits to crosslinker, and will prepare a
fixative/hydrogel composition that comprises a concentration of
hydrogel subunits that allows the passage of such macromolecules.
As another example, it may be desirable for the hydrogel to have a
particular stiffness, e.g., to provide stability in handling the
embedded specimen, e.g., a Young's Modulus of about 2-70
kN/m.sup.2, for example, about 2 kN/m.sup.2, about 4 kN/m.sup.2,
about 7 kN/m.sup.2, about 10 kN/m.sup.2 about 15 kN/m.sup.2, about
20 kN/m.sup.2, about 40 kN/m.sup.2, but typically not more than
about 70 kN/m.sup.2. The ordinarily skilled artisan will be aware
that the elasticity of a hydrogel network may be influenced by a
variety of factors, including the branching of the polymer, the
concentration of hydrogel subunits, and the degree of
cross-linking, and will prepare a fixative/hydrogel composition
that comprises a concentration of hydrogel subunits to provide such
desired elasticity. Thus, for example, the fixative/hydrogel
composition may comprise an acrylamide monomer at a concentration
of from about 1% w/v to about 20% w/v, e.g., about 2% to about 15%,
about 3% to about 10%, about 4% to about 8%, and a concentration of
bis-acrylamide crosslinker in the range of about 0.01% to about
0.075%, e.g., 0.01%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, or
0.075%; or, for example, the fixative/hydrogel composition may
comprise PEG prepolymers having a molecular weight ranging from at
least about 2.5K to about 50K, e.g., 2.5K or more, 3.5K or more, 5K
or more, 7.5K or more, 10K or more, 15K or more, 20K or more, but
typically not more than about 50K, at a concentration in a range
from about 1% w/w to about 50% w/w, e.g., 1% or more, 5% or more,
7.5% or more, 10% or more, 15% or more, 20% or more, 30% or more,
40% or more, and usually not more than about 50%. Concentrations of
hydrogel subunits and modifiers that provide desired hydrogel
characteristics may be readily determined by methods in the art or
as described in the working examples below.
[0050] The fixative hydrogel solution may be delivered to the
specimen by any convenient method, e.g., perfusion, injection,
instillation, absorption, application, immersion/submersion, etc.
The specimen will typically be fixed in the presence of the
hydrogel for 15 minutes or more, for example, for 30 minutes or
more, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or
more, 12 hours or more, in some instances, for 16 hours or more, 20
hours or more, or 24 hours or more.
[0051] Following fixation of the specimen, the hydrogel subunits
are polymerized, i.e., covalently or physically crosslinked, to
form a hydrogel network. Polymerization may be by any method
including, but not limited to, thermal crosslinking, chemical
crosslinking, physical crosslinking, ionic crosslinking,
photo-crosslinking, irradiative crosslinking (e.g., x-ray, electron
beam), and the like, and may be selected based on the type of
hydrogel used and knowledge in the art. For example, mixing of an
un-polymerized or partially polymerized resin with specific
crosslinking chemicals results in a chemical reaction that forms
cross-links. Crosslinking can be induced in materials that are
normally thermoplastic through exposure to a radiation source, such
as electron beam exposure, gamma-radiation, or UV light; for
example, electron beam processing is used to polymerize the C type
of crosslinked polyethylene. Other types of crosslinked
polyethylene are made by addition of peroxide during extruding
(type A) or by addition of a cross-linking agent (e.g. vinylsilane)
and a catalyst during extruding and then performing a
post-extrusion curing. Many polymers undergo oxidative
cross-linking, typically when exposed to atmospheric oxygen. In
some cases the reaction is more rapid than desired and thus
polymerization reactions may involve the use of an antioxidant to
slow the formation of oxidative cross-links. In other cases, e.g.,
when more rapid formation of cross-links by oxidation is desirable,
an oxidizer such as hydrogen peroxide may be used to speed up the
process. The length of time for polymerization will depend on the
type of hydrogel subunits used and the chosen polymerization
method, but will typically be about 15 minutes to about 48 hours,
for example, 15 minutes or more, 1 hour or more, 2 hours or more, 3
hours or more, 4 hours or more, 6 hours or more, 12 hours or more,
16 hours or more, 24 hours or more, or in some instances, 48 hours.
The optimal time and combination of reagents will be known to the
ordinarily skilled artisan or may be determined empirically or from
any number of publicly available resources (e.g., on the world wide
web at piercenet.com; see also, Macroporous Polymers: Production
Properties and Biotechnological/Biomedical Applications. Edited by
Bo Mattiasson, Ashok Kumar, and Igor Yu. Gaieaev. CRC Press 2010;
and Crosslinking Reagents Technical Handbook, Pierce Biotechnology,
inc., 2006).
[0052] Once polymerized, the hydrogel-embedded (i.e.,
hydrogel-hybridized) specimen may be cleared. By "clearing" a
specimen it is meant that the specimen is made substantially
permeable to light, i.e., transparent. In other words, about 70% or
more of the visual (i.e., white) light, ultraviolet light or
infrared light that is used to illuminate the specimen will to pass
through the specimen and illuminate only selected cellular
components therein, e.g., 75% or more of the light, 80% or more of
the light, 85% or more of the light, in some instances, 90% or more
of the light, 95% or more of the light, 98% or more of the light,
e.g. 100% of the light will pass through the specimen. This change
in the optical properties of the specimen provides for the
visualization of cellular and subcellular structures internal to
the tissue.
[0053] Any treatment that forces cellular components, e.g., lipids,
from the specimen, that draws cellular components, e.g., lipids,
from a specimen, or that causes cellular components, e.g., lipids,
to break down, i.e., dissolve, within a specimen may be used to
clear the specimen, including, without limitation, exposure to
organic solvents such as xylenes, ethanol or methanol, exposure to
detergents such as saponin, Triton X-100 and Tween-20, exposure to
ionic surfactants, e.g., sodium dodecyl sulfate (SDS),
electrophoresis, hydrodynamic pressure, ultrasonic vibration,
solute contrasts, microwave radiation, vascular circulation, and
the like. In some instances, clearing is performed using a solvent
that does not quench fluorescent proteins. Examples of organic
solvents that are known to quench fluorescent proteins include
tetrahydrofuran, hexane, benzylalcohol/benzylbenzoate (BABB), and
dibenzyl ether. Accordingly, in order to preserve the fluorescence
of various proteins, in some embodiments clearing is conducted
using solvents other than those listed above, e.g., is conducted
using non-organic solvents.
[0054] In some instances, clearing is conducted using an ionic
surfactant, e.g., SDS, in order to expedite the clearing process by
actively transporting charged ionic micelles out of the specimen
that is being cleared. Clearing may be performed in any convenient
buffer that is compatible with the selected clearance method, e.g.,
saline, phosphate buffer, phosphate buffered saline (PBS), sodium
borate buffer, boric acid buffer, citric acid buffer, etc., as
known in the art, and will typically take about 1-10 days per
centimeter thickness of specimen, i.e., usually about 1 day, in
some instances 2 days, sometimes 5 days, and typically no more than
10 days per cubic centimeter. Optimal time may be readily
determined by visual inspection of the specimen for clarity.
[0055] After clearing, a sample will generally be substantially
free of lipids. By "substantially free of lipids" is meant that the
original amount of lipids present in the sample before clearing has
been reduced by approximately 70% or more, such as by 75% or more,
such as by 80% or more, such as by 85% or more, such as by 90% or
more, such as by 95% or more, such as by 99% or more, such as by
100%.
[0056] Tissue specimens suitable for use with the methods and
systems described herein generally include any type of tissue
specimens collected from living or dead subjects, such as, e.g.,
biopsy specimens and autopsy specimens. Tissue specimens may be
collected and processed using the methods and systems described
herein and subjected to microscopic analysis immediately following
processing, or may be preserved and subjected to microscopic
analysis at a future time, e.g., after storage for an extended
period of time. In some embodiments, the methods described herein
may be used to preserve tissue specimens in a stable, accessible
and fully intact form for future analysis. For example, tissue
specimens, such as, e.g., human brain tissue specimens, may be
processed as described above and cleared to remove a plurality of
cellular components, such as, e.g., lipids, and then stored for
future analysis. In some embodiments, the methods and systems
described herein may be used to analyze a previously-preserved or
stored tissue specimen. For example, in some embodiments a
previously-preserved tissue specimen that has not been subjected to
the CLARITY process may be processed and analyzed as described
herein.
[0057] In some instances, no further manipulation of the specimen
will be necessary for microscopic analysis. For example, the
specimen may comprise biomolecules that can be directly visualized
by microscopy. By "biomolecules" it is generally meant proteins,
lipids, steroids, nucleic acids, etc. within a tissue or cell. One
example of this would be if the organism that was the source of the
specimen expressed a protein that possesses the ability to
fluoresce, i.e. a "fluorescent protein", or "FP". By "fluoresce" is
meant to absorb energy at one wavelength and emit it at another
wavelength. For example, a green fluorescent protein (GFP) refers
to a polypeptide that has a peak in the emission spectrum at 510 nm
or about 510 nm. A variety of FPs that emit at various wavelengths
are known in the art. FPs of interest include, but are not limited
to, a green fluorescent protein (GFP), yellow fluorescent protein
(YFP), orange fluorescent protein (OFF), cyan fluorescent protein
(CFP), blue fluorescent protein (BFP), red fluorescent protein
(RFP), far-red fluorescent protein, or near-infrared fluorescent
protein. As used herein, Aequorea OFF refers to GFPs from the genus
Aequorea and to mutants or variants thereof. Such variants and GFPs
from other species, such as Anthozoa, reef coral, Anemonia sea
anemone, Renilla sea pansy, Galaxea coral, Acropora brown coral,
Trachyphyllia and Pectimidae stony coral and other species are well
known and are available and known to those of skill in the art.
Exemplary OFF variants include, but are not limited to BFP, CFP,
YFP and OFF. Examples of florescent proteins and their variants
include GFP proteins, such as Emerald (Invitrogen, Carlsbad,
Calif.), EGFP (Clontech, Palo Alto, Calif.), Azami-Green (MBL
International, Woburn, Mass.), Kaede (MBL International, Woburn,
Mass.), ZsGreen1 (Clontech, Palo Alto, Calif.) and CopGFP
(Evrogen/Axxora, LLC, San Diego, Calif.); CFP proteins, such as
Cerulean (Rizzo, Nat Biotechnol. 22(4):445-9 (2004)), mCFP (Wang et
al., PNAS USA. 101(48):16745-9 (2004)), AmCyanl (Clontech, Palo
Alto, Calif.), MiCy (MBL International, Woburn, Mass.), and CyPet
(Nguyen and Daugherty, Nat Biotechnol. 23(3):355-60 (2005)); BFP
proteins such as EBFP (Clontech, Palo Alto, Calif.); YFP proteins
such as EYFP (Clontech, Palo Alto, Calif.), YPet (Nguyen and
Daugherty, Nat Biotechnol. 23(3):355-60 (2005)), Venus (Nagai et
al., Nat. Biotechnol. 20(1):87-90 (2002)), ZsYellow (Clontech, Palo
Alto, Calif.), and mCitrine (Wang et al., PNAS USA. 101(48):16745-9
(2004)); OFP proteins such as cOFP (Strategene, La Jolla, Calif.),
mKO (MBL International, Woburn, Mass.), and mOrange; and others
(Shaner N C, Steinbach P A, and Tsien R Y., Nat Methods.
2(12):905-9 (2005)). Another class of fluorescent proteins is the
red fluorescent protein Discosoma RFP (DsRed) that has been
isolated from the corallimorph Discosoma (Matz et al., Nature
Biotechnology 17: 969-973 (1999)), and red or far-red fluorescent
proteins from any other species, such as Heteractis reef coral and
Actinia or Entacmaea sea anemone, as well as variants thereof RFPs
include, for example, Discosoma variants, such as monomeric red
fluorescent protein 1 (mRFP1), mCherry, tdTomato, mStrawberry,
mTangerine (Wang et al., PNAS USA. 101(48):16745-9 (2004)), DsRed2
(Clontech, Palo Alto, Calif.), and DsRed-T1 (Bevis and Glick, Nat.
Biotechnol., 20: 83-87 (2002)), Anthomedusa J-Red (Evrogen) and
Anemonia AsRed2 (Clontech, Palo Alto, Calif.). Far-red fluorescent
proteins include, for example, Actinia AQ143 (Shkrob et al.,
Biochem J. 392 (Pt 3):649-54 (2005)), Entacmaea egFP611 (Wiedenmann
et al. Proc Natl Acad Sci USA. 99(18):11646-51 (2002)), Discosoma
variants such as mPlum and mRasberry (Wang et al., PNAS USA.
101(48):16745-9 (2004)), and Heteractis HcRed1 and t-HcRed
(Clontech, Palo Alto, Calif.).
[0058] Additionally or alternatively, it may be desirable to
contact the cells and intracellular structures of the specimen with
one or more macromolecules prior to microscopic analysis. For
example, macromolecules may be provided that promote the
visualization of particular cellular biomolecules, e.g., proteins,
lipids, steroids, nucleic acids, etc. and sub-cellular structures.
In some embodiments, the macromolecules are diagnostic. In some
embodiments, the macromolecules are prognostic. In some
embodiments, the macromolecules are predictive of responsiveness to
a therapy. In some embodiments, the macromolecules are candidate
agents in a screen, e.g., a screen for agents that will aid in the
diagnosis and/or prognosis of disease, in the treatment of a
disease, and the like.
[0059] For example, specimens may be contacted with nucleic acid
stains like DAPI and Hoechst, which bind the minor groove of DNA,
thus labeling the nuclei of cells. Drugs or toxins that bind
specific cellular structures and have been derivatized with a
fluorescent reporter may be employed, e.g., fluorescently
labelled-phalloidin, which is used to stain actin fibers in
mammalian cells. There are many fluorescent reported molecules,
called fluorophores or fluorochromes such as fluorescein, Alexa
Fluors or DyLight 488, which can be chemically linked to molecules
which bind the target biomolecules of interest within the
sample.
[0060] As another example, the specimen may be contacted with one
or more polypeptides, e.g. antibodies, labeled peptides, and the
like, that are specific for and will bind to particular cellular
biomolecules for either direct or indirect labeling by color or
immunofluorescence. By immunofluorescence it is meant a technique
that uses the highly specific binding of an antibody to its antigen
or binding partner in order to label specific proteins or other
molecules within the cell. A sample is treated with a primary
antibody specific for the biomolecule of interest. A fluorophore
can be directly conjugated to the primary antibody or peptide.
Alternatively a secondary antibody, conjugated to a detection
moiety or fluorophore, which binds specifically to the first
antibody can be used. See, for example, Buchwalow and Backer.
Immunohistochemistry: Basics and Methods, Springer-Verlag, Berlin
Heidelberg 2010, and Hayat, M. A. Microscopy, Immunohistochemistry,
and Antigen Retrieval Methods for Light and Electron Microscopy.
Kluwar Academic Publishers, New York 2002, for examples of
protocols that may be followed. Peptides that are specific for a
target cellular biomolecule and that are conjugated to a fluorophor
or other detection moiety may also be employed.
[0061] Another example of a class of agents that may be provided as
macromolecules is nucleic acids. For example, a specimen may be
contacted with an antisense RNA that is complementary to and
specifically hybridizes to a transcript of a gene of interest,
e.g., to study gene expression in cells of the specimen. As another
example, a specimen may be contacted with a DNA that is
complementary to and specifically hybridizes to genomic material of
interest, e.g., to study genetic mutations, e.g., loss of
heterozygosity, gene duplication, chromosomal inversions, and the
like. The hybridizing RNA or DNA is conjugated to detection
moieties, i.e. agents that may be either directly or indirectly
visualized microscopically. Examples of in situ hybridization
techniques may be found at, for example, Harris and Wilkinson. In
situ hybridization: Application to developmental biology and
medicine, Cambridge University Press 1990; and Fluorescence In Situ
Hybridization (FISH) Application Guide. Liehr, T, ed.,
Springer-Verlag, Berlin Heidelberg 1990.
[0062] As another example, the specimen may be contacted with small
molecules. For example, if the specimen comprises
.beta.-galactosidase or alkaline phosphatase, it may be desirable
to visualize cells and regions of the tissue expressing these
proteins. Towards this end, a specimen may be contacted with
substrates for .beta.-galactosidase (e.g. X-gal,
4-Trifluoromethylumbelliferyl-.beta.-D-galactopyranoside
(TFMU-Gal), Resorufin .beta.-D-qalactopyranoside (Res-gal),
4-Methylumbelliferyl .beta.-D-galactopyranoside (MUG),
di-.beta.-D-galactopyranoside (FOG), Carboxyumbelliferyl
.beta.-D-galactopyranoside (CUG)) or for alkaline phosphatase (e.g.
nitro-blue tetrazolium (NBT)/5-bromo-4-chloro-3'-indolyphosphate
(BCiP)) and other reagents that allow for visualization of
.beta.-galactosidase or alkaline phosphatase activity. As another
example, it may be desirous to visualize the dendritic arbors and
spins of neurons in, e.g., a CNS specimen. To do so, the specimen
may be exposed to chemicals used in Golgi-Cox impregnation, e.g.,
3% potassium bichromate followed by a 2% silver nitrate
solution.
[0063] In some instances, the biomolecules that are targeted by the
provided macromolecules are endogenous to the cell. In other
instances, the macromolecules may be provided to the specimen to
target/visualize biomolecules that were ectopically provided to the
cells of the specimen, e.g. agents that were introduced to the
specimen in vivo or ex vivo to label certain cell populations or
subcellular structures. For example, stereotactic surgery is often
used in the field of neuroscience to provide biomolecules such as
proteins, viruses, chemicals to neural tissue that label, or
"trace", the projections and/or the connectivity of subsets of
neurons in vivo or ex vivo. In this technique, a needle comprising
a labeling macromolecule is lowered into CNS tissue at a precise
location and the labeling molecule is released into the tissue. The
molecule will fill the neurons in the vicinity of the injection
site and, depending on the type of macromolecule delivered, may be
transported across synapses to label their efferent targets
("anterograde tracing") and/or across dendrites to label the
afferent neurons from which they are receiving signals ("retrograde
tracing"). Examples of agents that may be used to label neurons
stereotactically are well known in the art, including, for example,
nucleic acids that encode fluorescent proteins; viral tracers, e.g.
Herpes simplex virus type1 (HSV) and the Rhabdoviruses; wheat-germ
agglutinin (WGA); Phaseolus vulgaris leucoagglutinin (PHA-L);
horseradish peroxidase-conjugated lectins; biotinylated dextran
amines (BDA); cholera toxin B; NEUROBIOTIN Tracer.RTM. (Vector
labs). Specimens labeled in this way may be contacted with
macromolecules, e.g. polypeptides or chemicals, that promote the
visualization of these ectopically provided labels.
[0064] In some instances, the macromolecules that are used to
visualize the cellular biomolecules or subcellular structures are
passively transported into the specimen. In other words, the
macromolecules diffuse into the specimen. In other instances, the
macromolecules are actively transported into the specimen, e.g. by
electroporation, hydrodynamic pressure, ultrasonic vibration,
solute contrasts, microwave radiation, vascular circulation, or the
like. In some embodiments, the specimen is contacted with the
macromolecules after the specimen has been cleared. In other
embodiments, the hydrogel-embedded specimen may be contacted with
the macromolecules prior to clearing the specimen. In such
embodiments, contact with the macromolecules may be facilitated by
permeabilizing the specimen, that is, changing the properties of
the specimen to improve the permeability of the specimen to
macromolecules. By a "permeabilized" specimen it is meant that
about 50% or more of the macromolecules applied to the specimen
will penetrate to the deepest regions of the specimen, e.g. 60% or
more of the macromolecules, 70% or more of the macromolecules, or
80% or more of the macromolecules, in some instances 85% or more of
the macromolecules, 90% or more of the macromolecules, or 95% or
more of the macromolecules, for example 98% or more of the
macromolecules, e.g. 100% of the macromolecules will pass through
the specimen. Permeabilization of the specimen, and of the cells
therein, may be achieved by any of the protocols discussed above
for the removal of cellular components, e.g. lipids, from the
specimen or as known in the art for permeabilizing cells.
[0065] To microscopically visualize specimens prepared by the
subject methods, in some embodiments the specimen is embedded in a
mounting medium. Mounting medium is typically selected based on its
suitability for the reagents used to visualize the cellular
biomolecules, the refractive index of the specimen, and the
microscopic analysis to be performed. For example, for
phase-contrast work, the refractive index of the mounting medium
should be different from the refractive index of the specimen,
whereas for bright-field work the refractive indexes should be
similar. As another example, for epifluorescence work, a mounting
medium should be selected that reduces fading, photobleaching or
quenching during microscopy or storage. In certain embodiments, a
mounting medium or mounting solution may be selected to enhance or
increase the optical clarity of the cleared tissue specimen.
Nonlimiting examples of suitable mounting media that may be used
include glycerol, CC/Mount.TM., Fluoromount.TM. Fluoroshield.TM.,
ImmunHistoMount.TM., Vectashield.TM., Permount.TM., Acrytol.TM.,
CureMount.TM., FocusClear.TM., or equivalents thereof.
[0066] In some instances, the hydrogel-embedded specimen is
permanently mounted. In other words, once mounted in mounting
medium, the hydrogel-embedded specimen cannot be removed for
further manipulation. In other instances, the specimen is
temporarily, or reversibly, mounted. In other words, the
hydrogel-embedded specimen may be removed from the mounting medium
and re-stained after microscopy to visualize alternative/additional
biomolecules or subcellular structures. In such instances,
macromolecules that were previously added to the specimen, e.g. to
visualize certain biomolecules, may be removed after microscopic
analysis by, e.g., exposure to organic solvents such as xylenes,
ethanol or methanol, exposure to detergents such as sodium dodecyl
sulfate (SDS), saponin, Triton X-100 and Tween-20, electrophoresis,
hydrodynamic pressure, ultrasonic vibration, solute contrasts,
microwave radiation, vascular circulation, and the like. The
hydrogel-embedded specimen is then contacted with different
macromolecules specific for other biomolecules or subcellular
structures. As such, iterative staining may be performed on the
same specimen.
[0067] Specimens prepared using the subject methods may be analyzed
by any of a number of different types of microscopy, for example,
optical microscopy (e.g. bright field, oblique illumination, dark
field, phase contrast, differential interference contrast,
interference reflection, epifluorescence, confocal, etc.,
microscopy), laser microscopy, electron microscopy, and scanning
probe microscopy.
[0068] Bright field microscopy is the simplest of all the optical
microscopy techniques. Sample illumination is via transmitted white
light, i.e. illuminated from below and observed from above.
Limitations include low contrast of most biological samples and low
apparent resolution due to the blur of out of focus material. The
simplicity of the technique and the minimal sample preparation
required are significant advantages.
[0069] In oblique illumination microscopy, the specimen is
illuminated from the side. This gives the image a 3-dimensional
appearance and can highlight otherwise invisible features. A more
recent technique based on this method is Hoffmann's modulation
contrast, a system found on inverted microscopes for use in cell
culture. Though oblique illumination suffers from the same
limitations as bright field microscopy (low contrast of many
biological samples; low apparent resolution due to out of focus
objects), it may highlight otherwise invisible structures.
[0070] Dark field microscopy is a technique for improving the
contrast of unstained, transparent specimens. Dark field
illumination uses a carefully aligned light source to minimize the
quantity of directly-transmitted (unscattered) light entering the
image plane, collecting only the light scattered by the sample.
Dark field can dramatically improve image contrast (especially of
transparent objects) while requiring little equipment setup or
sample preparation. However, the technique suffers from low light
intensity in final image of many biological samples, and continues
to be affected by low apparent resolution.
[0071] Phase contrast is an optical microscopy illumination
technique that converts phase shifts in light passing through a
transparent specimen to brightness changes in the image. In other
words, phase contrast shows differences in refractive index as
difference in contrast. The phase shifts themselves are invisible
to the human eye, but become visible when they are shown as
brightness changes.
[0072] In differential interference contrast (DIC) microscopy,
differences in optical density will show up as differences in
relief. The system consists of a special prism (Nomarski prism,
Wollaston prism) in the condenser that splits light in an ordinary
and an extraordinary beam. The spatial difference between the two
beams is minimal (less than the maximum resolution of the
objective). After passage through the specimen, the beams are
reunited by a similar prism in the objective. In a homogeneous
specimen, there is no difference between the two beams, and no
contrast is being generated. However, near a refractive boundary
(e.g. a nucleus within the cytoplasm), the difference between the
ordinary and the extraordinary beam will generate a relief in the
image. Differential interference contrast requires a polarized
light source to function; two polarizing filters have to be fitted
in the light path, one below the condenser (the polarizer), and the
other above the objective (the analyzer).
[0073] Another microscopic technique using interference is
interference reflection microscopy (also known as reflected
interference contrast, or RIC). It is used to examine the adhesion
of cells to a glass surface, using polarized light of a narrow
range of wavelengths to be reflected whenever there is an interface
between two substances with different refractive indices. Whenever
a cell is attached to the glass surface, reflected light from the
glass and that from the attached cell will interfere. If there is
no cell attached to the glass, there will be no interference.
[0074] A fluorescence microscope is an optical microscope that uses
fluorescence and phosphorescence instead of, or in addition to,
reflection and absorption to study properties of organic or
inorganic substances. In fluorescence microscopy, a sample is
illuminated with light of a wavelength which excites fluorescence
in the sample. The fluoresced light, which is usually at a longer
wavelength than the illumination, is then imaged through a
microscope objective. Two filters may be used in this technique; an
illumination (or excitation) filter which ensures the illumination
is near monochromatic and at the correct wavelength, and a second
emission (or barrier) filter which ensures none of the excitation
light source reaches the detector. Alternatively, these functions
may both be accomplished by a single dichroic filter. The
"fluorescence microscope" refers to any microscope that uses
fluorescence to generate an image, whether it is a more simple set
up like an epifluorescence microscope, or a more complicated design
such as a confocal microscope, which uses optical sectioning to get
better resolution of the fluorescent image.
[0075] Confocal microscopy uses point illumination and a pinhole in
an optically conjugate plane in front of the detector to eliminate
out-of-focus signal. As only light produced by fluorescence very
close to the focal plane can be detected, the image's optical
resolution, particularly in the sample depth direction, is much
better than that of wide-field microscopes. However, as much of the
light from sample fluorescence is blocked at the pinhole, this
increased resolution is at the cost of decreased signal
intensity--so long exposures are often required. As only one point
in the sample is illuminated at a time, 20 or 3D imaging requires
scanning over a regular raster (i.e., a rectangular pattern of
parallel scanning lines) in the specimen. The achievable thickness
of the focal plane is defined mostly by the wavelength of the used
light divided by the numerical aperture of the objective lens, but
also by the optical properties of the specimen. The thin optical
sectioning possible makes these types of microscopes particularly
good at 3D imaging and surface profiling of samples.
[0076] In single plane illumination microscopy (SPIM), also known
as light sheet microscopy, only the fluorophores in the focal plane
of the detection objective lens are illuminated. The light sheet is
a beam that is collimated in one and focused in the other
direction. Since no fluorophores are excited outside the detectors'
focal plane, the method also provides intrinsic optical sectioning.
Moreover, when compared to conventional microscopy, light sheet
methods exhibit reduced photobleaching and lower phototoxicity, and
often enable far more scans per specimen. By rotating the specimen,
the technique can image virtually any plane with multiple views
obtained from different angles. For every angle, however, only a
relatively shallow section of the specimen is imaged with high
resolution, whereas deeper regions appear increasingly blurred.
[0077] Super-resolution microscopy is a form of light microscopy.
Due to the diffraction of light, the resolution of conventional
light microscopy is limited as stated by Ernst Abbe in 1873. A good
approximation of the resolution attainable is the FWHM (full width
at half-maximum) of the point spread function, and a precise
widefield microscope with high numerical aperture and visible light
usually reaches a resolution of .about.250 nm. Super-resolution
techniques allow the capture of images with a higher resolution
than the diffraction limit. They fall into two broad categories,
"true" super-resolution techniques, which capture information
contained in evanescent waves, and "functional" super-resolution
techniques, which use experimental techniques and known limitations
on the matter being imaged to reconstruct a super-resolution
image.
[0078] Laser microscopy uses laser illumination sources in various
forms of microscopy. For instance, laser microscopy focused on
biological applications uses ultrashort pulse lasers, or
femtosecond lasers, in a number of techniques including nonlinear
microscopy, saturation microscopy, and multiphoton fluorescence
microscopy such as two-photon excitation microscopy (a fluorescence
imaging technique that allows imaging of living tissue up to a very
high depth, e.g. one millimeter)
[0079] In electron microscopy (EM), a beam of electrons is used to
illuminate a specimen and produce a magnified image. An electron
microscope has greater resolving power than a light-powered optical
microscope because electrons have wavelengths about 100,000 times
shorter than visible light (photons). They can achieve better than
50 .mu.m resolution and magnifications of up to about
10,000,000.times. whereas ordinary, non-confocal light microscopes
are limited by diffraction to about 200 nm resolution and useful
magnifications below 2000.times.. The electron microscope uses
electrostatic and electromagnetic "lenses" to control the electron
beam and focus it to form an image. These lenses are analogous to
but different from the glass lenses of an optical microscope that
form a magnified image by focusing light on or through the
specimen. Electron microscopes are used to observe a wide range of
biological and inorganic specimens including microorganisms, cells,
large molecules, biopsy samples, metals, and crystals.
Industrially, the electron microscope is often used for quality
control and failure analysis. Examples of electron microscopy
include Transmission electron microscopy (TEM), Scanning electron
microscopy (SEM), reflection electron microscopy (REM), Scanning
transmission electron microscopy (STEM) and low-voltage electron
microscopy (LVEM).
[0080] Scanning probe microscopy (SPM) is a branch of microscopy
that forms images of surfaces using a physical probe that scans the
specimen. An image of the surface is obtained by mechanically
moving the probe in a raster scan of the specimen, line by line,
and recording the probe-surface interaction as a function of
position. Examples of SPM include atomic force microscopy (ATM),
ballistic electron emission microscopy (SEEM), chemical force
microscopy (CFM), conductive atomic force microscopy (C-AFM),
electrochemical scanning tunneling microscope (ECSTM),
electrostatic force microscopy (EFM), fluidic force microscope
(FluidFM), force modulation microscopy (FMM), feature-oriented
scanning probe microscopy (FOSPM), kelvin probe force microscopy
(KPFM), magnetic force microscopy (MFM), magnetic resonance force
microscopy (MRFM), near-field scanning optical microscopy (NSOM)
(or SNOM, scanning near-field optical microscopy, SNOM,
Piezoresponse Force Microscopy (PFM), PSTM, photon scanning
tunneling microscopy (PSTM), PTMS, photothermal
microspectroscopy/microscopy (PTMS), SCM, scanning capacitance
microscopy (SCM), SECM, scanning electrochemical microscopy (SECM),
SGM, scanning gate microscopy (SGM), SHPM, scanning Hall probe
microscopy (SHPM), SICM, scanning ion-conductance microscopy
(SICM), SPSM spin polarized scanning tunneling microscopy (SPSM),
SSRM, scanning spreading resistance microscopy (SSRM), SThM,
scanning thermal microscopy (SThM), STM, scanning tunneling
microscopy (STM), STP, scanning tunneling potentiometry (STP), SVM,
scanning voltage microscopy (SVM), and synchrotron x-ray scanning
tunneling microscopy (SXSTM).
Reagents and Kits
[0081] Also provided are reagents and kits thereof for practicing
one or more of the above-described methods. Reagents and kits may
include one or more of the following: fixative; hydrogel subunits;
clearing reagents; detection macromolecules, e.g., labeled and or
un-labeled antibodies, nucleic acid probes (oligonucleotides,
vectors, etc.), chemicals, etc.; buffers, e.g. buffer for fixing,
washing, clearing, and/or staining specimens; mounting medium;
embedding molds; dissection tools; etc. The subject reagents and
kits thereof may vary greatly.
[0082] Also provided are specimens that have been prepared by the
subject methods for use in, for example, studying tissue at the
cellular and subcellular level. For example, fixed and polymerized
specimens, or specimens that have been fixed, polymerized, and
cleared, are provided for use in studying the expression of genes
of interest, for screens to identify candidate agents that target
cells and/or subcellular structures of interest, etc. Such prepared
specimens may also be provided as a positive control in one of the
kits or systems as described herein.
[0083] In addition to the above components, the subject kits may
further include instructions for practicing the subject methods.
These instructions may be present in the subject kits in a variety
of forms, one or more of which may be present in the kit. One form
in which these instructions may be present is as printed
information on a suitable medium or substrate, e.g., a piece or
pieces of paper on which the information is printed, in the
packaging of the kit, in a package insert, etc. Yet another means
would be a computer readable medium, e.g., diskette, CD, digital
storage medium, etc., on which the information has been recorded.
Yet another means that may be present is a website address which
may be used via the Internet to access the information at a removed
site. Any convenient means may be present in the kits.
Devices and Systems
[0084] Also included are devices for performing aspects of the
subject methods. The subject devices may include, for example,
electrophoresis apparatus, ultrasounds, microwaves, needles,
tubing, perfusion pumps, etc., for fixing, clearing, and/or
staining specimens.
[0085] One device of particular interest is an electrophoresis
device suitable for use in removing cellular components from a
specimen, e.g., cellular components that are not crosslinked to the
hydrogel network. By "electrophoresis" it is meant the application
of an electric field to a sample, e.g., to a biological sample.
Electrophoresis is most commonly used to mobilize biological
macromolecules, e.g., nucleic acids, proteins, in a sample to
separate and analyze those macromolecules. Numerous electrophoretic
techniques have been developed including capillary electrophoresis,
gel electrophoresis, paper electrophoresis, and
immunoelectrophoresis. For example, in gel electrophoresis, a
hydrogel is formed using compounds such as agarose or
polyacrylamide. A mixture containing the desired macromolecules is
placed (or loaded) onto one end of the gel, and the gel is then
placed in contact with a liquid buffer. This liquid buffer contains
salts, which, when dissolved, form ions within the buffer.
Biological molecules are typically charged, for example when
contacted with electrophoresis buffer. For example, DNA is
negatively charged in common electrophoresis buffers due to the
phosphate groups in its backbone. Therefore, when electric current
is applied to the ends of the gel, the biological molecules move
through the gel from one end to the other. Examples of
electrophoresis devices and methods of gel electrophoresis may be
found in, for example, U.S. Pat. Nos. 3,129,158, 3,208,929,
3,346,479, 3,375,187, 3,616,454, 3,616,457, 3,616,454, 3,616,457,
3,563,880, 3,576,727, 3,674,678, 3,865,712, 4,088,561, 4,151,065,
4,292,161, 4,339,327, 4,375,401, 4,415,418, 4,479,861, and
4,588,491; and Martin, Robin. Gel electrophoresis: nucleic acids.
BIOS Scientific, 1996; Hames, B. D. Gel Electrophoresis of
Proteins: A Practical Approach, Oxford University Press 1998; and
Burmeister, M. and Ulanovsky, L. Pulsed-field Gel Electrophoresis,
The Humana Press Inc. 1992, the disclosures of which are
incorporated herein by reference.
[0086] Electrophoresis devices suitable for use in the subject
methods will generally comprise an electrophoresis chamber into
which a buffer solution and the hydrogel-embedded specimen may be
placed. See, for example, FIG. 2 and FIG. 8. The electrophoresis
chamber may generally be any suitable size to accommodate a
hydrogel-embedded sample of interest, and may be constructed of any
material that will retain solution within the chamber, for example
glasses and plastics, such as, for example, acrylics,
polycarbonates, polystyrenes, polymethyl methacrylates,
polyethylene, polyfluoroethylene, polypropylene, polyurethane,
polyethylene terephthalate, polytetrafluoroethylene and the like.
In some embodiments, a chamber may be molded or machined or
otherwise formed from a resin or hard plastic, as appropriate for
particular applications. In certain embodiments, an electrophoresis
chamber may further comprise a component that is configured to
support a hydrogel-embedded sample, such as, e.g., a platform,
within the electrophoresis chamber.
[0087] In some embodiments, an electrophoresis device may include a
lid that fits over the electrophoresis chamber to close the
chamber. Lids in accordance with embodiments of the invention may
include a seal that forms a liquid-tight and/or air-tight seal with
the body of the electrophoresis chamber when the lid is coupled to
the chamber. In some embodiments, one or more sealing components
may be attached to the lid, attached to the chamber, or attached to
both the lid and the chamber. When the lid is coupled to the
chamber, the sealing components may form a liquid and/or air-tight
seal.
[0088] Electrophoresis devices in accordance with some embodiments
of the invention may comprise two or more electrodes of opposite
polarity (i.e. "anode" (negatively charged) and at least one
"cathode" (positively charged)) operably associated with the
electrophoresis chamber to which an electric current may be applied
to create an electric field within the chamber. The electrodes may
be constructed of any material that will result in an electric
field being established upon the application of an electric current
to the electrodes, and may be configured within the chamber and
relative to the site where the specimen is to be placed in any
convenient way that will promote the establishment of an electric
field across a specimen positioned therein, for example as well
known in the art of nucleic acid or protein electrophoresis. See,
for example, U.S. Pat. Nos. 3,129,158, 3,208,929, 3,346,479,
3,375,187, 3,616,454, 3,616,457, 3,616,454, 3,616,457, 3,563,880,
3,576,727, 3,674,678, 3,865,712, 4,088,561, 4,151,065, 4,292,161,
4,339,327, 4,375,401, 4,415,418, 4,479,861, and 4,588,491; and
Martin, Robin. Gel electrophoresis: nucleic acids. BIOS Scientific,
1996; Hames, B. D. Gel Electrophoresis of Proteins: A Practical
Approach. Oxford University Press 1998; and Burmeister, M. and
Ulanovsky, L. Pulsed-field Gel Electrophoresis. The Humana Press
Inc. 1992. For example, the electrodes may be configured within the
chamber so as to substantially flank the specimen.
[0089] In some embodiments, one or more of the electrodes may
comprise an extension component that is used to enlarge the size of
the electric field that is generated between the electrodes. For
example, in certain embodiments, an electrode may comprise an
extension component in the form of a serpentine portion of the
electrode the doubles back on itself to form a plurality of
S-shaped bends. An example of an electrode comprising a serpentine
extension component can be seen in FIG. 2, Panel c (reference
numbers 103a and 103b). The extension component enlarges the size
of the electric field that is generated when a voltage is applied
to the electrodes so that an entire three-dimensional tissue
specimen can be placed inside the electric field. The length and
width of the extension component can be adjusted as needed to
accommodate tissue specimens of various dimensions. For example, in
certain embodiments the length and the width of an electrode
comprising an extension component are approximately equal, as
depicted in FIG. 8, Panel e.
[0090] In certain embodiments, an electrophoresis chamber may be
partitioned by, e.g., a solid divider or by air into two distinct
regions, where each region comprises one electrode in a buffer, and
the specimen is positioned within the buffer such that the specimen
spans, or straddles the two regions, such that the electric field
created by the electrodes is created through the specimen. In some
instances, the chamber may comprise a platform or support
structure, upon or into which the hydrogel-embedded specimen is
placed, e.g., a platform between two electrodes, a platform that
spans regions of the chamber comprising the electrodes, etc.
[0091] The electrophoresis apparatus may be operably linked to a
power source from which voltage may be applied to the electrodes.
In some instances, the power source may be separate from the
electrophoresis apparatus, i.e. the electrophoresis apparatus may
be a separate module from the power source. In other instances, the
power source may be integrated into the electrophoresis apparatus,
i.e., the electrophoresis apparatus will comprise the power
source.
[0092] In some instances, it may be desirable to replace or
recirculate buffer in the electrophoresis chamber. By "circulated"
or "recirculated" buffer it is meant that buffer is removed from
the chamber and then returned to the chamber, for example, after
passing through a cooling unit (refrigeration unit, ice bath,
etc.), a heating unit, a filter, etc. By "replaced" is meant that
buffer is removed from the chamber and fresh buffer is added in its
place. For example, it may be desirable to control the temperature
of the buffer inside the electrophoresis chamber (e.g., to prevent
the chamber from reaching temperatures that might cause the
hydrogel to depolymerize or the biomolecules in the specimen to
denature, e.g., 35.degree. C. or more, 40.degree. C. or more, or
50.degree. C. or more, 60.degree. C. or more, 70.degree. C. or
more, 80.degree. C. or more, 90.degree. C. or more, or 100.degree.
C. or more); to remove macromolecules from the buffer as they exit
the specimen; to vary the ionic strength of the buffer; etc.
Towards this end, the electrophoresis apparatus may optionally
comprise one or more ports through which buffer may enter and/or
exit the chamber. In some instances, the chamber may comprise two
or more ports, e.g. a first port through which buffer enters the
chamber and a second port through which buffer exits the
chamber.
[0093] Buffer may be added/removed/recirculated/replaced by the use
of the one or more ports and optionally, tubing, pumps, valves, or
any other suitable fluid handling and/or fluid manipulation
equipment, for example, tubing that is removably attached or
permanently attached to one or more components of a device. For
example, a first tube having a first and second end may be attached
to a first port and a second tube having a first and second end may
be attached to a second port, where the first end of the first tube
is attached to the first port and the second end of the first tube
is operably linked to a receptacle, e.g, a cooling unit, heating
unit, filtration unit, waste receptacle, etc.; and the first end of
the second tube is attached to the second port and the second end
of the second tube is operably linked to a receptacle, e.g. a
cooling unit, beaker on ice, filtration unit, waste receptacle,
etc.
[0094] As another example, one tube having a first and second end
may be removably attached to both a first and second port, i.e.,
the first end of the tube is removably attached to the first port
and the second end of the tube is removably attached to the second
port, where the tubing is operably linked to, for example, a
refrigeration unit (e.g., the tubing passes through the unit), a
filter (e.g. the tubing comprises a filter), a buffer reservoir
(e.g. the tubing receives replacement buffer from a reservoir via,
e.g., a splitter), etc. In some instances, the tubing will also be
operably connected to a pump, e.g. a peristaltic pump, an
electro-osmotic pump, an oscillatory pump, a diaphragm pump etc.,
that will facilitate the movement of liquid through the tubing,
facilitate the addition/removal/recirculation of buffer from the
electrophoresis chamber, etc. In this way, the electrophoresis
apparatus may be operably connected to a cooling unit, heating
unit, filtration unit, buffer reservoirs/receptacles, pump, etc. In
some embodiments, a refrigeration unit, heating unit, filtration
unit, buffer reservoirs/receptacles, pump, etc. will be integrated
into the electrophoresis apparatus. In other words, the
electrophoresis apparatus may comprise the refrigeration unit,
heating unit, filtration unit, buffer reservoirs/receptacles, pump,
etc. In other embodiments, the refrigeration unit, heating unit,
filtration unit, buffer reservoirs/receptacles, pump, etc., may be
a separate module from the electrophoresis apparatus.
[0095] Turning now to FIG. 2, a representative electrophoretic
tissue clearing device is shown. The example device 101 includes a
lid 102, a first electrode 103a, a second electrode 103b, a base
104, an outlet port 105, an inlet port 106, a first electrode
connector 107a and a second electrode connector 107b.
[0096] The present disclosure also provides systems for performing
the subject methods. Systems may include one or more of the modules
described herein, e.g. an electrophoresis apparatus, a power
supply, a refrigeration unit, a heating unit, a pump, etc. Systems
may also include any of the reagents described herein, e.g.
fixative; hydrogel subunits; clearing reagents; detection
macromolecules, antibodies, nucleic acid probes (oligonucleotides,
vectors, etc.), chemicals, etc.; buffers, e.g., buffer for fixing,
washing, clearing, and/or staining specimens; mounting medium;
embedding molds; etc. Systems in accordance with certain
embodiments may also include a microscope and/or related imaging
equipment, e.g., camera components, digital imaging components
and/or image capturing equipment, computer processors configured to
collect images according to one or more user inputs, and the
like.
Applications
[0097] Using the subject methods, reagents, kits, systems and
devices, the ordinarily skilled artisan will be able to prepare any
biological tissue for microscopic analysis. Methods, reagents,
kits, systems and devices may be used to prepare a specimen from
any plant or animal, including but not limited to transgenic
animals, e.g., vertebrate or invertebrate, e.g. insect, worm,
xenopus, zebrafish, mammal, e.g. equine, bovine, ovine, canine,
feline, murine, rodent, non-human primate or human. Tissue
specimens may be collected from living subjects (e.g., bipsy
samples) or may be collected from dead subjects (e.g., autopsy or
necrospsy samples). The specimens may be of any tissue type, e.g.
hematopoietic, neural (central or peripheral), glial, mesenchymal,
cutaneous, mucosal, stromal, muscle (skeletal, cardiac, or smooth),
spleen, reticulo-endothelial, endothelial, hepatic, kidney,
pancreatic, gastrointestinal, pulmonary, fibroblast, and other cell
types. In some instances, the specimen is the entire organism, e.g.
a worm, an insect, a zebrafish. In other instances, the specimen is
a whole organ, e.g., the whole brain of a rodent. In other
instances, the specimen is a portion of an organ, i.e. a biopsy,
e.g. a biopsy of a transplanted tissue. The specimen may be freshly
isolated or preserved, e.g. snap frozen. In some embodiments, the
specimen may be a previously preserved specimen, such as, e.g., a
preserved specimen from a tissue bank, e.g., a preserved specimen
of a human brain obtained from a tissue collection program. In some
instances, a specimen may be from a subject known to suffer from a
specified disease or condition, such as, e.g., a sample of brain
tissue from an autistic human. In other instances, a sample may be
from a "normal" subject that does not suffer from a specific
disease or condition. In some instances, a sample may be from a
transgenic subject, such as, e.g., a transgenic mouse.
[0098] Because the cells of the specimen are crosslinked to a
hydrogel that physically supports the ultrastructure of the tissue,
cellular components, e.g. lipids, that normally provide structural
support but that hinder visualization of subcellular proteins and
molecules may be removed while preserving the 3-dimensional
architecture of the cells and tissue. This removal renders the
interior of biological specimen substantially permeable to light
and/or macromolecules, allowing the interior of the specimen, e.g.
cells and subcellular structures, to be microscopically visualized
without time-consuming and disruptive sectioning of the tissue. The
procedure is also more rapid than procedures commonly used in the
art, as clearance and permeabilization, typically performed in
separate steps, may be combined in a single step of removing
cellular components. Additionally, the specimen can be iteratively
stained, unstained, and re-stained with other reagents for
comprehensive analysis.
[0099] The subject methods find many uses. For example, the subject
methods may be applied to preparing specimens for the study of the
connectivity of the central nervous system. "Connectivity" as used
herein generally means the connections between neurons, and
includes connections at the single cell level, e.g., synapses, axon
termini, dendritic spines, etc., as well as connections between
groups of neurons and regions of the CNS as major axon tracts,
e.g., corpus callosum (CC), anterior commissure (AC), hippocampal
commissure (HC), pyramidal decussation, pyramidal tracts, external
capsule, internal capsule (IC), cerebral peduncle (CP), etc. A
whole brain and/or spinal cord specimen or region thereof (e.g.
cerebrum (i.e. cerebral cortex), cerebellum (i.e. cerebellar
cortex), ventral region of the forebrain (e.g. striatum, caudate,
putamen, globus pallidus, nucleus accumbens; septal nuclei,
subthalamic nucleus); regions and nuclei of the thalamus and
hypothalamus; regions and nuclei of the deep cerebellum (e.g
dentate nucleus, globose nucleus, emboliform nucleus, fastigial
nucleus) and brainstem (e.g. substantia nigra, red nucleus, pons,
olivary nuclei, cranial nerve nuclei); and regions of the spine
(e.g. anterior horn, lateral horn, posterior horn)) may be prepared
post-mortem by the subject methods and the connectivity of the
neurons therein microscopically analyzed, e.g. obtained, stored,
rendered, used, and actuated, e.g. to provide the full connectivity
of a brain, e.g. a human brain, after death. Such studies will
contribute greatly to the understanding of how the brain develops
and functions in health and during disease, and of the
underpinnings of cognition and personality.
[0100] As another example, the subject methods may be employed to
evaluate, diagnose or monitor a disease, "Diagnosis" as used herein
generally includes a prediction of a subject's susceptibility to a
disease or disorder, determination as to whether a subject is
presently affected by a disease or disorder, prognosis of a subject
affected by a disease or disorder (e.g., identification of
cancerous states, stages of cancer, likelihood that a patient will
die from the cancer), prediction of a subject's responsiveness to
treatment for a disease or disorder (e.g., a positive response, a
negative response, no response at all to, e.g., allogeneic
hematopoietic stem cell transplantation, chemotherapy, radiation
therapy, antibody therapy, small molecule compound therapy) and use
of therametrics (e.g., monitoring a subject's condition to provide
information as to the effect or efficacy of therapy). For example,
a biopsy may be prepared from a cancerous tissue and
microscopically analyzed to determine the type of cancer, the
extent to which the cancer has developed, whether the cancer will
be responsive to therapeutic intervention, etc.
[0101] As another example, a biopsy may be prepared from a diseased
tissue, e.g. kidney, pancreas, stomach, etc., to determine the
condition of the tissue, the extent to which the disease has
developed, the likelihood that a treatment will be successful, etc.
The terms "treatment", "treating" and the like are used herein to
generally mean obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse effect attributable to the disease. "Treatment" as
used herein covers any treatment of a disease in a mammal, and
includes: (a) preventing the disease from occurring in a subject
which may be predisposed to the disease but has not yet been
diagnosed as having it; (b) inhibiting the disease, i.e., arresting
its development; or (c) relieving the disease, i.e., causing
regression of the disease. The therapeutic agent may be
administered before, during or after the onset of disease or
injury. The treatment of ongoing disease, where the treatment
stabilizes or reduces the undesirable clinical symptoms of the
patient, is of particular interest. Such treatment is desirably
performed prior to complete loss of function in the affected
tissues. The subject therapy will desirably be administered during
the symptomatic stage of the disease, and in some cases after the
symptomatic stage of the disease. The terms "individual,"
"subject," "host," and "patient," are used interchangeably herein
and refer to any mammalian subject for whom diagnosis, treatment,
or therapy is desired, particularly humans. Examples of diseases
that are suitable to evaluation, analysis, diagnosis, prognosis,
and/or treatment using the subject methods and systems include, but
are not limited to, cancer, immune system disorders,
neuropsychiatric disease, endocrine/reproductive disease,
cardiovascular/pulmonary disease, musculoskeletal disease,
gastrointestinal disease, and the like.
[0102] Similarly, the subject methods may be used to monitor tissue
grafts to determine how well the subject has accepted a
transplanted organ/tissue, e.g. heart, kidney, liver, or other
organ. In such instances, a biopsy of the transplanted organ may be
prepared by the subject methods, and the specimen microscopically
analyzed for, e.g., tissue integrity, tissue vascularization, the
infiltration of immune cells, etc.
[0103] The subject methods may also be used to evaluate normal
tissues, organs and cells, for example to evaluate the
relationships between cells and tissues of a normal tissue
specimen, e.g., a tissue specimen taken from a subject not known to
suffer from a specific disease or condition. The subject methods
may be used to investigate, e.g., relationships between cells and
tissues during fetal development, such as, e.g., during development
and maturation of the nervous system, as well as to investigate the
relationships between cells and tissues after development has been
completed, e.g., the relationships between cells and tissues of the
nervous systems of a fully developed adult specimen. In some
embodiments, the subject methods may be used on samples collected
from transgenic animals to investigate the effects of genetic
changes on the development and/or function of specific cells,
tissues, and/or organs.
[0104] The subject methods also provide a useful system for
screening candidate therapeutic agents for their effect on a tissue
or a disease. For example, a subject, e.g. a mouse, rat, dog,
primate, human, etc. may be contacted with a candidate agent, an
organ or a biopsy thereof may be prepared by the subject methods,
and the prepared specimen microscopically analyzed for one or more
cellular or tissue parameters. Parameters are quantifiable
components of cells or tissues, particularly components that can be
accurately measured, desirably in a high throughput system. A
parameter can be any cell component or cell product including cell
surface determinant, receptor, protein or conformational or
posttranslational modification thereof, lipid, carbohydrate,
organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc.
or a portion derived from such a cell component or combinations
thereof. While most parameters will provide a quantitative readout,
in some instances a semi-quantitative or qualitative result will be
acceptable. Readouts may include a single determined value, or may
include mean, median value or the variance, etc. Characteristically
a range of parameter readout values will be obtained for each
parameter from a multiplicity of the same assays. Variability is
expected and a range of values for each of the set of test
parameters will be obtained using standard statistical methods with
a common statistical method used to provide single values. Thus,
for example, one such method may comprise detecting cellular
viability, tissue vascularization, the presence of immune cell
infiltrates, efficacy in altering the progression of the disease,
etc. In some embodiments, the screen includes comparing the
analyzed parameter(s) to those from a control, or reference,
sample, e.g., a specimen similarly prepared from a subject not
contacted with the candidate agent. Candidate agents of interest
for screening include known and unknown compounds that encompass
numerous chemical classes, primarily organic molecules, which may
include organometallic molecules, inorganic molecules, genetic
sequences, etc. Candidate agents of interest for screening also
include nucleic acids, for example, nucleic acids that encode
siRNA, shRNA, antisense molecules, or miRNA, or nucleic acids that
encode polypeptides. An important aspect of the invention is to
evaluate candidate drugs, including toxicity testing; and the like.
Evaluations of tissue samples using the subject methods may
include, e.g., genetic, transcriptomic, genomic, proteomic, and/or
metabolomics analyses.
[0105] The subject methods may also be used to visualize the
distribution of genetically encoded markers in whole tissue at
subcellular resolution, for example, chromosomal abnormalities
(inversions, duplications, translocations, etc.), loss of genetic
heterozygosity, the presence of gene alleles indicative of a
predisposition towards disease or good health, likelihood of
responsiveness to therapy, ancestry, and the like. Such detection
may be used in, for example, diagnosing and monitoring disease as,
e.g., described above, in personalized medicine, and in studying
paternity.
Materials and Methods
[0106] The following materials and methods were used in the
examples below.
CLARITY Process for Mouse Brain:
[0107] Adult mice (4-12 weeks old) were anesthetized with
Beuthenasia-D and perfused transcardially with a mixture of 4 wt %
paraformaldehyde/4% acrylamide (wt/vol)/0.025% bis-acrylamide
(wt/vol)/0.25% VA044 (wt/vol)/PBS. The brains were then extracted
and incubated in the same solution at 4.degree. C. for three days.
In the next step, the temperature of the solution was increased to
37.degree. C. to initiate polymerization. After three hours
incubation at 37.degree. C., the hydrogel-embedded brains were
placed in an electrophoresis chamber. While circulating sodium
borate buffer (200 mM, pH 8.5) containing 4% SOS (wt/vol) through
the chamber, 10-40V was applied across the brains at 50.degree. C.
for two days. After clearing, the brains were incubated in PBS
buffer at 37.degree. C. for two days to remove SOS from the brain.
To make 1 mm-thick coronal blocks of mouse brain for
immunostaining, the hydrogel-embedded and cleared brains were cut
into 1 mm-thick blocks using a mouse brain matrix (cat. no. 15003,
Ted Pella, inc., Redding, Calif.). The blocks were then cleared by
electrophoresis for one day as described above.
CLARITY Process for Postmortem Human Brain Tissue:
[0108] The frontal lobe (Brodmann's area 10) of postmortem human
brain tissue (autism case, #AN13961; age, 7 years; sex, male;
storage, 82 months in 10% formalin at room temperature) was sliced
into 500 .mu.m-thick blocks using a vibratome. The blocks were
incubated in a mixture of 4 wt % paraformaldehyde/4% acrylamide
(wt/vol)/0.025% bis-acrylamide (wt/vol)/0.25% VA044 (wt/vol)/PBS at
4.degree. C. for one week. Next, the temperature of the solution
was increased to 37.degree. C. to initiate polymerization. After
three hours of incubation at 37.degree. C., the hydrogel-embedded
tissues were placed in the custom-built electrophoresis chamber
(see FIG. 2 and FIG. 8). While circulating sodium borate buffer
(200 mM, pH 8.5) containing 4% SDS (wt/vol) through the chamber,
10-40V was applied across the tissues at 50.degree. C. for one day.
After clearing, the cleared tissues were incubated in PBS buffer at
37.degree. C. for one day to remove SDS.
Immunostaining of the CLARITY-Processed Mouse Brain Tissues:
[0109] For OFF staining, the cleared 1 mm-thick block of Thy1-EYFP
H line mouse (two months old) brain was incubated at 37.degree. C.
for two days in 0.1% Triton X-100 (wt/vol)/anti-GFP antibody
conjugated with Alexa Fluor 594 (Cat. no. A21312, Invitrogen,
Carlsbad, Calif., 1:50 dilution)/1M sodium borate buffer (pH 8.5)
solution, followed by wash at 37.degree. C. for one day with 0.1%
Triton X-100 (wt/vol)/1M sodium borate buffer (pH 8.5). For the
other staining, the cleared 1 mm-thick block of Thy1-EYFP H line
mouse (two months old) was incubated at 37.degree. C. for two days
in 0.1% Triton X-100 (wt/vol)/primary antibody (dilution,
1:50-1:100) 1M sodium borate buffer (pH 8.5) solution, followed by
wash at 37.degree. C. for one day with 0.1% Triton X-100
(wt/vol)/1M sodium borate buffer (pH 8.5). The tissue was then
incubated at 37.degree. C. for one day in 0.1% Triton X-100
(wt/vol)/secondary antibody (dilution, 1:50-1:100)/1M sodium borate
buffer (pH 8.5) solution, followed by wash at 37.degree. C. for one
day with 0.1% Triton X-100 (wt/vol) 1M sodium borate buffer (pH
8.5).
Immunostaining of the CLARITY-Processed Postmortem Human Brain
Tissues:
[0110] The cleared 500 .mu.m-thick blocks were incubated at
37.degree. C. for one day in 0.1% Triton X-100 (wt/vol)/primary
antibody (dilution, 1:50-1:100) 1M sodium borate buffer (pH 8.5)
solution, followed by wash at 37.degree. C. for half day with 0.1%
Triton X-100 (wt/vol)/1M sodium borate buffer (pH 8.5). The tissue
was then incubated at 37.degree. C. for one day in 0.1% Triton
X-100 (wt/vol)/secondary antibody (dilution, 1:50-1:100)/1M sodium
borate buffer (pH 8.5) solution, followed by wash at 37.degree. C.
for half day with 0.1% Triton X-100 (wt/vol)/1M sodium borate
buffer (pH 8.5).
Imaging of the CLARITY Processed Mouse Brain Tissues:
[0111] For imaging the intact Thy1-EYFP H line mouse brain, the
cleared brain (three months old) was incubated in FocusClear.TM., a
water-based immersion medium, for two days. The brain was then
enclosed between two coverglass-bottom petri dishes. The dorsal
half of the brain was first imaged (stack size=3.4 mm, step-size=10
.mu.m) using a Leica SP5 system equipped with the 10.times.
water-immersion objective (Leica HCX AQP L, NA=0.30, working
distance=3.6) and 514 nm excitation. The mounted brain was then
flipped and the ventral half was imaged in the same way.
[0112] To obtain the 3.4 mm-thick volume image visualizing from the
cortex to the thalamus, the intact H line mouse brain was mounted
as described and imaged using the 10.times. objective and 514 nm
excitation (stack size=3.4 mm, step-size=2 .mu.m).
[0113] The immunostained 1 mm-thick coronal blocks were incubated
in FocusClear.TM. for one day and enclosed between the
coverglass-bottom petri dish and slideglass. The mounted samples
were imaged using the 10.times. objective and one-photon
excitations (514 nm, 591 nm, and 654 nm).
Imaging of the CLARITY-Processed Postmortem Human Brain Tissue:
[0114] The cleared and immunostained tissues were incubated in
FocusClear.TM. for one day and mounted as described above. The
tissues were then imaged using the Leica, SP5 system equipped with
the 25.times. water-immersion objective (Leica HCX IRAPO L,
NA=0.95, working distance=2.4) (stack size-500 .mu.m, step-size=0.5
.mu.m). Alexa Fluor 594 was excited with a 780 nm laser.
Protein Loss Measurement:
[0115] Six PFA-fixed adult mouse (four weeks old) brains (for 4%
SDS, Scale, and Triton X-100 treatments) and two (four weeks old)
PFA-fixed/hydrogel-embedded brains (for CLARITY) were cut into 1
mm-thick coronal blocks. One half of each PFA-fixed brain was
weighed and placed in 2.5 ml of 4% SDS, ScaleU2 (a mixture of 4M
urea and 30% glycerol) or 0.1% Triton X-100 solution. One half of
each PFA-fixed/hydrogel-embedded brain was placed in 2.5 ml of 4%
SDS solution. The tissues were allowed to clear for one week at
37.degree. C. in the respective solutions and quantity of protein
lost from tissue by diffusing into solution was measured using the
BOA (bicinchoninic acid) protein assay; total protein in mouse
brain estimated at 10 wt %.
Neurite Tracing:
[0116] Manual tracing of individual neurons was performed using
Imaris software (Bitplane, Inc). Neurons whose cell bodies were
located in the middle 150 .mu.m of the z-stack were randomly
sampled and chosen for tracing. The cell body was reconstructed
semi-automatically through a user-defined threshold which included
as much as the cell body as possible but less than 5 .mu.m of any
dendrite. All neurites originating from the cell body were traced
manually in short segments in the "Surpass" mode, and each segment
was automatically centered (with the opportunity for user
corrections) before being connected. The number of interconnections
between filaments of the same cell and the number of
interconnections between a filament from the traced cell and that
of any other cell were manually counted. Neurons exhibiting
interesting structures were chosen non-randomly and traced using
the same method described above.
Hydrogel Solution Preparation:
[0117] 1. Combine and mix the following with special attention to
temperature and safety precautions. Keep all components on ice at
all times to prevent polymerization.
[0118] Caution: paraformaldehyde (PFA) is an irritant, sensitizer,
carcinogen and toxin, Acrylamide is a potent neurotoxin, a
respiratory and skin sensitizer, carcinogen, irritant, mutagen,
teratogen and reproductive hazard. Many of the chemical
constituents of hydrogels which could be used for CLARITY would
fall into one or more of these categories. Therefore, to avoid skin
contact or inhalation of monomers and/or crosslinkers (e.g.
acrylamide or PFA), solution preparation and all subsequent
handling of hydrogel solution and polymer must be conducted in a
fume hood with personal protective equipment (PPE) including face
shield, lab coat, gloves, and closed-toe shoes.
For 400 mL of Hydrogel Monomer Solution:
TABLE-US-00001 [0119] Ingredient Add: Final Concentration
Acrylamide (40%) 40 mL 4% Bis (2%) 10 mL 0.025% VA-044 Initiator 1
g 0.25% 10X PBS 40 mL 1X 16% PFA 100 mL 4% dH.sub.2O 210 mL NA
Saponin (optional) 200 mg 0.05% Total Volume 400 mL NA
[0120] Saponin is a widely-used mild non-ionic surfactant often
used to permeabilize cellular membranes in conventional
immunohistochemistry. In CLARITY, saponin can be employed in the
hydrogel monomer infusion process to facilitate diffusion of the
hydrogel monomer and initiator into the tissue, particularly for
samples in which cardiac perfusion is not feasible, such as
postmortem human tissues and zebrafish brains. Saponin shortens
incubation time required in the hydrogel monomer infusion process.
However, bubbles may form that could be linked to saponin use, so
routine saponin is not suggested.
[0121] 2. Distribute 40 mL aliquots into 50 mL conical tubes on
ice. Each tissue sample will require the use of one 40 mL tube: 20
mL for perfusion and the remaining 20 mL for sample embedding.
[0122] 3. Seal tubes tightly and keep in secondary containment (on
ice) before removing them from the hood. Transfer aliquots from ice
to -20.degree. C. Store these solutions at -20.degree. C. until
they are ready to be used. They can be stored at -20.degree. C.
indefinitely if all components were kept sufficiently cold during
the preparation process.
Clearing Solution Preparation:
[0123] 1. To avoid skin contact or inhalation, prepare solution in
a fume hood in proper PPE. Paying special attention to safety,
combine water, boric acid, and Sodium Dodecyl Sulfate (SDS) while
stirring. Add NaOH until the pH has reached 8.5. This solution can
be made, stored, and used at room temperature. Caution: SDS is a
toxin and irritant to the skin and respiratory system.
For 10 L of Clearing Solution:
TABLE-US-00002 [0124] Ingredient Add: Final Concentration Boric
Acid 123.66 g 200 mM Sodium Dodecyl Sulfate 400 g 4% dH.sub.2O Fill
to 10 L NA NaOH To pH 8.5 NA Total Volume 10 L NA
Transcardial Perfusion with Hydrogel Solution:
[0125] 1. Prior to perfusing, thaw the frozen hydrogel monomer
solution in the refrigerator or on ice.
[0126] 2. When the solution is completely thawed and transparent
(but still ice cold), gently invert to mix. Make sure that there is
no precipitate, and avoid introducing any bubbles into the
solution.
[0127] 3. Prepare perfusion materials within a fume hood.
[0128] 4. Deeply anesthetize adult mouse with Beuthanasia-D.
[0129] 5. Perfuse the animal transcardially first with 20 mL of ice
cold 1.times.PBS and then 20 mL of the ice cold hydrogel solution.
Maintain a slow rate of perfusion (about 2 minutes for the 20 mL of
each solution).
[0130] 6. Immediately place the tissue (e.g. brain) in 20 mL of
cold hydrogel solution in a 50 mL conical tube. Keep the sample in
solution on ice until it can be moved to a 4.degree. C.
refrigerator.
[0131] 7. Cover sample in aluminum foil if it contains
fluorophores, and incubate at 4.degree. C. for 2-3 days to allow
for further diffusion of the hydrogel solution into the tissue.
Hydrogel Tissue Embedding:
[0132] 1. De-gas the 50 mL conical tube containing your sample in
the desiccation chamber (in a fume hood) to replace all of the gas
in the tube with nitrogen (as oxygen impedes hydrogel formation),
as follows: [0133] a. Place the 50 mL conical tube on a rack in the
desiccation chamber. [0134] b. Twist the 50 mL conical tube open
sufficiently to allow gas exchange. [0135] c. Turn on the nitrogen
tank and adjust the control valve such that the inlet to the
desiccation chamber fills with nitrogen gas. [0136] d. Switch the
desiccation chamber valve from nitrogen gas flow to the vacuum.
[0137] e. Turn on the vacuum pump. Verify that the chamber is under
full vacuum by testing the chamber lid. Keep vacuum on for 10
minutes. [0138] f. Switch the vacuum off and slowly turn the valve
to fill the chamber with nitrogen gas. [0139] g. Carefully open the
chamber just enough to reach the tubes while purging with nitrogen
gas. Taking great care to minimize exposure to air, and quickly and
tightly close the sample tube.
[0140] 2. Submerge the tube in 37.degree. C. water bath in a
37.degree. C. room or incubator on the rotator. Incubate for 3
hours or until solution has polymerized.
[0141] 3. In a fume hood, extract the embedded sample from the gel
(carefully take the sample out and remove extra gel pieces with
gloved fingers). Hydrogel waste disposal should be conducted in
accordance with all institutional, state and country regulations
for hydrogel monomers and crosslinkers (e.g. acrylamide and
PFA).
[0142] 4. Wash the sample with 50 mL of clearing solution for 24
hours at room temperature to dialyze out extra PFA, initiator, and
monomer. Wash the sample two more times with 50 ml for 24 hours
each at 37.degree. C. to further reduce residual PFA, initiator,
and monomer. Take care to dispose of this waste solution carefully
as a biohazard.
Electrophoretic Tissue Clearing (ETC):
[0143] 1. Construct the ETC chamber. [0144] a. Place the sample in
the chamber. [0145] b. Circulate the clearing solution through the
chamber using the temperature controlled circulator, with 10-60V
applied across the tissue (e.g., brain) at 37-50.degree. C. for
several days to clear the sample. The clearing process is faster at
higher voltage and temperature, but requires more power, limiting
the number of clearing setups simultaneously operable by one power
supply (typical power output maximum, 300 W). For example, four
setups can be run simultaneously at 37.degree. C. and 30V, whereas
only 2 setups can be run at 50.degree. C. and 60V, so circulator
temperature and voltage should be chosen to meet practical
considerations.
[0146] 2. After several days, wash the sample two times with PBST
(0.1% TritonX in 1.times. PBS) twice for 24 hours each.
Preparing the Sample for Imaging:
[0147] 1. Incubate the sample in FocusClear.TM. or 80% glycerol
solution for 2 days before imaging. These solutions have refractive
indices matching that of CLARITY-processed tissue. Ensure there is
sufficient solution surrounding the sample, and that evaporative
losses do not occur. Protect the sample from light.
[0148] 2. Take a clean glass slide and gently place it on a
dust-free surface.
[0149] 3. Take a small piece of BluTack putty and prepare
constant-diameter worm-shapes using gloved hands. Make the
thickness uniform and about 1.5.times. the thickness of the sample
(e.g. if the sample is 1 mm, make the putty tube diameter 1.5
mm).
[0150] 4. Place two tubes of putty horizontally across the vertical
slide, leaving space in between for the tissue sample. Cut excess
putty that protrudes off the slide.
[0151] 5. Using a spatula, carefully take the sample and place it
between the putty tubes in the middle of the slide. Pipette
.about.20 .mu.l of FocusClear.TM. medium on top of the sample.
[0152] 6. Carefully place a Willco dish (with the lipped side
facing up) on top of the putty tubes. Press down on the glass part
of the dish (keeping fingers over the putty to avoid glass
shattering) carefully and slowly until contact is made with the
sample and FocusClear.TM. medium. Ensure that there are no bubbles
between the sample, medium, slide, and dish.
[0153] 7. Now using a P200 pipette, carefully introduce more
FocusClear.TM. to either side of the sealed chamber (from the
liquid that surrounded the sample for incubation as it has been
optically matched). Take great care not to introduce any
bubbles.
[0154] 8. Now that the whole chamber is filled with FocusClear.TM.,
use PDMS sealant (Kwik-Sil) across both vertical openings between
the putty, dish, and slide to fully seal the chamber and prevent
evaporation.
[0155] 9. Place aluminum foil on top of the chamber and place it on
a level surface (shielded from light to minimize photodamage).
Leave the sample for 10-15 minutes to allow the PDMS sealant to
polymerize fully.
[0156] 10. Preparation is now ready for imaging.
Examples
[0157] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1: Process for Imaging Whole Tissue
[0158] A process was developed to image whole tissue (including
whole organs/organisms) for precise three-dimensional (3-D)
imaging, immunohistology, clinical diagnostics, and
functionalization. By embedding a tissue in nano-porous hydrogel
that physically supports the ultrastructure of the tissue and then
electrophoretically removing only membrane lipids, the whole tissue
becomes highly transparent and permeable to macro-molecules. This
technology preserves proteins, small peptides, small molecules, and
nucleic acids in the tissue in their three-dimensional
distribution, secured by the fine hydrogel mesh. By bypassing
destructive sectioning of the tissue, subcellular structures may be
analyzed. In addition, tissue can be iteratively stained,
unstained, and restained with other reagents for comprehensive
analysis. An illustration of the process, termed "CLARITY," is
provided in FIG. 1. Panel a: tissue is crosslinked using
formaldehyde (red) in the presence of hydrogel monomers (blue). In
this process, formaldehyde covalently links the monomers to
biomolecules, such as proteins, nucleic acids, and small molecules.
Panel b: next, biomolecule-bound monomers are polymerized into a
hydrogel mesh. This hydrogel-tissue hybridization physically
supports the structure of the tissue and chemically incorporates
the biomolecules into the hydrogel mesh. Panel c: next, an electric
field is applied across the hybrid immersed in an ionic detergent
solution. Panel d: electrophoresis is used to actively transport
ionic micelles into the hybrid and extract only membrane lipids out
of the tissue, leaving structures and crosslinked biomolecules in
place and available for imaging and molecular phenotyping. Panel e:
whole tissue immunostaining can be conducted using antibodies.
Detergent-mediated removal of the antibodies can be used to conduct
multiple rounds of immunostaining on the same sample.
Example 2: Devices and Systems for CLARITY Process
[0159] An electrophoretic tissue clearing (FTC) chamber and
associated components (including a temperature-controlled buffer
circulator, buffer filter, and electrophoresis power supply) were
developed to carry out portions of the CLARITY process described in
Example 1. Illustrations of the devices and systems are provided in
FIG. 2. Panel a: ETC setup consists of the custom ETC chamber, a
temperature-controlled buffer circulator (RE415, Lauda, Germany), a
buffer filter (Cat. No. 4422K61, McMaster, Robbinsville, N.J.), and
a power supply (Cat. no. 164-5052, Biorad, Hercules, Calif.). The
sample is electrophoresed by applying 20-60V to the electrodes.
Buffer solution is circulated through the chamber to maintain
temperature and the composition of the buffer solution constant
throughout the clearing process. Panels b-d: ETC chamber design.
Panel b shows various views of a representative device showing a
cylindrical plastic housing, inlet/outlet ports (Cat. No. 5463K245,
McMaster, Robbinsville, N.J.) and two platinum electrodes (Cat. No.
267201, Sigma, St. Louis, Mo.). The buffer inlet and outlet are
located such that buffer flow through the chamber effectively
removes air bubbles generated by electrolysis of the buffer
solution. Panel c shows components of the assembly. Panel d shows a
top cut-through view. The hydrogel-embedded tissue is placed in a
sample holder (Cell Strainer, BD Biosciences, Durham, N.C.) located
in the middle of the chamber between the two electrodes. The end of
each electrode exposed outside the chamber is connected to a power
supply. Examples of spatial dimensions of a chamber in accordance
with embodiments of the invention are included in FIG. 8.
Example 3: Imaging of Intact Adult Mouse Brain Samples
[0160] Intact adult mouse brain samples were subjected to the
CLARITY process and imaged. FIG. 3 shows the results. Panels a-d:
images of whole mouse brains (three months old) with a Ramon y
Cajal quote in the background. Panel a: before CLARITY. Panel b:
after CLARITY: Thy1-EYFP H line brain after hydrogel-tissue
hybridization, ETC, and refractive index (RI) matching with
FocusClear.TM.. Panel c: fluorescent image of the same brain shown
in Panel b. Panel d: mounting of the whole cleared mouse brain for
imaging. The brain is enclosed between two coverglasses. The dorsal
half of the brain is first imaged using single photon (1P)
excitation microscopy, and then the brain is inverted and the
ventral half is imaged. Panel e: 3D reconstruction of the intact
CLARITY-processed mouse brain (three months old) that was imaged
using a 10.times. water immersion objective (numerical aperture
(NA)=0.3, working distance=3.6 mm) and 514 nm excitation. Left,
reconstruction of the dorsal half (dorsal to ventral, stack
size=3100 .mu.m, step size=20 .mu.m). Right, reconstruction of the
ventral half (ventral to dorsal, stack size=3400 .mu.m, step
size=20 .mu.m). Scale bar, 1 mm. Panel f: 3D reconstruction of the
non-sectioned mouse brain showing visualization through all layers
of cortex, hippocampus, and thalamus (10.times. objective, stack
size=3400 .mu.m, step size=2 .mu.m, 514 nm excitation). Scale bar,
400 .mu.m. Panels g-l: three optical sections from Panel f at
different depths showing negligible resolution loss even at the
depth of .about.3400 .mu.m. Scale bar, 100 .mu.m. Panel g:z=446
.mu.m. Panel h: z=1683 .mu.m. Panel i: z=3384 .mu.m. Panels j-l:
boxed regions in Panels g, h, and i, respectively. Panel m: image
of a cross section of axon bundles in the striatum of
Thy1-ChR2-EYFP line showing membrane-localized ChR2-EYFP after the
CLARITY process. 1 mm-thick coronal block was CLARITY-processed and
imaged using a 63.times. glycerol immersion objective (NA=1.3,
working distance=280 .mu.m). Scale bar, 5 .mu.m. Panel n:
high-resolution image showing well-preserved dendrites and
dendritic spines of pyramidal neurons in the cortex of the
CLARITY-processed Thy1-EYFP H line mouse. 1 mm-thick coronal block
was imaged using the 63.times. glycerol objective. Scale bar, 5
.mu.m.
Example 4: Molecular Phenotyping in Intact Tissue Volumes
[0161] Intact tissue volumes were processed using CLARITY and
subjected to molecular phenotyping analysis. The results are shown
in FIG. 4. Panel a: protein loss measurement (see Methods); error
bars, s.e.m.; n=4 for each condition. Panel b: volume rendering of
a 1 mm-thick coronal block of Thy1-EYFP mouse immunostained for OFF
in intact non-sectioned form, showing uniform immunostaining. The
intact block was ETC-cleared for one day and immunostained for
three days (two days in GFP antibody conjugated with Alexa594 and
one day wash) at 37.degree. C. The block was then imaged using the
10.times. water-immersion objective and 1p excitation (514 nm and
591 nm). Left, EYFP (green). Middle, anti-GFP staining (red).
Right, overlay. Scale bar, 500 .mu.m. Panel c: enlarged 3D
rendering of the boxed region in the cortex showing complete
overlap of EYFP and anti-GFP staining. Panel d: co-localization
analysis of the GFP staining. Manders Overlap Coefficient (MOC) was
plotted as a function of tissue depth. Right, two optical sections
at different depths from the 3D rendering on the left. Scale bar,
100 .mu.m. Panels e-f: identification of individual synapses. A 500
.mu.m-thick coronal block of H line mouse brain (two months old)
was cleared for one day and immunostained for synapsin I (Red) and
PSD-95 (green) for 3 days: primary (1 day)-wash (0.5 day)-secondary
(1 day)-wash (0.5 day). The hippocampus was then imaged using the
63.times. glycerol objective and single photon (1P) excitation (514
nm, 591 nm, 651 nm). Panel e: left, two optical sections (z=20
.mu.m and 200 .mu.m). Right, enlarged images of the boxed regions.
Individual synaptic puncta can be resolved throughout the depth.
EYFP in white. Panel f: average immunofluorescence cross-section of
PSD-95 puncta at z=20 .mu.m (top) and z=200 .mu.m (bottom). Error
bars indicate 95% confidence interval of mean. Panel g: full width
at half maximum (FWHM) of average immunofluorescence cross-section
of PSD-95 puncta as a function of imaging depth. Nearly constant
FWHM across depths suggests loss of resolution is negligible.
Insets show average puncta at z=20 .mu.m and 200 .mu.m, Panels h-i:
small molecules, proteins, and nucleic acids are well preserved,
localized, and can be visualized using molecular probes in
CLARITY-transformed tissue. Panel h: gamma-aminobutyric acid (GABA)
and parvalbumin (PV) staining in the hippocampus showing
co-localization of GABA and PV. Left, GABA. Middle, PV. Right,
overlay. A 500 .mu.m-thick coronal block of wild-type mouse brain
(three months old) was cleared for one day and immunostained for 3
days: primary (1 day)-wash (0.5 day)-secondary (1 day)-wash (0.5
day). The block was then imaged using a 25.times. water-immersion
objective (NA=0.95, working distance=2.5 mm) and single photon
excitation (488 nm and 591 nm). Scale bar, 20 .mu.m. Panel i: in
situ hybridization (ISH) on CLARITY-transformed mouse brain block
showing dopamine receptor D2 (drd2) mRNA localization around the
cell body in the striatum (AP: -1.7; DV: -2.3; ML: 2.6), LV,
lateral ventricle. Blue, DAPI. 50 bp RNA probes for drd2 were
hybridized with a 500 .mu.m-thick CLARITY-processed coronal block
and visualized with FastRed. The block was imaged using the
25.times. water-immersion objective, single photon excitation (555
nm) for FastRed, and two photon excitation for DAPI (720 nm). Scale
bar: left, 100 .mu.m; right, 20 .mu.m. Panels j-k: axonal fibers of
TH-positive neurons in the nucleus accumbens (NAc) and
caudate-putamen (CPu) of mouse brain. Panel j: 3D rendering of 1
mm-thick mouse brain block (Bregma 1.10->0.10) stained for TH
(red) and DAPI (green). aca, anterior commissure; scale bar 500
.mu.m. Panel k; maximum projection of 50 .mu.m volume of the NAc
and aca in Panel j. Scale bar, 50 .mu.m.
Example 5: Multi-Round Molecular Phenotyping of Intact Tissue
[0162] Intact tissue samples were processed using CLARITY and
subjected to multi-round molecular phenotyping analysis. Results
are shown in FIG. 5. Panel a: 1st round staining. Volume rendering
of a 1 mm-thick coronal block (bregma-2.5 mm) of Thy1-EYFP mouse
immunostained for tyrosine hydroxylase (TH) in intact non-sectioned
form. The block was ETC-cleared for 1 day and immunostained for six
days: primary (2 day)-wash (1 day)-secondary (2 day)-wash (1 day).
Scale bar, 500 .mu.m. Panel b: antibody elution. The imaged
antibodies were eluted from the block shown in Panel a by
incubating the block in 4% SDS solution at 60.degree. C. for 0.5
days. Note that the TH signal was completely removed while the
fluorescence signal of EYFP was retained. Panel c: 2nd round
staining, 3D rendering of the same block immunostained for PV (red)
and GFAP (blue). DAPI (white) was used to counterstain nuclei.
Panels d-f: maximum projections of 100 .mu.m-volume of yellow-boxed
region in Panels a, b, and c, respectively. Note that patterns of
EYFP-positive neurons, as well as cellular morphologies, are very
similar; tissue architecture and cellular structures are well
preserved throughout the process of multi-round staining. SNR,
substantia nigra; cp, cerebral peduncle; scale bar, 100 .mu.m.
Panel g: optical section of white-/dotted-boxed region in Panel c
showing strong DAPI signal. DG, dentate gyrus; scale bar, 100
.mu.m. Panels h-i: TH channel of white-boxed region in Panel a
(Panel h) and Panel j (Panel i). The pattern and signal intensity
of TH staining is similar between the 1st round and 3rd round;
antigens and antigenicity are well preserved after two sequential
antibody elution processes. Scale bar, 100 .mu.m. Panel j: 3.sup.rd
round staining. The same block shown in Panels a-c was
immunostained for TH (red) and ChAT (blue). Panel k: enlarged 3D
view of the hippocampus in Panel c showing distribution of
EYFP-expressing neurons (green), PV-positive neurons (red), and
GFAP (blue). Alv, alveus of hippocampus; scale bar, 200 .mu.m.
Example 6: Structural Mapping and Molecular Phenotyping of Human
Brain Samples
[0163] Human brain samples were processed using CLARITY and
subjected to structural mapping and molecular phenotyping analysis.
Results are shown in FIG. 6. Panels a-g, i, k: immunostaining of
CLARITY-processed frontal lobe (BA 10) of postmortem human brain
(Autism Tissue Program case #AN13961; age, 7 years; sex, male; PMI,
25; storage, 82 months in 10% formalin at room temperature). 500
.mu.m-thick intact blocks were cleared for 1 day and immunostained
for 3 days as follows: primary (1 day)-wash (0.5 day)-secondary (1
day)-wash (0.5 day). Stained blocks were imaged using the 25.times.
water immersion objective. Panel a: optical section showing myelin
basic protein (MBP) and PV staining. White arrowheads indicate
membrane-localized MBP wrapping around PV-positive projections.
Scale bar, 10 .mu.m. Panel b: TH and PV staining. Maximum
projection of a 120 .mu.m-thick volume image. Step size, 0.5 .mu.m;
scale bar, 50 .mu.m. Panel c: optical section showing Neurofilament
(NP) and GFAP staining. Scale bar, 20 .mu.m. Panel d: somatostatin
and PV staining. Maximum projection of a 63 .mu.m-thick volume
image. Step size, 0.5 .mu.m; scale bar, 20 .mu.m. Panel e: 3D
reconstruction of NP-positive axonal fibers. Red, traced axon
running across the volume. Scale bar, 500 .mu.m; Inset, enlarged
image of the boxed region (scale bar, 20 .mu.m). Panel f:
visualization of PV-positive neurons in the neocortex of the autism
case. A 500 .mu.m-thick intact block was cleared and stained as
described above, and subcortical layers were identified. Scale bar,
500 .mu.m. Panel g: yellow-boxed region in Panel f showing
PV-positive neuronal cell bodies and fibers in layers IV, V, and
VI. Three representative PV-positive interneurons in layer VI with
ladder-shaped hetero- or iso-neuronal connections were traced
(green, purple, blue). Scale bar, 100 .mu.m. Panel h: 3D
reconstruction of the three abnormal neurons in Panel g. Scale bar,
80 .mu.m. Panel i; zoomed-in maximum projection of 8 .mu.m volume
image showing morphological details of the ladder-shaped structure
formed by neurites from a single neuron. Scale bar, 10 .mu.m. Panel
j: tracing of the structure shown in Panel i. Panel k: maximum
projection of 18 .mu.m volume image showing a ladder-shaped
structure formed by neurites from two different neurons. Scale bar,
10 .mu.m. Panel l: tracing of the structure shown in Panel k. Panel
m: bar graph showing iso- or hetero-neuronal dendritic bridges per
neuron. Neurons were selected randomly and traced in software (see
Methods); dendritic bridges were manually counted. **P.ltoreq.0.05;
error bars, s.e.m.; n=6 for both superficial and deep layers of the
autism case. n=4 for both superficial and deep layers of the
control case (#AN10251; age 10 years; sex, male; PMI, 19.83). Panel
n: 3D reconstruction of a neuron in layer II (superficial) of the
autism case; typical avoidance of iso-dendritic contact was
observed.
Example 7: Preservation of GFP and TdTomato Signals
[0164] Transgenic mouse brain samples were processed using CLARITY
and subjected to imaging analysis. The results are shown in FIG. 7.
Panel a: 3D rendering of a 1 mm-thick Thy1-EGFP M line mouse brain
block processed by CLARITY (Bregma -1.6->-2.6) showing
distribution of EGFP-expressing neurons and projections. Scale bar,
1 mm. Panel b: 3D rendering of a 1 mm-thick mouse coronal block of
PV-Cre mouse line (B6; 129P2-Pvalb.sup.tm1(cre)Arbr/J) crossed with
TdTomato reporter line (B6;
129S6-Gt(ROSA)26Sor.sup.tm9(CAG-tdTomato)Hze/J) Scale bar, 1 mm.
Panel c: maximum projection of 300 .mu.m volume of the hippocampus
in Panel a showing EGFP-expressing neurons and their neurites.
Scale bar, 50 .mu.m. Panel d: high resolution optical section from
the tissue in Panel b showing TdTomato-expressing neurons in the
cortex. Scale bar, 25 .mu.m. The results demonstrate that GFP and
TdTomato signals are preserved during the CLARITY process.
Example 8: Electrophoretic Tissue Clearing (ETC) Device
[0165] An ETC device was designed and constructed to carry out the
CLARITY process. An example of an ETC device in accordance with
embodiments of the invention is shown in FIG. 8. Panels a-d:
various views of a representative device showing a cylindrical
plastic housing, inlet/outlet ports (Cat. No. 5463K245, McMaster,
Robbinsville, N.J.) and two platinum electrodes (Cat. No. 267201,
Sigma, St. Louis, Mo.). The buffer inlet and outlet are located
such that buffer flow through the chamber effectively removes air
bubbles generated by electrolysis of the buffer solution. Panels
e-h: components of the assembly and example dimensions of a
representative device. All dimensions are in millimeters. Panels
i-j: sections through the device in Panel i (isometric) and Panel j
(top view) indicate component positions in the assembled chamber.
The hydrogel-embedded tissue is placed in the sample holder (Cell
Strainer, BD Biosciences, Durham, N.C.) located in the middle of
the chamber between the two electrodes. The single end of each
electrode that is exposed outside the chamber is connected to a
power supply.
Example 9: Optical Tissue Clearing of Intact Adult Mouse Brain
Using FocusClear.TM. and Glycerol
[0166] Intact adult mouse brain samples were processed using
CLARITY and FocusClear.TM. and glycerol solutions. The results are
shown in FIG. 9. Panel a: an image of a
PFA-fixed/hydrogel-embedded/non-ETC cleared mouse brain (four weeks
old) incubated in FocusClear.TM. for two days at room temperature.
Panel b: the same mouse brain shown in Panel a incubated in
FocusClear.TM. for eight days at room temperature. Panel c: an
image of a PFA-fixed hydrogel-embedded/non-ETC cleared mouse brain
(four weeks old) incubated in 85% glycerol for four days at
37.degree. C. Panel an image of PFA-fixed/hydrogel-embedded/ETC
cleared mouse brain (four weeks old) incubated in 85% glycerol for
two days at room temperature.
Example 10: Electron Microscope (EM) Imaging of CLARITY-Processed
Samples
[0167] Mouse brain tissue samples were processed using CLARITY and
then imaged using electron microscopy. The results are shown in
FIG. 10, and demonstrate EM-compatibility of the CLARITY process. A
Thy1-EYFP H line mouse (4 months old) was perfused transcardially
with a mixture of 2 wt % paraformaldehyde/2 wt % glutaraldehyde/4%
acrylamide (wt/vol)/0.05% bis-acrylamide (wt/vol)/0.25% VA044
(wt/vol)/PBS. Hydrogel-embedded brains were ETC-cleared for four
days. Cleared tissue was imaged using the 10.times. water-immersion
objective (3D rendering shown in Panel c). After imaging, the
tissue was post-fixed in 1% Osmium tetroxide (EMS Cat#19100) for 1
hr at RT, washed 3.times. with ultra-filtered water, and then
en-bloc stained for 2 hrs at RT. Samples were then dehydrated in a
series of ethanol washes for 15 minutes each at 4.degree. C. (50%
70% 95% (whereupon samples were allowed to warm to RT) 100%
(2.times.) acetonitrile for 15 min). Samples were infiltrated with
EMbed-812 resin (EMS Cat#14120) mixed 1:1 with acetonitrile for 2
hrs followed by a 2:1 mixture of EMbed-812:acetonitrile for 2
hours. Samples were then placed into EMbed-812 for 2 hours,
followed by TAAB capsules filled with fresh resin, then placed into
65.degree. C. oven overnight. Sections were cut between 75 and 90
nm on a Leica Ultracut S (Leica, Wetzlar, Germany), and picked up
on formvar/carbon coated slot grids (EMS Cat# FCF2010-Cu) or 100
mesh Cu grids (EMS Cat#FCF100-Cu). Grids were contrast-stained for
30 seconds in 3.5% UrAcetate/50% acetone followed by staining in
0.2% lead citrate for 30 seconds. Observation was in the JEOL
JEM-1400 TEM at 120 kV and photos were taken using a Gatan Orius
digital camera. Panel a: TEM image showing myelinated axons in the
hippocampus. Scale bar, 100 nm. Panel b: TEM image showing
post-synaptic densities (yellow arrowheads) in the hippocampus.
Scale bar, 200 nm. Panel c: 3D rendering of a cleared 1 mm-thick
Thy1-EYFP H line mouse block before EM sample preparation. Mouse
brain tissues fixed using 2% glutaraldehyde for EM were cleared and
imaged using CLARITY.
Example 11: Molecular Phenotyping of Whole Mouse Brain Samples
[0168] Whole mouse brain samples were processed using CLARITY and
subjected to molecular phenotyping analysis. The results are shown
in FIG. 11. Panel a: 3D immunohistological visualization of the
TH-positive neurons and their fibers in the intact mouse brain. The
intact brain was ETC-cleared for three days and stained for six
weeks: primary (2 weeks) wash (1 week)-secondary (2 weeks)-wash (1
week), and imaged at 2500 .mu.m from ventral side using the
10.times. water immersion objective. Scale bar, 700 .mu.m. Panels
b-d: optical sections at different depths. Note that TH-positive
neurons are well labeled and clearly visible even at 2500
.mu.m-deep in the intact brain. CPu, caudate putamen; PO, preoptic
nucleus; VTA, ventral tegmental area; SNR, substantia nigra; RR,
retrorubral nucleus; DR, dorsal raphe; Scale bar, 100 .mu.m.
Example 12: Imaging of Axonal Fibers in the Prefrontal Cortex of
Mouse Brain Samples
[0169] Mouse brain samples were processed using CLARITY and the
axonal fibers of the TH-positive neurons in the prefrontal cortex
were imaged. Results are shown in FIG. 12. Panel a: 3D rendering of
1 mm-thick mouse brain block (Bregma 2.68->1.68) stained for TH
(red) and DAPI (green). The block was ETC-cleared for 1 day and
immunostained for six days: primary (2 day) wash (1 day) secondary
(2 day) wash (1 day). PrL, prelimbic cortex; IL, infralimbic
cortex; Scale bar, 500 .mu.m. Panel b: zoom-in 3D rendering showing
TH fibers in the IL/PrL. Scale bar, 200 .mu.m. Panels c-e: maximum
projection of 50 .mu.m-volume of the IL/PrL in Panel a. Scale bar,
200 .mu.m. Panel c: DAPI; Panel d: TH; Panel e: DAPI and TH; Panel
f: yellow-boxed region in Panel e showing a heavily invested cell
(white arrowhead) and non-innervated cells (yellow arrowheads).
Scale bar, 100 .mu.m.
Example 13: Imaging of Axonal Fibers in the Nucleus Accumbens and
Striatum of Mouse Brain Samples
[0170] Mouse brain samples were processed using CLARITY and the
axonal fibers of the TH-positive neurons in the nucleus accumbens
and striatum were imaged. Results are shown in FIG. 13. Panel a:
maximum projection of 20 .mu.m-volume of the CPu in FIG. 4, Panel
j. The 1 mm-thick mouse brain block (Bregma 1.10->0.10) was
ETC-cleared for 1 day and immune-stained for six days: primary (2
day)-wash (1 day)-secondary (2 day)-wash (1 day). CPu, caudate
putamen; aca, anterior commissure; NAc, nucleus accumbens; Scale
bar, 50 .mu.m. TH (red) and DAPI (green). Panel b: optical section
of the CPu in Panel a showing cells massively invested by TH
fibers. Scale bar, 50 .mu.m.
Example 14: Imaging of Axonal Fibers in the Amygdala of Mouse Brain
Samples
[0171] Mouse brain samples were processed using CLARITY and the
axonal fibers of the TH-positive neurons in the amygdala were
imaged. Results are shown in FIG. 14. Panels a-d: a 1 mm-thick
mouse brain block (Bregma -1.46.->-2.46) was ETC-cleared for 1
day and immune-stained for six days: primary (2 day)-wash (1
day)-secondary (2 day)-wash (1 day). Panels a-b: 3D rendering of
the tissue stained for TH (red) and DAPI (blue). BLA, basolateral
amygdaloid nucleus; CeA, central amygdale; Fir, piriform cortex;
Scale bar, 300 .mu.m. Panel b: TH only. Panels c-d: maximum
projection of 100 .mu.m-volume in Panel a. Scale bar, 200 .mu.m.
Panel d: TH only.
Example 15: Measurement of Average Immunofluorescence Cross-Section
of PSD-95 Puncta at Varying Depths in Tissue Volumes Processed
Using CLARITY
[0172] Intact tissue volumes were processed using CLARITY and the
average immunofluorescence cross-section of post-synaptic density
(PSD)-95 puncta was measured at different depths (0-200 .mu.m, 20
.mu.m interval). The results are shown in FIG. 15. The average
fluorescence intensity of PSD-95 puncta was estimated at each depth
by applying a circular Hough transform to the gradient field of the
PSD-95 images (custom Matlab software). This located the center of
all puncta with radii ranging from 114 nm to 684 nm (N between 31
and 108). The centers were aligned to compute the average puncta at
each depth and the cross-section and the full width at half maximum
was calculated (see graph shown in FIG. 4, Panel g).
Example 16: Measurement of Average PSD-95 Puncta at Varying Depths
in Tissue Volumes Processed Using CLARITY
[0173] Intact tissue volumes were processed using CLARITY and the
average post-synaptic density (PSD)-95 puncta was measured at
different depths (0-200 .mu.m, 20 .mu.m interval). The results are
shown in FIG. 16. Scale bar, 200 .mu.m. The method described in
Example 14 was used.
Example 17: MAP2 Staining of Dendritic Fibers and Neuronal Cell
Bodies in Mouse Brain Tissue
[0174] Mouse brain tissue samples were processed using CLARITY and
were stained for microtubule-associated protein 2 (MAP2). The
results are shown in FIG. 17, and demonstrate uniform labeling of
dense dendritic fibers and neuronal cell bodies throughout the 1
mm-thick mouse brain tissue. Panel a: 3D rendering of 1 mm-thick
mouse brain tissue stained for MAP2, which is expressed in neuronal
cell bodies and their dendritic projections. The block was
ETC-cleared for 1 day and immune-stained for six days: primary (2
day)-wash (1 day)-secondary (2 day)-wash (1 day). hf, hippocampal
fissure; DG, dentate gyrus; scale bar, 300 .mu.m. Panel b: an
optical section from the 3D rendering. Scale bar, 250 .mu.m. Panels
c-d: high resolution optical sections at two different depths in
the DG showing cross sections of the labeled fibers. Note that
individual cross-sections of the dendritic fibers and
weakly-labeled neuronal cell bodies (yellow arrowhead) can be
clearly identified throughout the imaging depth. The images were
taken using the 63.times. glycerol-immersion objective. Scale bar,
25 .mu.m.
Example 18: Molecular Imaging of Whole Adult Zebrafish Brain
[0175] A whole adult zebrafish brain was processed using CLARITY
and subject to molecular phenotyping analysis. The results are
shown in FIG. 18. Adult zebrafish brain was extracted from a 213
dpf (days post fertilization) fish, hydrogel-hybridized, and
cleared by incubating in 4% SDS solution for 15 days. The cleared
brain was stained with anti-tyrosine hydroxylase (TH) primary for
four days and Alexa fluor 594 secondary for three days. The brain
was then imaged with the 25.times. water immersion objective. 3D
volume data (3,406.times.2,589.times.1,108 .mu.m; step-size=5
.mu.m). Red, TH; Blue, autofluorescence; scale bar, 100 .mu.m.
Example 19: Tracing of PV-Positive Neurons in a Brain Sample from
an Autistic Subject
[0176] A brain sample from an autistic subject was processed using
CLARITY and PV-positive neurons in the neocortex were traced. The
results are shown in FIG. 19. A 500 .mu.m-thick intact block of
frontal lobe (BA 10) of post-mortem human brain (autism case,
#AN13961; age, 7 years; sex, male; PMI, 25; storage, 82 months in
10% formalin at room temperature) was ETC cleared for a day and
immune-stained for parvalbumin (PV) for three days: primary (1
day)-wash (0.5 day)-secondary (1 day)-wash (0.5 day). The stained
block was imaged using the 25.times. water immersion objective.
Neurons were selected randomly, with the stipulation that the cell
body be located in the middle 125 .mu.m of the block, and traced in
Imaris software. Panel a: top view showing cortical laminar
structure and traced PV-positive neurons. PV, red; scale bar, 200
.mu.m. Panel b: traced neurons only (top view) showing relative
positions of the neurons (1-13) in the cortical lamina. Number of
iso- or hetero-neuronal dendritic bridges for each neuron can be
found in Table 1, below. Panel c: side view. Panel d: side view,
traced neurons only.
TABLE-US-00003 TABLE 1 Raw quantification data for the fourteen
PV-positive neurons in autistic brain tissue shown in FIG. 6, Panel
f and FIG. 19. # of heteroneuronal # of isoneuronal Neuron Layer
bridges bridges 1 1/2 1 0 2 1/2 0 0 3 1/2 0 0 4 3 3 0 5 3 0 0 6 3 0
0 7 4 2 0 8 4 6 1 9 4 3 4 10 5/6 14 0 11 5/6 4 4 12 5/6 4 2 m* 5/6
31 21 n* 5/6 4 9
Example 20: Tracing of PV-Positive Neurons in a Brain Sample from a
Normal Subject
[0177] A brain sample from a normal subject was processed using
CLARITY and PV-positive neurons in the neocortex were traced. The
results are shown in FIG. 20. A 500 .mu.m-thick intact block of
frontal lobe (BA 10) of post-mortem human brain (normal control
case for #AN13961 shown in FIG. 19, #AN10251; age, 10 years; sex,
male; PMI, 19.83) was ETC cleared for a day and immune-stained for
parvalbumin (PV) for three days: primary (1 day)-wash (0.5
day)-secondary (1 day)-wash (0.5 day). The stained block was imaged
using the 25.times. water immersion objective. Neurons were
selected randomly, with the stipulation that the cell body be
located in the middle 125 .mu.m of the block, and traced in Imaris
software. Panel a: top view showing cortical laminar structure and
traced PV-positive neurons. PV, red; scale bar, 200 .mu.m. Panel b:
the traced neurons only (top view) showing relative positions of
neurons (1-8) in the cortical lamina. Number of iso- or
hetero-neuronal dendritic bridges for each neuron can be found in
Table 2, below. Panel c: side view. Panel d: side view, traced
neurons only.
TABLE-US-00004 TABLE 2 Raw quantification data for the eight
PV-positive neurons in normal control human brain tissue shown in
FIG. 20 # of heteroneuronal # of isoneuronal Neuron Layer bridges
bridges 1 1/2 0 0 2 1/2 0 0 3 3 0 0 4 3 0 0 5 5/6 0 0 6 5/6 0 0 7
5/6 0 0 8 5/6 1 1
* * * * *