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WO2017031249A1 - Stabilisation de tissu de tout le corps et extractions sélectives par le biais d'hybrides hydrogel-tissu pour un phénotypage et un appariement de circuit intact à haute résolution - Google Patents

Stabilisation de tissu de tout le corps et extractions sélectives par le biais d'hybrides hydrogel-tissu pour un phénotypage et un appariement de circuit intact à haute résolution Download PDF

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Publication number
WO2017031249A1
WO2017031249A1 PCT/US2016/047430 US2016047430W WO2017031249A1 WO 2017031249 A1 WO2017031249 A1 WO 2017031249A1 US 2016047430 W US2016047430 W US 2016047430W WO 2017031249 A1 WO2017031249 A1 WO 2017031249A1
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tissue
clearing
solution
hydrogel
sample
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Viviana Gradinaru
Jennifer TREWEEK
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California Institute of Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the present invention generally relates to the field of tissue preparation and characterization.
  • the worm Caenorhabditis elegans and the zebrafish Danio rerio provide scientists with an unobstructed, organism-wide view of tissue anatomy and cellular activity (e.g. via cell-type specific fluorescent labeling and genetically encoded calcium indicators) using conventional imaging techniques.
  • their whole-body transparency enables rigorous, high throughput investigations into how environmental, cellular, and genetic alterations influence biological processes from cellular signaling and apoptosis, to organism development and survival.
  • the comparatively large size and optical opacity of mammalian models generally has limited researchers to imaging snapshots of cellular organization on thin- sectioned tissue samples.
  • the invention teaches a method for clearing and expanding tissue.
  • the method includes the steps of: (1) applying a fixing solution to the tissue, thereby forming fixed-tissue; (2) applying a surfactant to the fixed-tissue, thereby forming permeabilized-tissue; (3) incubating the permeabilized-tissue in a solution that includes aciylate-acrylamide copolymer (AcAm) and one or more polymerizing agent; (4) incubating the tissue in a solution that includes sodium dodecyl sulfate (SDS), thereby forming SDS-treated cleared tissue; (5) incubating the SDS-treated cleared tissue in a solution that includes collagenase, thereby forming collagenase-treated tissue; and (6) incubating the collagenase-treated tissue in water, thereby forming cleared and expanded tissue.
  • AcAm aciylate-acrylamide copolymer
  • SDS sodium dodecyl sulfate
  • the fixing solution is comprised of 1-15% paraformaldehyde (PFA) and/or 0.1-5% glutaraldehyde.
  • the method further includes applying a quenching solution to the fixed tissue.
  • the quenching solution includes glycine.
  • the surfactant includes Triton X-100.
  • the solution that includes surfactant further includes phosphate buffered saline (PBS).
  • the solution that includes AcAm includes 0-4 % acrylamide, 4-10 % sodium acrylate, and 0-1 % bis-acrylamide.
  • the solution that includes SDS includes SDS at a concentration of 4-10 %.
  • the pH of the solution that includes SDS is 6.5-9.5.
  • the solution that includes collagenase includes collagenase at a concentration of 1-mg/ml.
  • the tissue includes animal tissue.
  • the tissue includes mammalian tissue.
  • the tissue includes brain tissue.
  • the tissue is immunolabeled and/or fluorescently labeled.
  • the method further includes incubating the tissue in a refractive index matching solution (RIMS).
  • RIMS refractive index matching solution
  • the invention teaches a method for visualizing and/or imaging a cleared and expanded tissue.
  • the invention includes utilizing a microscope to visualize and/or image a tissue treated according to any of the methods described above.
  • the tissue includes fluorescently labeled cells.
  • the refractive index of the tissue has been homogenized.
  • the method further includes counting the fluorescently labeled cells.
  • the fluorescently labeled cells are automatically counted.
  • one or more nucleic acids within the tissue are labeled with a marker that can be visualized and/or imaged with a microscope.
  • one or more of the nucleic acids are mRNA.
  • the one or more nucleic acids are labeled using single-molecule fluorescence in-situ hybridization (smFISH).
  • the method further includes quantifying one or more species of mRNA in the tissue based on a unique fluorescent signature.
  • FIG. 1 depicts, in accordance with various embodiments of the invention, applications of whole-organ and whole-organism clearing protocols.
  • A-E PARS-based whole-body clearing for assessing cellular level AAV tropism (see Supplementary Methods). Three weeks after systemic injection of AAV9:CAG-GFP, mice were PARS-cleared and their organs excised and sectioned for imaging.
  • A-B Projection images show GFP + transduced cells in the adrenal gland. Arrow highlights a GFP + cell near the surface of the adrenal gland with neuronal morphology, which is shown in higher magnification in B.
  • C Projection images show GFP + cells in the stomach from the surface to the lumen. GFP expression is particularly high in the myenteric plexus.
  • D-E AAV9 transduces cells in several layers within the intestine (duodenum).
  • D Projection image of GFP fluorescence. Double colored lines correspond to the positions of 50 ⁇ maximum projection images extracted from the data set and presented in E.
  • E GFP + cells in the intestinal crypt (top), submucosal plexus (middle), and myenteric plexus (bottom).
  • F-G Islet distribution within human pancreatic tissue.
  • F A 2 mm thick section of an adult human pancreas (top) was rendered transparent (bottom) with the PACT method.
  • a 2 mm thick section was cut from a 4% PFA- fixed human pancreas, incubated in 0.5% PFA, 4% acrylamide at 4 °C overnight, and then polymerized in fresh A4P0 hydrogel monomer with 0.25% VA-044 thermal initiator for 2 hours at 37 °C.
  • the tissue was cleared with 4% SDS-PBS (pH 7.5) for 48 hours, immunostained, and mounted in sRIMS (-50% sorbitol in 0.02 M PB, refractive index of 1.44).
  • the islet distribution was visualized by immunostaining for insulin (red), somatostatin (green) and DAPI (cyan) (see Table 4 for details on antibodies and nuclear stain); panels represent an imaging stack of 70 um. Sparsely distributed islets are easily located with only 5 ⁇ magnification (middle panel). A group of islets were identified at 10x magnification (right, top) and a 3D image of a single islet was captured with at 25 ⁇ magnification (right, bottom). All images were collected on a Zeiss LSM 780 confocal with the Fluar 5* 0.25 N.A. M27 air objective (w.d. 12.5 mm), Plan-Apochromat 10* 0.45 N.A. M27 air objective (w.d 2.0 mm), and the LD LCI Plan-Apochromat 25* 0.8 N.A. Imm Corr DIC M27 multi-immersion objective (w.d 0.57 mm).
  • FIG. 2 depicts, in accordance with various embodiments of the invention, PACT set-up and procedure.
  • the sample and hydrogel solution was incubated at 37 °C in an oxygen-depleted environment. This is accomplished within an air-tight container that permits sample degassing.
  • Supplies for PACT chamber left: 50 ml conical tube (large sample) or vacutainer (small sample), size 7 stoppers that fit the 50 ml conical tube, PTFE tubing, needles, syringes, and a razor blade or scissors to cut syringe in half.
  • Figure 3 depicts, in accordance with various embodiments of the invention, PACT protein loss and tissue expansion for different hydrogel and clearing conditions.
  • Figure 4 depicts, in accordance with various embodiments of the invention, clearing time course and antibody penetration of PACT-processed samples. Quantitative comparison of the effect of different hydrogel embedding conditions and clearing buffers on time to clear and antibody penetration during immunostaining. 1 mm thick mouse coronal slices were hybridized and cleared with the array of previously used PACT conditions (Fig. 3). Slices were monitored for the time they took to become transparent. Once cleared, slices were washed and then immunostained. (A) Representative images of two 1-mm thick coronal brain slices (-1.0-0.0 mm anterior to bregma) through the time course for PACT clearing and a comparison of time to clear (mean ⁇ s.e.m.) for each PACT hydrogel composition.
  • Labeling intensities for A4P0, A4P1, A4P4 and unhybridized samples cleared with 8% SDS-PBS (pH 7.5), as a representative sample of all the different buffers, are plotted on a logarithmic scale. The amount of PFA contained in the hydrogel-tissue matrix is inversely proportional to immunohistochemical staining efficiency.
  • Figure 5 depicts, in accordance with various embodiments of the invention, preservation of tissue architecture during delipidation.
  • A-C Mice that received bilateral intracranial injections in the lateral septum of AAV expressing the tdTomato transgene were perfusion-fixed with 4% PFA and a subset of 1 mm thick unhybridized coronal brain sections were prepared for microscopy without clearing (control, first column), or were first rendered transparent via the CUBIC method (second column).
  • the second subset of 1 mm thick sections underwent PACT processing: A4P0- (third column) or A4P4-embedding (fourth column) and clearing with 8% SDS-PBS (pH 7.5), followed by preparation for ultrastructural study or RIMS mounting.
  • A Brain sections were photographed after fixation (control) or immediately after clearing (CUBIC, A4P0, A4P4) to illustrate the degree of tissue swelling that occurred for each condition.
  • CUBIC, and PACT-cleared (A4P0, A4P4) tissues were then processed identically for ultrastructural examination using electron microscopy and tomography (see Supplementary Methods).
  • the images correspond to a 50 ⁇ thick maximum intensity projection over the dentate gyrus; Top: A4P0-PACT cleared, Bottom: uncleared smaller panels are high magnification images of the boxed areas showing myelinated axons.
  • IACUC Institutional Animal Care and Use Committee
  • Figure 6 depicts, in accordance with various embodiments of the invention, assembling and working with the PARS chamber.
  • A A completed PARS chamber used for whole-body tissue clearing.
  • B Individual parts to build a PARS chamber: (1) three 1 ⁇ 2" ⁇ 1 ⁇ 2" barbed connectors, (2) two 3 ⁇ 4 2 " barbed male Luers with locking nut, (3) a 1000 ⁇ pipette tip box, (4) a 1 -gallon Ziploc freezer bag, (5) a 3-way stopcock with Luer lock, (6) a 3 ⁇ 4 2 " barbed female Luer with full tread, (7) a roll of lab tape, (8) a 22 G ⁇ 1" gavage needle, (9) a 1 ⁇ 2" barbed male slip Luer, (10) a female Luer tee with locks, (11) clay, and (12) Tygon E-lab tubing.
  • Ruler shown is 5 cm in length.
  • C Three 1 ⁇ 2" holes are drilled into the pipette tip box: two into the box front and one into its side, all approximately 2 cm below the top rim of the box. The three 1 ⁇ 2" ⁇ 1 ⁇ 2" barbed connectors are placed into the drilled holes. To connect the outflow line (blue tape bands on outflow line tubing), a piece of Tygon tubing is connected from the bottom inside of the pipette box to the single 1 ⁇ 2" barbed connector that was inserted through the box side.
  • a second, longer piece of blue-taped tubing is attached to the outer fitting of this same barbed connector (on the outside of the pipette tip box side) and then the other end of this tubing is threaded through the peristaltic pump, pulled back over toward the pipette box, and finally (D) connected to a 3-way stopcock with a 3 ⁇ 4 2 " barbed male Luer with locking nut (rightmost blue-banded tubing in D).
  • a short length of tubing green tape band
  • the solute flushing line and nitrogen bubbling line which are subserved by the same tubing (white tape band), are formed by another short length of tubing that joins the third port of the stopcock to the front left 1 ⁇ 2" barbed connector.
  • the inflow line is continued inside the pipette box, with the tubing coiled several times around the base of the box so that the solute will be re-heated before it passes through the feeding gavage into the subject.
  • the solute flushing line and nitrogen bubbling line is continued inside the pipette tip box and taped to the bottom of the chamber (not shown).
  • the Tygon tubing is reconnected from the outside of the bag and surrounded with clay to make an air-tight seal.
  • H The animal is placed onto the pipette tip box and the 22 G ⁇ 1" gavage needle is used to catheterize the heart.
  • I The chamber is placed into a 37 °C waterbath. A female Luer tee, which is taped onto the lid of the pipette tip box, is punctured through the Ziploc bag and this joint is sealed with clay to ensure an airtight seal.
  • a vacuum line is connected to the female Luer tee to remove oxygen (orange arrow) and a nitrogen gas line (white arrow) is connected to the 1 ⁇ 2" barbed connector to deliver a steady flow of nitrogen into the bagged system.
  • the solute is continually circulated through the animal from the outflow line (blue arrow, which also indicates the direction of flow through blue-taped tubing) and inflow line (green arrow, which also indicates the direction of flow through green-taped tubing).
  • Figure 7 depicts, in accordance with various embodiments of the invention, whole- body clearing of mice with PARS.
  • A A4P0-hybridized organs shown before the start of clearing (left) and after 5 days of clearing with 8% SDS-PBS (pH 8.5) and overnight washing with l x PBS at pH 7.5 (right). Numbers correspond to the extracted organs in panel (B).
  • B Extracted organs from the cleared mouse of panel (A), pictured before (top) and after (bottom) R MS incubation for 3 days. Black pointers correspond to the adrenal gland on the kidney and to the ovaries on the fallopian tubes. Each square represents 0.5 cm 2 .
  • Figure 8 depicts, in accordance with various embodiments of the invention, light sheet microscopy enables fast and high-resolution imaging of cleared samples.
  • the scientific CMOS camera (Zyla 4.2 sCMOS, Andor) is running in a light sheet mode, in which the readout direction of the camera is unidirectional and synchronized with the scanning direction and speed of the light-source. In this configuration, only the pixels that are illuminated will be recorded thus improving the signal to noise ratio of the image.
  • the function generator, the camera, and the oscilloscope are controlled using a custom MATLAB program.
  • C A volume rendering (Imaris, Bitplane) and cross sections at different depths of a cleared Thyl-YFP mouse brain section (1 mm thick), taken with the light sheet microscope, the intensity of the layers was normalized. The images were acquired at 45 frames per second (voxel size: 0.117 ⁇ x 0.117 ⁇ x 0.25 ⁇ , bit depth: 12). The cross sections at different depths, which are perpendicular to the scan direction, are maximum intensity projections (Imaris) across a 5 ⁇ volume. A parts list for this set-up is available in Table 7.
  • Figure 9 depicts, in accordance with various embodiments of the invention, two different workflows for cell tracing in neuTube and Imaris.
  • A Tracing using neuTube.
  • B Tracing using Imaris 7.1 (Bitplane). Results shown here took 25 minutes for a novice user with ⁇ 5 hours of total experience using each tracing tool. Total tracing time to achieve similar results was generally comparable but we found neuTube to be more efficient for quickly tracing isolated neurites.
  • Al neuTube 3D visualization, A2. neuTube semi- automated tracing result, A3. Tracing error, A4. Manual correction, Bl . Imaris ROI selection, B2. Imaris Autopath seeding, B3. Manual correction of tracing error, B4. Trace extension using Autopath.
  • Figure 10 depicts, in accordance with various embodiments of the invention, effects of bis-acrylamide crosslinker on clearing time and swelling of PACT-cleared sections.
  • A Representative images of the timecourse for PACT clearing of four 2 mm thick rat coronal brain slices, (displayed anterior to posterior, from left to right). Slices were embedded in either A4P0B0.05 or A4P0 and then cleared with 8% SDS-PBS (pH 7.5). The A4P0 slices were completely clear by 144 hours. Although some heavily myelinated brain sections seemed to resist clearing in A4P0B0.05-embedded sections initially, this effect did not persist, resulting in similar overall clearing time as slices embedded without bis-acrylamide.
  • RIMS RIMS formulation guide to optimize the RI to that of the cleared sample.
  • RFMS formulated with 82% HistodenzTM should be broadly applicable to cleared brain tissue, while RIMS with a higher RI of 1.48-1.49 is suggested for denser cleared tissue such as bone.
  • Figure 11 depicts, in accordance with various embodiments of the invention, protein loss over the course of PACT clearing.
  • the amount of protein lost while clearing was measured by performing a BCA on the clearing buffer, which was collected and replaced periodically while 1 mm tissue samples were undergoing PACT.
  • a standard curve of BSA protein concentration in each of the four different clearing buffers was generated. Standard curves were fit with a third order polynomial and used to extrapolate all protein loss measurements.
  • A A representative case, shown here for BSA concentrations in 8% SDS- PBS (pH 7.5).
  • Figure 12 depicts, in accordance with various embodiments of the invention, PACT compatibility with histological staining.
  • A-C Representative images of thick section clearing with addition of CuS0 4 or 0.2% SB compared to regular PACT. 0.5 mm and 1 mm coronal Thy 1 -YFP mouse sections are shown after A4P1 hydrogel polymerization (A) and during clearing with 8% SDS-BB (pH 8.5) and subsequent 24 hour incubation in RIMS (B and C for 0.5 mm and 1 mm, respectively).
  • A A4P1 hydrogel polymerization
  • B and C for 0.5 mm and 1 mm, respectively.
  • D The control, CuS0 4 , and 0.2% SB treated 0.5 mm slices from (A-B) were immunostained for parvalbumin (see Table 4) and then transferred to RIMS, degassed, and mounted.
  • Endogenous YFP (cyan) and immunolabeled PV (red) were imaged throughout the slice (left) in a region of the cortex.
  • a 100 ⁇ thick maximum intensity projection (right) was taken at a depth of 500 ⁇ to show representative imaging in the middle of the section.
  • Signal range of the red channel was adjusted for better visualization of PV staining at depth. All sections were imaged on a Zeiss LSM 780 confocal with the Plan-Apochromat 10* 0.45 N.A. M27 air objective (w.d 2.0 mm).
  • Figure 13 depicts, in accordance with various embodiments of the invention, ePACT: a protocol for tissue clearing through expansion.
  • A Fluorescence image of Thy 1 -YFP expression prior to expansion-clearing. A 70 ⁇ thick maximum intensity projection of five cells expressing YFP represents the standard for imaging pre-expansion. A bright-field image of the pre-expansion 100 ⁇ brain slice is shown in the top right, with the location of the cells being imaged indicated by the red arrowhead. Noteworthy features that may differ between pre- and post- expansion-cleared tissue, such as cell bodies, branching processes, and large projections, are numbered 1, 2, and 3, respectively.
  • B Fluorescence image of Thy-YFP expression after 4x expansion-clearing.
  • a 340 ⁇ thick maximum intensity projection of the same five cells in (A) is shown, with the same features labeled again 1, 2, and 3.
  • a cell body (1) and the neuronal processes of an adjacent cell (2) are both partially obstructed by tissue lipids in (A), but can be easily identified in (B) after clearing and expansion.
  • the 4x expansion that contributes to this increased visibility through tissue also causes some tissue destruction, as apparent in the multiple severed processes (such as (2)).
  • a bright-field image of the expanded slice embedded in agarose is shown at the top right.
  • C YFP fluorescence from the same cell in pre-expanded (blue box) and post-expanded (yellow box) tissue is shown.
  • Figure 14 depicts, in accordance with various embodiments of the invention, whole body PARS clearing with borate-buffered detergent.
  • A Mice were perfusion-fixed, A4P0- embedded, PARS-cleared for 5 days with 8% SDS-BB (pH 8.5), and washed with l x PBS at pH 7.5. Numbers correspond to the extracted organs in panel (B).
  • B Extracted organs from the cleared mouse in panel (A), pictured before (top) and after (bottom) RIMS incubation for 3 days. Black pointers correspond to the adrenal gland on the kidney and to the ovaries on the fallopian tubes. Each square represents 0.5 cm 2 .
  • Rodent husbandry and euthanasia conformed to all relevant governmental and institutional regulations; animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and by the Office of Laboratory Animal Resources at the California Institute of Technology.
  • Figure 15 depicts, in accordance with various embodiments of the invention, small- format antibodies for thick-tissue labeling.
  • camelid nanobodies are a promising alternative to traditional antibodies, either full immunoglobulins or their engineered formats (single-chain variable fragment (scFv), Fab, and F(ab') 2 ).
  • the stained section was then washed 3 times in PBST over 1 hour, followed by a 1-hour incubation in RIMS.
  • the transparent sections were RIMS- mounted and imaged on a Zeiss LSM 780 confocal with the Plan-Apochromat 10x 0.45 N.A. M27 air objective (w.d 2.0 mm).
  • (B, left) 850 ⁇ thick 3D rendering of mouse internal capsule stained with GFAP nanobody.
  • B, right Side view showing uniform labeling of GFAP nanobody throughout the entire 850 ⁇ slice.
  • Figure 16 depicts, in accordance with various embodiments of the invention, user interface elements for image analysis.
  • A neuTube.
  • B Imaris.
  • Computer screenshots depict the image processing workspace for each software during the 3D visualization of labeled cells in Figure 9, panels Al and Bl .
  • Figure 17 depicts, in accordance with an embodiment of the invention, (A) an immersion chamber and (B) a sample holder that can be used in conjunction with the light sheet microscope described in Fig. 8.
  • PACT is an acronym for PAssive CLARITY Technique.
  • PARS is an acronym for Perfusion-assisted Agent Release in situ.
  • RIMS Refractive Index Matching Solution
  • ePACT is an acronym for expansion-enhanced PACT.
  • “Mammal,” as used herein, refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domesticated mammals, such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult, newborn subjects, and unborn subjects whether male or female, are intended to be included within the scope of this term.
  • Peripheral organs can include but are in no way limited to muscles, heart, lungs, kidneys, colon, gut, intestines, and the like.
  • the invention teaches a method for clearing and expanding tissue for improved visualization of various constituents of the tissue (e.g., cells, nucleic acids, and other small molecules).
  • the method includes applying a fixing solution that includes paraformaldehyde (PFA) to the tissue, thereby forming fixed tissue.
  • the fixing solution includes PFA at a concentration of from 1- 15%.
  • the fixing solution includes glutaraldehyde at a concentration of from 0.1-5%).
  • the fixing solution includes glutaraldehyde at a concentration of from 0.1-5% and PFA at a concentration from 1-15%.
  • the fixing solution includes PFA at a concentration of 4%.
  • the free aldehydes present in fixed tissue are quenched before subsequent processing steps.
  • the quenching solution may include glycine.
  • the tissue is subsequently rinsed with Tris-glycine (0.1-0.3 M glycine with pH adjusted to pH 7.2-8 using Tris-base).
  • the quenching solution may include ammonium chloride.
  • the tissue is rinsed for 5 minutes to 5 hours in 0.1 M - 1 M glycine and 10 mM - 100 mM ammonium chloride in buffer (l x PBS or l x TBS (tris-buffered saline)).
  • the quenching solution is 1%> sodium borohydride in l x PBS.
  • the method further includes permeabilizing the fixed tissue in a solution that includes a surfactant.
  • the surfactant is a nonionic surfactant.
  • the nonionic surfactant is Triton X-100.
  • the solution includes PBST (l x PBS containing 0.1% Triton X-100 (vol/vol)).
  • the solution may further include 0.1 M - 1M glycine.
  • the solution may include 0.05 - 5% Triton X-100 and 0.1 M - 1 M glycine.
  • the solution may include 0.1 M - 1M lysine in place of glycine.
  • the tissue is subsequently rinsed with a buffer solution.
  • the tissue is rinsed with PBS.
  • the tissue is rinsed with lx PBS.
  • R MS refractive index matching solution
  • any appropriate RIMS solution described in the examples set forth herein may be used for this step of the method.
  • the tissue before or after incubation with RIMS, or in the absence of incubation in RIMS, the tissue is incubated in a solution that includes acrylate- acrylamide copolymer (AcAm).
  • the solution that includes AcAm includes 0-4%> acrylamide, 4-10%> sodium acrylate, and 0-l%> bis-acrylamide.
  • the solution that includes AcAm includes a buffer.
  • the solution that includes AcAm is prepared in lx PBS with 2 M NaCl.
  • the solution that includes AcAm includes 2.5% acrylamide, 8.625%) sodium acrylate, and 0.15 %> bis-acrylamide in lx PBS with 2M NaCl.
  • the solution that includes AcAm further includes a catalyst of polymerization such as TEMED (Tetramethylethylenediamine), and an initiator of polymerization such as the oxidant APS (ammonium persulfate) at approximately equimolar concentrations of 1-10 mM.
  • a polymerization inhibitor such as 4-hydroxy TEMPO (4-hydroxy-2,2,6,6- tetramethylpiperidin-l-oxyl) is included in the AcAm solution to allow adequate diffusion of hydrogel monomers throughout the sample (i.e., tissue or fixed cells).
  • the polymerization of the AcAm solution is accomplished via including the following (w/w): 0.01-0.1% 4-hydroxy TEMPO , 0.05-0.2% TEMED, and 0.05-0.2% APS.
  • potassium persulfate (KPS) or riboflavin (5-10 ⁇ g/ml) may be included in the AcAm solution instead of APS.
  • the solution that includes AcAm further includes the following (w/w): 0.01% 4-hydroxy TEMPO, 0.2% TEMED, and 0.2% APS.
  • the solution that includes AcAm contains a water-soluble azo initiator such as the thermoinitiator VA-44 (2,2'-Azobis[2-(2-imidazolin-2- yl)propane]dihydrochloride) in place of APS and TEMED.
  • the solution that includes AcAm further includes 0.5% VA-044.
  • the tissue is incubated in the solution containing AcAm, polymerization inhibitors (e.g., 4- hydroxy TEMPO) and polymerization initiators for 10-60 minutes or longer at 1-10 °C.
  • the tissue immediately following this first incubation, the tissue undergoes a second incubation in fresh solution containing AcAm, polymerization inhibitors (4-hydroxy TEMPO) and polymerization initiators for 10-60 minutes or longer at 1-10 °C.
  • the tissue is incubated in the solution containing AcAm and thermoinitiator for 10-60 minutes or up to 48 hours or longer at 1-10 °C.
  • the tissue is incubated in AcAm and VA-044 for 20-40 minutes at 4 °C.
  • the tissue is further incubated at 35-45 °C until the AcAm is polymerized.
  • the tissue is purged of free oxygen via degassing under nitrogen for 1-15 minutes at 4-25 °C, or incubation in an inert gas atmosphere for 1-24 hours at 4-25 °C.
  • the oxygen-purged tissue is incubated at 37-42 °C until the AcAm is polymerized.
  • the tissue is incubated for 1-8 hours or longer.
  • excess gel is removed from around the tissue after polymerization.
  • after polymerization (and optionally after excess gel has been removed) the tissue is incubated in a 4-10%) SDS solution.
  • the SDS solution is borate-buffered.
  • the SDS solution includes 4-10%> SDS and 0.2 M boric acid buffer.
  • the boric acid buffer is prepared according to the description in the examples set forth herein.
  • the pH of the SDS solution is 6.5-9.5.
  • the pH of the SDS solution is 8.5.
  • the tissue is then incubated in boric acid wash buffer (BBT).
  • BBT boric acid wash buffer
  • the BBT includes 0.2 M boric acid buffer (prepared as described in the examples set forth herein) and 0.1-0.25% Triton X-100 (vol/vol).
  • the tissue is incubated in 0.2M boric acid buffer with 0.1% Triton X-100 (vol/vol).
  • the pH of the BBT is 6.5- 9.5. In some embodiments, the pH of the BBT is 8.5. In some embodiments, after incubation in BBT, the tissue is washed in TESCA buffer. In some embodiments, the buffer includes 50 mM TES and 0.36 mM calcium chloride solution. In some embodiments, the TESCA buffer includes 50mM TES and 0.36mM calcium chloride solution. In some embodiments, the pH of the buffer is 6.5-9.5. In some embodiments, the pH of the TESCA buffer is 7.4 at 37 °C. In some embodiments, the tissue is subsequently incubated in a solution that includes collagenase.
  • the concentration of collagenase in the solution is 1-10 mg/ml.
  • the solution that includes collagenase further includes a buffer.
  • the collagenase is in TESCA buffer.
  • the tissue is incubated in the solution containing collagenase for 1-48 hours or longer. In some embodiments, the tissue is incubated in a solution that includes collagenase for 12-24 hours. In certain embodiments, after incubation in a solution that includes collagenase, the tissue is soaked in H 2 0. In some embodiments, the tissue is soaked in dd H 2 0.
  • the tissue is soaked in H 2 0 at a temperature of 20-37 °C for a period of 10-60 minutes, or until expanded to a desired extent. In some embodiments, the tissue is soaked at 23 °C. In some embodiments, the tissue is soaked in the absence of light, or with reduced exposure to light. In some embodiments, the tissue used in connection with the aforementioned ePACT methods is animal tissue. In some embodiments, the tissue is mammalian tissue. In certain embodiments, the tissue is brain tissue. In some embodiments, the tissue thickness is 10- 2000 ⁇ . In some embodiments, the tissue thickness is 50-150 ⁇ . In certain embodiments, the tissue is 100 ⁇ .
  • the invention teaches imaging a tissue prepared according to the aforementioned ePACT methods.
  • the tissue embedded in AcAm (described above) is mounted to prevent sample drift during imaging.
  • the tissue is embedded in agarose.
  • the mounted sample is sealed between a coverslip and glass slide, so that the water content of the agarose and of the expanded AcAm tissue -hydrogel remains at a steady-state.
  • one or more cells, cellular components, and other molecules within the tissue are labeled (e.g. with a fluorescent label or by any other means of labeling described in the examples set forth herein) prior to imaging the tissue.
  • neural circuits are mapped (as described in greater detail in the example section) by imaging the prepared tissue.
  • one or more nucleic acids e.g. DNA and/or RNA
  • RNA within the tissue is visualized using single-molecule fluorescence in-situ hybridization (smFISH) (see Skinner, S. O., et al.
  • quantitative analysis of multiple transcripts isolated to their subcellular locations, and visualized using smFISH is performed.
  • super-resolution microscopy is used to visualize one or more labeled transcripts within tissues that have been prepared according to the ePACT methods described above and in the ensuing examples.
  • Exemplary super-resolution technologies include but are not limited to I 5 M microscopy, 4Pi-microscopy, Stimulated Emission Depletion microscopy (STEDM), Ground State Depletion microscopy (GSDM), Spatially Structured Illumination microscopy (SSIM), Photo-Activated Localization Microscopy (PALM), Reversible Saturable Optically Linear Fluorescent Transition (RESOLFT), Total Internal Reflection Fluorescence Microscope (TIRFM), Fluorescence-PALM (FPALM), Stochastical Optical Reconstruction Microscopy (STORM), Fluorescence Imaging with One-Nanometer Accuracy (FIONA), and combinations thereof.
  • the invention teaches a method for masking autofluorescence of a tissue.
  • the method includes applying a fixing solution to the tissue, thereby forming fixed tissue, and applying an autofluorescence masking solution to the fixed tissue, thereby forming a masked tissue.
  • the fixing solution includes PFA.
  • the autofiouroescence masking solution includes CuS0 4 or Sudan Black (SB).
  • CuS0 4 is included at a concentration of 1-10 mM.
  • the autofluorence masking solution is lOmM CuS0 4.
  • the autofluorescence masking solution includes 0.01- 1.0 % SB.
  • the autofluorescence masking solution is 0.2% SB.
  • the foregoing autofluorescence masking solution is applied by incubating the tissue in the autoflourosecence masking solution.
  • the tissue is incubated for 1-72 hours at from 0-23 °C.
  • the tissue is incubated for 48 hours at 4 °C.
  • the masked tissue is then washed or dipped with water to remove excess stain, thereby forming water-treated tissue.
  • the water-treated tissue is then rinsed in phosphate buffered saline (PBS), thereby forming rinsed tissue.
  • PBS phosphate buffered saline
  • the rinsed tissue is then incubated in hydrogel monomer solution that includes acrylamide, thereby forming a hydrogel-treated tissue.
  • the hydrogel monomer solution includes 1-10 % acrylamide and 0-4 % paraformaldehyde and 0-1% bisacrylamide.
  • the hydrogel monomer solution includes 1-10% acrylamide and 1-10% paraformaldehyde.
  • the hydrogel monomer solution includes 4% acrylamide and 1% paraformaldehyde.
  • the hydrogel-treated tissue is subsequently incubated in 1-20% sodium dodecyl sulfate (SDS).
  • the hydrogel-treated tissue is subsequently incubated in 5-15% SDS, thereby forming cleared tissue. In certain embodiments, the hydrogel-treated tissue is incubated in 8% SDS. In some embodiments, the pH of the SDS solution is 6.5-9.5. In some embodiments, the pH of the SDS solution is 8-9. In some embodiments, the pH of the SDS solution is 8.5. In some embodiments, the tissue is brain tissue. In some embodiments, the tissue is any animal tissue. In some embodiments, the tissue is 0.01-5 mm thick. In some embodiments, the hydrogel- treated tissue is incubated in SDS, as described above, for 1-240 hours. In certain embodiments, the hydrogel-treated tissue is incubated in SDS for 12-15 hours.
  • the hydrogel-treated tissue is incubated in SDS for 24-48 hours. In some embodiments, the hydrogel-treated tissue is incubated in SDS for 72-240 hours.
  • the cleared tissue is immunostained and/or labeled (before or after clearing) with fluorescent markers, including any immunostains or fluorescent markers described in the examples set forth herein.
  • the tissue or any component thereof is visualized with microscopy. In some embodiments, the tissue is visualized and/or imaged by any form of microscopy described or referenced herein (e.g. confocal microscopy, light sheet microscopy, super-resolution microscopy, etc.).
  • the invention teaches a method for immunostaining tissue prepared according to any of the methods described herein.
  • the method includes applying a solution that includes a primary antibody to the cleared and washed tissue of the methods described above, thereby forming an antibody-bound tissue. Any suitable antibodies (including small format) and antibody types described or referenced herein can be used in conjunction with the inventive methods.
  • the method further includes rinsing the antibody-bound tissue with a buffer solution.
  • the buffer solution includes PBS.
  • the method further includes applying a solution that includes a secondary antibody to the antibody-bound tissue that has been washed with buffer solution, wherein the secondary antibody is labeled with a visualizable marker.
  • the visualizable marker is fluorescent.
  • the primary antibody is labeled with a visualizable marker.
  • the tissue is obtained from a biopsy.
  • the invention teaches a method for visualizing and/or imaging immunostained tissue.
  • the method includes utilizing a microscope to visualize and/or image immunostained tissue prepared according to any of the methods described herein.
  • the microscope is utilized to implement a form of microscopy that may include, but is in no way limited to epi-fluorescence microscopy, confocal microscopy, multi-photon microscopy, spinning disk confocal microscopy, light-sheet microscopy, light-field microscopy (including, but not limited to the formats for light sheet microscopy referenced and described in the examples), and Fluorescence Talbot Microscopy (FTM).
  • FTM Fluorescence Talbot Microscopy
  • PARS, PACT, ePACT, autofluorescence masking, and related methods described herein could be used on any animal, and are in no way limited to those examples specifically set forth herein. Further, the methods described herein can be used for tissues and cells of organisms ranging from embryos to adults.
  • tissue stabilization and clearing methods described herein use gentle delivery of structural supportive hydrogels and removal of light obstructing lipids through, importantly, either passive clearing (PACT) or through the vasculature of intact post-mortem organisms (PARS).
  • PACT passive clearing
  • PARS vasculature of intact post-mortem organisms
  • the hydrogel mesh itself is transparent and secures proteins and nucleic acids into place so they can be later detected with fluorescent labels under a microscope.
  • tissue clearing protocols available that combine the use of "chemical” clearing methods (i.e. the modification and/or removal of a tissue components) and “optical” clearing methods (i.e.
  • PACT and PARS are notable for their versatility in preparing a variety of tissue types for high-resolution imaging at depth.
  • the PACT hydrogel formulation and clearing process is modified to render difficult-to-image tissues transparent (e.g. PACT-deCAL, for PACT delipidation and decalcification of bone, as described in PCT/US2015/059600), to expand tissues for better separation of compact structures (e.g. ePACT, for PACT-based expansion clearing of dense cells or projections), and to preserve tissue integrity in fragile samples through varying the degree of paraformaldehyde-tissue crosslinking.
  • PARS is positioned to tackle a variety of scientific problems that would benefit from a comprehensive, whole-body view of gene expression patterns, cellular organization, and/or structural composition.
  • PACT- or PARS- based preparation and clearing of tissue can preserve the signal from native fluorescent proteins (Fig. 1A) and improve the efficacy of post-clearing immunofluorescent labeling (Fig. 1G). Fluorescence signal intensity is also maintained through month-long storage periods post fixation.
  • Other brain-specific tissue clearing protocols (Table 2) have at least one functional drawback, such as incompatibility with endogenous fluorescent labels.
  • the procedures described below include 7 main stages: tissue preparation (steps 1-5); formation of a tissue-hydrogel matrix (step 6); tissue clearing (step 7- 8); staining (steps 9, optional); enhancement of optical clarity using RIMS (refractive index matching solutions; steps 10-13); imaging (step 14); and image visualization and analysis (steps 15-17).
  • tissue preparation steps 1-5
  • tissue clearing step 7- 8
  • staining steps 9, optional
  • enhancement of optical clarity using RIMS reffractive index matching solutions
  • steps 10-13 imaging
  • imaging step 14
  • image visualization and analysis steps 15-17.
  • PACT and PARS including their respective tissue-specific variations (PACT-deCAL, PARS-CSF)
  • PACT-deCAL, PARS-CSF tissue-specific variations
  • the decision to proceed with PACT or PARS is generally made prior to commencing the procedure.
  • the primary goal is to stabilize soft and/or amorphous samples (e.g. thymus, spleen, pancreas) for experimentation and sectioning,
  • steps 1-5) require that the scientist be approved for working with laboratory animals and/or possess the surgical dexterity to establish an intravascular route for delivery of PARS reagents.
  • the scientist should be proficient in conducting animal euthanasia via transcardial perfusion and/or basic animal surgical techniques and practices.
  • the composition of the PARS/PACT hydrogel monomer solution bares a few major changes from the originally proposed CLARITY hydrogel, which consists of 4% acrylamide, 4% PFA, and 0.05% bis-acrylamide (A4P4B0.05).
  • the crosslinker bis- acrylamide should be excluded from the PARS hydrogel formulation to prevent hydrogel blockages in vasculature and perfusion lines. Its exclusion from the PACT hydrogel as well, and the reduced exposure of tissues to PFA in both protocols accelerates clearing and immunolabeling steps.
  • the resulting minimal polymeric scaffold of the PARS and PACT tissue-hydrogel matrices suffices not only to retain tissue proteins (Fig. 3 A, Fig. 11) and stabilize tissue macrostructure during clearing, but it also allows SDS micelles to diffuse more freely through tissue for efficient clearing (Fig. 4A, Fig. 11C, Fig. 7).
  • a lower crosslink density ensures that antibodies can better access tissue epitopes during immunolabeling (Fig. 4B-D, Fig. 12E).
  • tissue clearing protocols have aimed to render samples transparent via homogenizing the refractive indices (RI) of the various tissue components, and matching their RI with the lens and mounting set-up (e.g. glass coverslip interfaces). This has often been accomplished via exchanging the aqueous fraction of tissue (RI ⁇ 1.33) with a mounting medium of higher refractive index, which includes organic solvents such as BABB (RI ⁇ 1.53-1.57), dibenzyl ether (RI ⁇ 1.56), methyl salicylate (RI ⁇ 1.52-1.54), and 2,2'thiodiethanol (RI ⁇ 1.52); polyol and saturated sugar solutions such as glycerol (RI ⁇ 1.43-1.47), sucrose and fructose (RI ⁇ 1.49-1.50); and amides such as formamide (RI ⁇ 1.44) and urea (RI ⁇ 1.38).
  • organic solvents such as BABB (RI ⁇ 1.53-1.57), dibenzyl ether (RI ⁇ 1.56), methyl salicy
  • samples can be processed in parallel, and adjacent areas can be directed either to TEM or to A4P0-4 clearing to obtain both ultrastructural and volume information respectively.
  • the denaturing anionic detergent sodium dodecyl sulfate (SDS) used for lipid removal in PACT/PARS is also very effective in dissociating DNA from proteins (e.g. for cell nuclei removal) and disrupting extracellular matrices to facilitate protein removal (e.g. ionic interactions of SDS with membrane proteins allow for their removal and purification).
  • SDS solubilization of lipid bilayers via a micellar mechanism, is a slower process.
  • the CLARITY protocol featured a hydrogel monomer solution composed of 4% acrylamide, 4% PFA and 0.05% bis-acrylamide (A4P4B0.05), which confers dense tissue- hydrogel crosslinking.
  • the advanced CLARITY protocol suggests decreasing acrylamide concentrations to as low as 0.5% (A0.5P4B0.0125) when clearing is performed passively rather than with ETC-based rate enhancement.
  • A0.5P4B0.0125 acrylamide concentrations to as low as 0.5%
  • PACT and PARS tissues are infused with A4P0 monomer.
  • the inventors have not found the addition of bis-acrylamide to be beneficial in preventing protein loss (Fig.
  • the inventors describe two modes of detergent-based tissue clearing: passive lipid removal (PACT: step 6 option A for hydrogel permeation and embedding, step 7 option A for PACT clearing), and active delipidation (PARS: step 6 option B for hydrogel perfusion and embedding, step 7 option C for PARS clearing).
  • PACT passive lipid removal
  • PARS active delipidation
  • Several factors, including the chemical properties of the detergent solution, the pH of the detergent solution, and the tissue components to be extracted i.e., peptide, lipid, nucleic acid
  • the role of pH is elevated in scenarios, such as tissue clearing, where relatively high SDS concentrations (4-8% SDS) are employed.
  • a slightly basic clearing solution will help to counteract proton build-up at the negatively charged surface of SDS micelles.
  • a clearing solution that becomes too acidic has the potential to impair lipid extraction via disrupting the structure of the ionic micelles, as well as to encourage protein extraction via their denaturation and release from membranes.
  • Temperature represents a second important factor that influences the solubilization process, and in particular, the micellular composition.
  • SDS in aqueous medium the average micelle volume decreases but the total number of micelles increases as the temperature rises. It is hypothesized that smaller micelles may more readily diffuse through the tissue-hydrogel matrix, and so increasing the temperature of the clearing bath will accelerate lipid extraction. Higher temperatures (-50 °C), which may enhance clearing efficiency will promote protein denaturation, which has the potential to damage relevant protein epitopes or incur fluorescent protein signal loss. Thus, both PACT and PARS clearing steps are performed at 37 °C. To accelerate lipid extraction, the concentration of SDS is raised from 4% to 8% SDS relative to CLARITY, which has a similar effect as raising the clearing temperature.
  • PACT and PARS-prepared tissues are amenable to most standard histological techniques, including those which employ immunohistochemical, small-molecule, and fluorescent protein-based labels, as well as brightfield stains.
  • Small-molecule dyes such as nuclear stains rapidly distribute throughout thick tissue sections, such that hour-long to overnight incubations are sufficient for most samples.
  • Infusing and mounting cleared tissues in RIMS helps to minimize the mismatch between the refractive indices of the sample and the microscope objective.
  • This so-called "optical clearing" which is detailed in steps 10-13, greatly enhances the optical clarity of cleared samples (see Fig. 1A-E, Fig. 2B, Fig. 4, Fig. 5A, Fig. 5C-D, Fig. 7, Fig. 8C, Fig. 12, Fig.14, Fig. 15B).
  • RF S e.g. sRF S (see Fig. 1F-G), cRF S, glycerol dilution, FocusClear, Cargille Labs optical liquids, 2,2'- thiodiethanol).
  • RF S e.g. sRF S (see Fig. 1F-G)
  • cRF S glycerol dilution
  • FocusClear gille Labs optical liquids, 2,2'- thiodiethanol
  • the microscope set-up must be capable of acquiring high-resolution image stacks through thick, cleared samples.
  • an objective that has been optimized to the RI range of the RIMS-mounted tissue and immersion media (RI ⁇ 1.46-1.49) will minimize spherical aberrations, maximize lateral and axial resolution, and help to preserve fluorescent signal intensity while imaging through thick, cleared tissues.
  • Imaging cleared tissues via two-photon or confocal microscopy can generate extremely high resolution data sets.
  • these imaging modalities are time-consuming, particularly when scanning a large field of view at depth.
  • Light sheet fluorescence microscopy permits rapid scanning through comparatively large sample volumes, which alleviates the imaging bottleneck that can occur with the high-throughput preparation of cleared samples.
  • LSFM minimizes sample photobleaching, a major drawback in using point-scanning confocal systems to image large fluorescently labeled samples.
  • PACT and PARS provide a means for efficiently acquiring information on the spatial position of neurons within large tissue volumes at high resolution.
  • LSFM long-working-depth objectives and high-throughput imaging
  • CLARITY Optimized Light sheet Microscopy CLARITY Optimized Light sheet Microscopy
  • Fig. 8 the custom-made, economical system
  • PACT and PARS provide a means for efficiently acquiring information on the spatial position of neurons within large tissue volumes at high resolution.
  • these gigabytes or even terabytes of raw image data e.g. for a whole mouse brain at 25 ⁇ magnification
  • Many available software tools and image file formats were not designed with terra-scale datasets in mind and assume that entire image volumes fit in computer RAM.
  • Confocal and light sheet microscopes equipped with motorized stages usually support tiled acquisition, which is essential for imaging large volumes at cellular resolution. These tiles can then be aligned to pixel accuracy and blended together using microscope acquisition software: e.g. Leica Application Suite (Leica Microsystems) (see Bria, A. & Iannello, G. TeraStitcher - a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images.
  • Vaa3D i Stitch plugin see Yu, Y. & Peng, H. Automated high speed stitching of large 3D microscopic images in 2011 IEEE International Symposium. 238-241 (2011)
  • ImageJ stitching plugin see Preibisch, S., Saalfeld, S. & Tomancak, P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25, 1463-1465 (2009)
  • XuvTools see Emmenlauer, M. et al. XuvTools: free, fast and reliable stitching of large 3D datasets. J. Microsc.
  • CIDRE see Smith, K. et al. CIDRE: an illumination-correction method for optical microscopy. Nat. Methods 12, 404-406 (2015), and then to apply the estimated correction to each acquired image tile. This so-called “flat field” or shading correction from a reference image is often supported by acquisition software: e.g.
  • Image stacks can be visualized using commercial software such as Imaris (Bitplane), Amira (FEI), MetaMorph (Molecular Devices) and others (Zen (Zeiss), Leica Application Suite (Leica Microsystems), NIS Elements (Nikon Instruments), cell Sense (Olympus), MetaMorph (Molecular Devices), Volocity (PerkinElmer), Huygens (SVI), Arivis (see http://vision ⁇ dot>arivis ⁇ dot>com/en/arivis-Vision4D) (see Dominguez, E. et al. Noninvasive in vivo measurement of cardiac output in C57BL/6 mice using high frequency transthoracic ultrasound: evaluation of gender and body weight effects. Int. J.
  • BioImageXD see Kankaanpaa, P. et al. BioImageXD: an open, general-purpose and high-throughput image-processing platform. Nat. Methods 9, 683-689 (2012)), Vol View (Kitware, see http://www ⁇ dot>kitware ⁇ dot>com/opensource/volview ⁇ dot>html), or Bioview3D (see
  • Table 3 indicates which software tools support "out of core” visualization, utilizing read on demand, caching and multi-resolution representations to process and visualize datasets that are too large to fit in memory while preserving interactivity.
  • TeraFly see Bria, A. & Iannello, G.
  • TeraStitcher - a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images.
  • Morphometric Analysis Tracing of neurites can be carried out using plugins provided in general processing tools: e.g. Imaris Filament Tracer (BitPlane) (see Myatt, D. R., Hadlington, T., Ascoli, G. A. & Nasuto, S. J. Neuromantic - from semi-manual to semi-automatic reconstruction of neuron morphology. Front. Neuroinform. 6, 4 (2012)), Amira Skeletonization Plugin (FEI) (see Gleeson, P. et al. NeuroML: a language for describing data driven models of neurons and networks with a high degree of biological detail. PLoS Comput. Biol.
  • Imaris Filament Tracer (BitPlane) (see Myatt, D. R., Hadlington, T., Ascoli, G. A. & Nasuto, S. J. Neuromantic - from semi-manual to semi-automatic reconstruction of neuron morphology. Front. Neuroinform. 6,
  • Vaa3D-Neuron2 see Peng, H. et al. Virtual finger boosts three-dimensional imaging and microsurgery as well as terabyte volume image visualization and analysis. Nat. Commun. 5, 4342 (2014)); or via special purpose software: e.g. Neurolucida (see Glaser, J. R. & Glaser, E. M. Neuron imaging with Neurolucida ⁇ a PC-based system for image combining microscopy. Comput. Med. Imaging Graph. 14, 307-317 (1990)), neuTube (see Feng, L., Zhao, T. & Kim, J.
  • neuTube 1.0 a New Design for Efficient Neuron Reconstruction Software Based on the SWC Format, eneuro, DOI: 10.1523/ENEURO.0049-1514.2014 (2015)), Neural Circuit Tracer (see Chothani, P., Mehta, V. & Stepanyants, A. Automated tracing of neurites from light microscopy stacks of images. Neuroinformatics 9, 263-278 (2011)), flNeuronTool (see Ming, X. et al. Rapid reconstruction of 3D neuronal morphology from light microscopy images with augmented rayburst sampling. PLoS One 8, DOI: 10.1371/journal.
  • NeuroML a language for describing data driven models of neurons and networks with a high degree of biological detail.
  • L- Measure a web-accessible tool for the analysis, comparison and search of digital reconstructions of neuronal morphologies. Nat. Protoc. 3, 866-876 (2008)), assembly and simulation of biophysical models (see Gleeson, P., Steuber, V. & Silver, R. A. neuroConstruct: A tool for modeling networks of neurons in 3D space. Neuron 54, 219-235 (2007)) and deposition in online searchable databases (e.g. http://www ⁇ dot>neuromorpho ⁇ dot>org/).
  • PACT, PARS, and RIMS clear a variety of tissues, from laboratory mice and rats (organs and adult whole-bodies) to human primates (Fig. 1F-G, tumor biopsy) and are compatible with endogenous-fluorescence, immunohistochemistry, long-term sample storage, smFISH, and microscopy with cellular and subcellular resolution.
  • PARS the clearing and staining of large, isolated whole-organs when the vasculature is preserved during organ excision.
  • Akin to paraffin embedding the increased rigidity of hydrogel-embedded, uncleared samples can allow unstructured soft tissues (e.g. pancreas, thymus) and amorphous biological samples (e.g.
  • tissue-hydrogel hybrids are PACT- or PARS-cleared rather than thin-sectioned for imaging, whole organs and thick tissue blocks become amenable to visualization with modern microscopy methods such as light sheet fluorescence microscopy (LSFM, which rapidly scans large sample volumes, thereby minimizing photobleaching but maximizing the phenotypic content within the image stack) and super-resolution microscopy.
  • LSFM light sheet fluorescence microscopy
  • PARS and PACT enable detailed structural information from peripheral tissue and organ samples to be obtained, aiding in the study of distinct cellular populations/environments within their unsevered tissue milieu.
  • stem cell niches that are embedded within relevant tissue environments can be studied, such as the intestinal stem cells located in small intestinal crypts and within the bone marrow niche.
  • Tumor architecture and morphology can be mapped, including tumor margins, tumor vascularization, cellular heterogeneity, and metastatic foci across the entire organism, for both research and diagnostic purposes.
  • Whole-body optical clearing by PARS and imaging could facilitate obtaining better peripheral nerve maps which can then facilitate understanding of the neural processing that accompanies peripheral nerve/organ function and dysfunction.
  • PARS may also facilitate whole-body screening of therapeutics for off- target and on-target binding, and for imaging the biodistribution of administered agents as a method for the qualitative determination of their pharmacokinetic-pharmacodynamic (PK/PD) properties.
  • PARS can be employed to expedite the slow, labor-intensive process of screening novel viral vector variants for specific tropism characteristics. Typically researchers perform conventional tissue slicing and histology on numerous tissues across multiple samples, an exceedingly laborious process. Whole-body screening through PARS can improve throughput and reduce the risk of sampling errors.
  • tissue stabilization and lipid removal allow for rapid phenotyping of whole-organs and whole-organisms and therefore could advance biomedical research with respect to the study of changing tissue pathology during aging or during disease progression.
  • One obstacle to studying the progression of cell death that occurs during neurodegeneration e.g. in Parkinson's, Alzheimer's, epilepsy, stroke) is the inability to visualize cells that have already died and have been removed by macrophages before the tissue was dissected for histological analysis.
  • a similar cellular mapping confound exists in ablation experiments, wherein toxins are used to damage cells for studies that aim to causally link the function of a defined neuronal population (compact or sparsely distributed) to brain activity and behavior.
  • tissue-hydrogel microstructure including the ordering of monomelic units within a polymerized hydrogel, the degree of crosslinking, and the mechanical rigidity of the embedded tissue.
  • tissue clearing as detergent gradually solubilizes and extracts tissue biomacromolecules, not only can water migrate into this additional space in the tissue-hydrogel matrix, but also there is less mechanical resistance from tissue components to polymer swelling as water continues to diffuse in.
  • tissue size changes in mounting media Upon their initial immersion in RIMS, tissue samples contract during the first hour (-20% for A4P0-embedded coronal rodent brain sections), followed by a gradual rebound back to their pre-RFMS size. Imaging during this time window should be avoided as these slight size fluctuations could introduce apparent tissue deformities or sample drift issues during image acquisition. With adequate equilibration in REVIS (e.g. hours to days, depending on sample size, tissue permeability, etc.), sample size and transparency will reach a steady- state for high-resolution, deep imaging.
  • REVIS e.g. hours to days, depending on sample size, tissue permeability, etc.
  • swelling - if isotropic can be advantageous.
  • dense cell populations can be distributed spatially for cell counting or for analyzing local cell contacts (Fig.13, Supplementary Methods); likewise, dense cell and/or fiber tracts, such as the corpus callosum, the spinal cord, and individual muscles may be expanded for easier anatomical study.
  • scientists may quickly adjust the overall volume occupied by the hydrogel-embedded tissues, shrinking tissues to fit within the working distance of an objective, or swelling tissues for facile high-resolution imaging of diffraction-limited spots (see Dodt, H. U. Microscopy. The superresolved brain. Science 347, 474-475 (2015)).
  • tissue deformation is expected with all tissue clearing protocols (see examples in Table 2), wherein the tendency for tissue to expand and/or shrink moderately during sample clearing and/or mounting is frequently noted. Whether these volume changes cause structural damage that would confound the interpretation of sample images is widely debated. Although some tissue swelling has been observed during PACT and PARS clearing, tissues subsequently contract to approximately their original size in RIMS media. Although difficult to test exhaustively by individual efforts, the net impact of these changes on overall cellular architecture appears to be minimal, as demonstrated by the preservation of fine cellular morphology, including that of fragile dendritic processes, across a range of tissue types. However, such changes in tissue volume do potentially complicate the process of image registration.
  • tissue stains that can help with registration include: nissl or Golgi stain for the brain; membrane and organelle stains such as H&E stain for dual hematoxylin-based nucleic acid staining and eosin-labeling of red blood cells, cytoplasmic material, cell membranes, and extracellular structures and protein; fuchsin to stain collagen, smooth muscle, or mitochondria.
  • the image data file sizes will be tera- scale; thus, it is important to employ a computational workstation with substantial RAM (this will be highly dependent on the individual software requirements, user-specific variables such as the average file size and the desired image analysis capabilities.
  • Our experience showed that as much as 64-256 GB might be needed, depending on data and analysis type), multi-core CPUs and an excellent graphics card (e.g. Windows platform: AMD Radeon R9 290X 4.0 GB; MAC platform: AMD FirePro D700 6 GB).
  • REAGENTS Sample to be imaged This protocol describes imaging of brain and body samples prepared from wild-type mice (C57BL/6N and FVB/N, both males and females), Thyl-YFP mice (line H), and TH-cre rats.
  • Sodium nitrite (Sigma-Aldrich, cat. no. 237213-500G). As a vasodilator, 0.5% sodium nitrite is added to the heparinized saline perfusion buffer to facilitate thorough blood removal from vasculature and perfusion ease. Alternatively, nitroglycerin may be substituted for sodium nitrite.
  • RIMS Refractive Index Matching Solution
  • Sodium azide (Fisher Scientific, cat. no. 71448-16) To prevent microbial growth, sodium azide should be added to all mounting medias (RIMS and sRIMS), as well as to all immunostaining dilutions and wash buffers that are used in extended incubations.
  • Glycerol (87%, vol/vol): Prepare 80-90% (vol/vol) glycerol (Sigma-Aldrich, cat. no.
  • Aminosilane-treated coverslips ((3-Aminopropyl) triethoxysilane, Sigma-Aldrich, cat. no.
  • Nitrogen gas supply any
  • PTFE tubing McMaster-Carr
  • Masterflex L/S 14 tubing Cold Palmer
  • Rubber Stoppers 31.4 mm diameter (Spectrum Chemical Mfg. Corp, cat. no.142-55179) or TwistitTM Rubber stopper size 6 (Fisher Scientific, cat. no. 14-13 ID; Sigma-Aldrich, cat. no. Z164364; eBay, various)
  • Air-Tite Vet premium hypodermic needles 22 G x 4", (Air-Tite Products Co., Lot: 14- 11563, SKU N224)
  • Peristaltic pump or circulator any, e.g. Cole Palmer Masterflex L/S, cat. no. 77800-60; or Cole Palmer Masterflex L/S Easy Load II head and pump drive, cat. nos. 77200-62 and 7557-12
  • Peristaltic pump or circulator any, e.g. Cole Palmer Masterflex L/S, cat. no. 77800-60; or Cole Palmer Masterflex L/S Easy Load II head and pump drive, cat. nos. 77200-62 and 7557-12
  • Silicon aquarium sealant any, e.g. 3MTM Marine Grade Silicone Sealant Clear, PN08019)
  • Vacuum grease (Sigma-Aldrich, cat. no. Z273554)
  • Refractometer (Rei chert AR200 Digital Handheld Refractometer, cat. no.
  • Microscope objectives for thick-section imaging such as the CLARITY-optimized objectives now produced by major microscopy companies, including Leica and Olympus.
  • Image handling software such as Imaris (Bitplane) (see Uygun, B. E. et al.
  • l x PBS For 10 L of the 10x stock, dissolve 800 g NaCl, 20 g KC1, 144 g Na 2 HP0 4 dihydrate, 24 g KH 2 P0 4 in 8 L of distilled water. Add additional water to a total volume of 10 L; sterile filter or autoclave. Upon dilution to l x PBS, the pH should approach 7.4. The pH may be adjusted with hydrochloric acid or sodium hydroxide, as needed. The resultant l x PBS should have a final concentration of 10 mM P0 4 3" , 137 mM NaCl, and 2.7 mM KC1. Alternatively, purchase 10x PBS pre-made solution (any, such as Lonza, cat. no. 17-517Q) from a commercial supplier.
  • PBST may be stored at RT for a few months when sterile-filtered; vortex or stir on a stirplate for several minutes prior to use.
  • Boric Acid Buffer (BB)
  • BBT boric acid wash buffer
  • Triton X-100 (vol/vol), pH 8.5
  • dilute the 1 M boric acid stock to 0.2 M boric acid in dd H 2 0, adding 1 ml of Triton X-100 per litre of BBT and stirring on a stirplate for 10 minutes.
  • BBT may be stored at RT for several weeks, barring contamination; vortex or stir on a stirplate for several minutes prior to use.
  • Hydrogel monomer solutions must remain cold prior to use to prevent premature polymerization; we generally prepare solutions fresh on ice, but they may be stored short-term (several hours) at 4 °C or on ice, or long-term (several months) at -20 °C, protected from light.
  • hydrogel monomer formulations have been tested, including combinations of 2% or 4% acrylamide with 0% or 4% PFA and/or 0.05%-0.25% bis-acrylamide. It was determined that A4P0 without bis-acrylamide granted rapid clearing and good antibody penetration during IHC without compromising the macromolecular content and cellular structure of tissue samples. In comparison to CLARITY, 4% PFA was eliminated from the hydrogel monomer solution, however, thorough PFA-mediated crosslinking of tissue proteins was ensured prior to hydrogel monomer incubation via 4% PFA transcardial perfusion and 4% PFA post-fixation steps.
  • PACT and PARS tissue clearing is accomplished via exposing tissue to an 8% SDS detergent solution, or in special cases (PACT-deCAL, ePACT), to a 10% SDS detergent solution. All initial validation of PACT and PARS was performed using a range of SDS concentrations (4%-16% SDS), prepared in a range of buffers (l x PBS at pH 7.5, l PBS at pH 8.0 (for PACT-deCAL), l x PBS at pH 8.5, and in 0.2 M sodium borate buffer at pH 8.5).
  • an appropriate antimicrobial agent should be added to the buffer (e.g. a final concentration of 0.01%) sodium azide in buffer solutions).
  • a final concentration of 0.01% sodium azide in buffer solutions.
  • the dilution of antibodies used in PACT and PARS will be highly dependent on, among other things, the quality of the antibody, the size and tissue type of the sample to be labeled, the cellular location and concentration (i.e., expression level) of the target biomolecule, etc.
  • Glass vacutainers work well for degassing and hydrogel-embedding small rodent organs and tissue samples. However, for rat whole-brains and larger tissue samples, a larger container is sometimes useful.
  • One solution is to purchase rubber stoppers that are compatible with 50 ml conical tubes and replace the conical screw-cap with an air-tight rubber stopper during degassing and hydrogel polymerization steps (see Fig. 2).
  • a PARS chamber was constructed using components that are readily found in most biological research laboratories (see Fig. 6).
  • the necessary components of a PARS set-up are: 1) a feeding needle catheter to deliver PARS reagents to vasculature, 2) a perfusate catch-basin (pipette box) where recirculating PARS reagents may pool once they exit the vasculature, 3) Tygon tubing threaded through a peristaltic pump so that pooling reagents may be collected from the catch-basin and recirculated back into a subject's vasculature, 4) Luer-to-tubing couplers, and finally 5) a Ziploc bag to contain the entire PARS chamber set-up.
  • the PARS chamber To construct the PARS chamber, drill two 1 ⁇ 2" holes into the front and one 1 ⁇ 2" hole into the left side wall of an empty 1000 ⁇ pipette tip box. The holes are drilled just below the tip wafer (in Fig 6, the holes are ⁇ 2 cm below the top rim). Next, snap 1 ⁇ 2" ⁇ 1 ⁇ 2" barbed connectors into each of the drilled holes. The outflow line will circulate solvents from the pipette box chamber to the 3-way stopcock. To join the outflow line to a 3-way stopcock, use a 10 cm piece of Tygon tubing and connect one end to the inner left side 1 ⁇ 2" x 1 ⁇ 2" barbed connector and tape the other end to the inside bottom of the pipette tip box.
  • a line linking the outflow line back to the pipette tip box is connected by joining a piece of 15 cm Tygon tubing to the 3-way stopcock with a full thread 3 ⁇ 4 2 " barbed female Luer to the outer left front 1 ⁇ 2" ⁇ 1 ⁇ 2" barbed connector.
  • This line is continued inside the pipette tip box and taped to the bottom.
  • thread the inflow line through the top-left corner of the tip wafer and connect it to a feeding tube with a 1 ⁇ 2" barbed male slip Luer.
  • SDS and salt precipitate will begin to accumulate within these narrow lines over time. It is important to flush the lines (e.g. with dd H 2 0) between subjects, and to replace occluded lines with new Tygon tubing (e.g. after every few subjects).
  • the chamber must be enclosed inside a Ziploc freezer bag. To do this, disconnect the outer Tygon tubing that connects to the barbed connectors of the pipette tip box and puncture three holes into the Ziploc bag to accommodate the 1 ⁇ 2" ⁇ 1 ⁇ 2" barbed connectors. Reconnect the Tygon tubing to their original 1 ⁇ 2" x 1 ⁇ 2" barbed connector. To connect a vacuum line to this bagged PARS box for withdrawing oxygen, tape a female Luer tee onto the lid of the pipette box and puncture one hole through the Ziploc. Finally, make the Ziploc airtight by placing clay around the punctured regions in the Ziploc.
  • a 1000 ⁇ tip box has a volume of approximately 750 ml.
  • 200-300 ml solution may be placed in the pipet box for recirculation without risk of the pipet box overflowing, or solution splashing out during its transport.
  • a 200 ⁇ tip box may be used to construct the PARS chamber; only 100 ml reagent is necessary to fill such chamber ⁇ 1 ⁇ 2 full (see Fig. 6).
  • the light sheet microscope we use was built based on the laser-scanning single-side illumination method (see Huisken, J. & Stainier, D. Y. R. Selective plane illumination microscopy techniques in developmental biology. Development 136, 1963-1975 (2009)).
  • objectives that offer a long working distance of eight millimeters while maintaining numerical aperture (N.A.) of 1.0 (e.g. CLARITY objectives).
  • N.A. numerical aperture
  • CLARITY objectives e.g. CLARITY objectives.
  • the system described below provides a cost-effective and relatively easy-to-replicate alternative to COLM (see Tomer, R., Ye, L., Hsueh, B. & Deisseroth, K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat. Protoc.
  • the microscope is built onto a 4 ⁇ 6 foot optical table (see Fig. 8B).
  • the various lasers are combined using dichroic filters, to one beam which is then expanded using a Galilean telescope, and shaped with an iris to match the Galvanometer scanner mirror size (6 mm in diameter).
  • the Galvanometer scanner coupled with a f-theta lens, is then used to generate the scanning light sheet, which is projected to the sample holder using two achromatic doublet lenses.
  • the resulting light sheet has a full-width-half-maximum of 5-7 ⁇ , depending on the wavelength of illumination (473-632 nm).
  • the detection objective lens (25 x, 1.0 N.A. CLARITY objective, Olympus) is inserted into the immersion chamber.
  • the immersion chamber is printed using a 3D printer (ABS plastic) and is filled with glycerol to prevent evaporation-induced aberrations in RIMS medium.
  • magnification is used to digitally sample the acquired images in lieu with the high NA of the detection lens.
  • a camera with a light sheet mode feature is used (Zyla 4.2 sCMOS, Andor), where the scanning light sheet and the camera pixel readout are synchronized to improve the signal to noise ratio (see Baumgart, E. & Kubitscheck, U. Scanned light sheet microscopy with confocal slit detection. Opt. Express 20, 21805-21814 (2012)).
  • the sample In order to rapidly scan large volumes, the sample is constantly translated using a xyz- theta stage, while the light sheet remains stationary.
  • the xyz-theta stage is mounted on heavy-duty stainless steel bars to prevent sample vibration during data acquisition.
  • To connect the sample holder to the xyz-theta stage we first place the sample in a quartz cuvette filled with RIMS solution. The cuvette is then attached to a 3D printed cap that has a Luer lock female connector mounted on top, and laboratory parafilm is used to seal the connector- cuvette interface. The sample holder is then attached to a xyz-theta stage via the Luer lock male connector.
  • Tissue Preparation for PACT and PARS 1. Prepare the perfusion and hydrogel monomer solutions, including l x PBS containing 0.5% NaN0 2 (optional, for vasodilation) and 10 U/ml heparin (optional, for anticoagulation) (hPBS), 4% PFA in l PBS, and the A4P0 hydrogel solution. 4% PFA should be prepared fresh. A4P0 may be prepared fresh or stored at -20 °C until use. For the latter, thaw A4P0 on ice prior to use. Perfusion solutions should be ice-cold. Discard PFA and hydrogel stock solutions if precipitate is observed.
  • PFA perfusate should exit the right atrium and drain into the pipette box. This perfusate is then drawn up through tubing and recirculated through the subject. If necessary, add additional 4% PFA to the pipette box so that there is always enough PFA pooled in the pipette box to be recirculated through the tubing and subject vasculature.
  • the amount of solution required for continuous recirculation will depend on the individual set-up (size of pipette box, liquid volume to fill tubing, evaporation from PARS chamber, species of subject, etc.).
  • perfuse l x PBS for 45 minutes at RT.
  • tissue components with hydrogel monomers are crucial as it ensures that SDS micelles preferentially solubilize and remove tissue lipids during clearing.
  • a minimal acryl ami de-based network which supports more rapid clearing, was nevertheless sufficient for stabilizing proteins and nucleic acid.
  • the hydrogel-infused tissue should be carried through a rigorous degassing step.
  • tissue-hydrogel matrix which imparts superior tissue crosslinking and only minor slowing of clearing and immunostaining steps.
  • the A4P0 solution With rigorous degassing, the A4P0 solution will form a hydrogel the consistency of honey or tacky silicon sealant that is somewhat difficult to remove from the tissue. With 1- minute nitrogen gas exchange, the A4P0 solution will form a hydrogel the consistency of syrup that may be poured off easily.
  • Hydrogel-embedded samples will have increased rigidity and structural integrity, and indeed this may be the primary goal for some users.
  • hydrogel-embedded soft tissues e.g. pancreas, spleen, thymus
  • amorphous biological samples e.g. sputum, mucus, organoid cell masses
  • the nitrogen gas can be bubbled directly into 200 ml of 0.25% VA-044 initiator in l x PBS already loaded into the pipette box while slowly degassing the chamber as shown in Figure 71. This requires disassembly of the PARS chamber to make an airtight environment with the Ziploc.
  • the rate of tissue clearing depends on several parameters, including the inherent structural and biochemical properties of the tissue sample, the volume of the tissue sample, the hydrogel pore size and the density of tissue-hydrogel crosslinking, and the clearing set-up (SDS concentration, incubation temperature, pH of clearing buffer). It is important for users to determine the clearing parameters for their specific tissue samples empirically, using these guidelines as a starting point for further optimization. Likewise, because the rate of clearing may vary greatly, tissues embedded in minimal hydrogel monomer compositions, such as the A4P0 hydrogel suggested here, are more susceptible to deteriorating when samples are left unattended in SDS.
  • tissue-hydrogel sample Place each tissue-hydrogel sample into a 50 ml conical containing clearing buffer; gently rock the sample in a 37-42 °C shaking waterbath until tissue is optically transparent.
  • tissue mounting in RIMS will lend an additional degree of optical transparency to tissues, it is crucial to remove tissues from SDS when the majority of tissue, or the portion of interest, is transparent, even if some regions appear under-cleared. This will help to ensure that the tissue macromolecular content is preserved.
  • the following steps have been optimized for clearing the dissected tibia of an adult mouse. It is important to tune the parameters of PACT-deCAL, such as the duration of bone incubations in clearing and decalcifying buffers, and the concentration of EDTA. Temperature fluctuations (e.g. from performing SDS or EDTA buffer changes with RT solutions rather than with pre-warmed 37 °C solutions, or from a waterbath that is unable to maintain a constant 37 °C environment) may adversely affect bone tissue morphology. i. Place each bone-hydrogel sample into a 50 ml conical containing 10% SDS-PBS (pH 8.0) clearing buffer; gently rock the sample in a 37 °C shaking waterbath for 2 days.
  • SDS-PBS pH 8.0
  • hydrogel-perfused whole organs may be excised following hydrogel polymerization (step 6B) or following the initiation of PARS clearing (step 7C(i)), and then stored in 4-8% SDS at 37 °C for up to one month.
  • This allows whole organs to clear slowly during storage; their clearing progress must be monitored, albeit infrequently (e.g. weekly), as smaller, porous organs may become completely transparent in less than one month, wherein they should be transferred into l x PBS (or PBST or BBT) containing 0.01% sodium azide at RT.
  • step 7A Ensure that all storage solutions contain 0.01% sodium azide, and when ready to resume processing tissue, follow the protocol steps for PACT-based clearing and labeling (step 7A). Although this PACT-based clearing of PARS prepared whole organs conserves reagents and minimizes the constant oversight required during PARS clearing, it negates the principal benefits of PARS: efficiency and uniform sample preparation.
  • the sample can be continuously perfused for up to 2 weeks until all desired organs have cleared, even if some organs appear clear within the first 24-48 hours.
  • the 1-2 semi-opaque excised organs are transferred into 8% SDS to finish clearing via PACT (see step 7A), while the organs that cleared more rapidly are immediately promoted to passive immunostaining (optional) and mounting (step 10) without further delay.
  • perfuse 8 buffer changes of 200 ml BBT or
  • the hydrogel matrix will be required to support the cleared tissue for several rounds of washing and multi-day incubations with gentle shaking. If the already delicate tissue-hydrogel matrix seems precariously fragile after clearing (this usually only occurs with thin-sectioned tissue), it is advisable to repeat the hydrogel embedding and polymerization steps (step 5 option A). This will stabilize tissue architecture during immunolabeling, prevent tissue loss or disintegration, and counteract expansion in mounting media. For cleared samples that will not undergo any immunohistochemical labeling steps prior to imaging, skip steps 8-9. Single-cell Phenotyping of Cleared Tissues
  • PACT and PARS prepared tissues are amenable to most standard immunohistochemical protocols; a list of validated small-molecule dyes, primary antibodies, and secondary fluorescent labels is provided in Table 4.
  • This PARS -histology protocol is sufficient to label molecular targets in the peripheral organs of mice and rats, with antibody amounts adjusted for body size. Individual users may need to adjust the incubation times and/or lengthen wash steps.
  • 9. Prepare the primary antibody cocktail in IHC buffer. An antibody dilution of 1 :200-400 is recommended, however a more or less concentrated antibody dilution may be required, depending on the tissue identity and bimolecular target.
  • the duration of primary antibody incubation must be determined on a case-specific basis (see antibody penetration guidelines, Fig. 4D). It is highly recommended to use smaller antibody formats for thick-tissue staining, when available.
  • A4P0-embedded rodent brain tissue a full IgG will penetrate approximately 500 ⁇ over a 3-day incubation at RT with shaking. This length of time is often sufficient for 1 mm tissue slices if the tissue can be imaged from either side.
  • a full IgG will penetrate approximately 200 ⁇ over a 3 -day incubation at RT with shaking.
  • RI refractive index
  • RIMS For imaging thick tissue using immersion objectives corrected for immersion media with a refractive index between 1.38-1.42, it is sometimes beneficial to match the refractive index of RFMS to that of the immersion media.
  • RIMS when using the LD-Plan Apochromat 20x 1.0 N.A. Scale objective (Zeiss), RIMS with RI ⁇ 1.42 will help to reduce image distortion in the Z-direction.
  • Samples may be stored long-term ( ⁇ 3 months) in RIMS.
  • RIMS-submerged samples should be kept in an airtight container at RT and protected from light.
  • samples may be mounted in cRIMS; store in a dry, air-tight container.
  • cleared tissue Upon RIMS immersion, cleared tissue will shrink over the course of a few hours (e.g.
  • RIMS outperforms sREVIS in our hands
  • the primary ingredient of sRIMS - sorbitol not only offers a cost advantage over HistodenzTM, but it is also commonly available in research laboratories owing to its broad use as a cell culture reagent.
  • sREVIS grants superior imaging resolution over glycerol.
  • RIMS or other mounting media be prepared with 0.01% sodium azide to prevent microbial growth in mounted tissue. Limit the number of air bubbles in sealed slides.
  • Auto Z provides an automatic gradual adjustment of the detector gain, amplifier offset, amplifier gain, and laser intensity setting between the first and last optical slice of a Z Stack. This will help to insure that signal intensity is uniform throughout the sample since even clear tissue will scatter at depth.
  • viii Set the image acquisition parameters (e.g. laser power, scan depth and the scan resolution in Z) and initiate the acquisition sequence.
  • image acquisition parameters e.g. laser power, scan depth and the scan resolution in Z
  • the 3D view window will now display the whole image dataset and progressively load in higher resolution sub-volumes as the user zooms in to particular parts of the image. Utilize Vaa3D's color map, annotation and analysis tools on selected sub-volumes.
  • Vaa3D TeraConvert plugin to convert an already stitched image to tiled, multi-res format (steps (i)-(ii)) and then use the Vaa3D TeraFly plugin to visualize the resulting image by following steps (iii)-(iv).
  • the 3D view window will now display the whole image dataset and progressively load in higher resolution sub-volumes as the user zooms in to particular parts of the image. Utilize Vaa3D's color map, annotation and analysis tools on selected sub-volumes.
  • a typical workflow is to first run automated tracing to generate initial estimates of morphology and then perform more detailed semi-automated editing to refine the tracing.
  • Automated tracing is computationally intensive so it is essential to restrict processing to small regions-of-interest (ROIs) or cropped out sub-volumes and manually merge the traces afterwards.
  • ROIs regions-of-interest
  • semi-automated and manual editing of traces can be greatly accelerated by taking time to learn keyboard shortcuts for a given software tool rather than clicking on graphical user interface elements such as menus or buttons.
  • neuTube includes a fully automatic tracing option but we found a semi-automatic tracing approach (which requires 1-2 clicks per neurite) to be faster and more stable.
  • PACT, PARS and RIMS collectively form a tissue clearing toolkit that is versatile, user-friendly and sample-friendly across tissue types.
  • Fig. 1A-E rodent
  • Fig. 1F-G human tissue samples alike, whether through the visualization of natively expressed fluorescent markers (Fig. 1 A-E, Fig. 5C, Fig. 8C, and Fig. 12D-E) or through immunolabeling whole-organ and thick tissue samples (Fig. 1F-G, Fig. 4B-C, Fig. 5D, Fig. 6A, and Fig. 15B) after clearing.
  • RIMS also serves to preserve the molecular content of mounted samples: no protein was measured to leach out of mounted samples after a one week incubation and YFP fluorescence was readily detected in cleared samples that were stored in RIMS for 3 months.
  • the enhanced optical transparency of delipidized and refractive-index matched tissues permits high-resolution detection of endogenously expressed fluorescent proteins, antibody-labeled proteins, and nucleic acid transcripts at the single molecule level (FISH), usually with similar intensity and lower background signals than are seen in uncleared tissues (Fig. 5C-D, confocal images for control versus cleared samples; Fig. 13).
  • camelid nanobodies Fig. 15
  • protein affinity tags i.e., SNAP- tag
  • Halo-tag see Los, G. V. et al. HatoTag: A novel protein labeling technology for cell imaging and protein analysis.
  • CLIP -tag see Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128-136 (2008)
  • TMP-tag see Miller, L. W., Cai, Y.
  • a frame rate of 45 frames per second shows -10-100 times improvement in image acquisition speeds and thus allows for rapid imaging of large cleared samples, in addition to its recognized utility for live-cell imaging (see Keller, P. J. & Ahrens, M. B. Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy. Neuron 85, 462-483 (2015)).
  • light sheet microscopy significantly reduces photobleaching (1/300-1/5000 photon energy exposure), which is critical for imaging dim samples and especially for conducting smFISH experiments, where the 20-100 single fluorophore-labeled probes are used to visualize individual transcripts.
  • NPS neuronal positioning system
  • biofilms characterizing samples (e.g. PACT-hydrogel formulated biofilm structure and the interaction of with paraformaldehyde and/or bis- different cellular layers), the acrylamide) so that bacterial colonies are heterogeneity and distribution of retained in tissue/biofilm samples during microbes that occupy the same niche clearing
  • DTI Diffusion Tensor Imaging
  • tissue macromolecular components e.g. lipids, heme
  • IHC Instrumental Component Identities: Identities: Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Spectra-Specific Specification, some restrictions in immunofluorescence (e.g. rapid signal decay) and/or some reservations about harsh tissue treatments that may adversely affect tissue integrity or labeling; IHC/F: Compatible with IHC and immunofluorescent labeling, validated for (> 0.5 mm) depth of antibody penetration and for a wide range of fluorophore wavelengths; IHC:
  • BABB a mixture of benzyl-alcohol and benzyl-benzoate
  • Image Analysis and Visualization Tools A list of image analysis tools appropriate for processing cleared tissue volumes including functionality for stitching, visualization, and tracing.
  • Guinea pig anti-insulin DAKO (A0564) Carpinteria, CA
  • Atto fluorescent dyes that possess an NHS ester moiety (available from Sigma Aldrich) may be conjugated to the primary antibody; this eliminates the need to perform a secondary antibody incubation when imaging tissues via fluorescence microscopy.
  • IgG available as Cy2 c , West Grove, PA
  • Cyanine dyes are traditionally better able to withstand dehydration and embedding in nonpolar, plastic media, whereas DyLight and Alexa Fluor® dyes are perceived as brighter than Cyanine dyes in aqueous media. Both seem to work well in labeling thick, cleared tissue samples.
  • Transcardial Incomplete Catheter is not stably Use a single suture (loop perfusion (Step 3, exsanguination, or placed in heart in order thread around aorta) or clip to
  • HM solution large tissue samples such as embedding (Step unnecessarily fragile throughout tissue whole rat organs in hydrogel 6) monomer for > 12 hours so that the monomer may fully penetrate the tissue.
  • Tissue is structurally Consider including PFA (1- fragile or delicate 4%) in HM formulation for subsequent sample preparations; extend post- fixation step.
  • Embedded tissue or Insufficient density of Increase the concentration of biological sample is too tissue crosslinking PFA (1-4%) and/or include fragile for non-clearing bisacrylamide (0.05%) in the applications (e.g. thin- hydrogel monomer sectioning and imaging) formulation.
  • Tissue clearing Clearing rate appears to Clearing may slow Buffer-exchange the clearing (Step 7) slow down before the down as the clearing solution,
  • Tissue appears to Bacterial Buffer-exchange the clearing degrade contamination solution, adding 0.01%-0.05% sodium azide to PBS-based clearing solutions.
  • Tissue becomes white Salts and, in particular and nearly opaque residual SDS will precipitate upon transfer to 4 °C in tissue if it is moved to 4
  • a specific organ does Vasculature becomes Identify and try to fix not clear well via compromised during leakages in the vasculature; if whole-body PARS clearing process not successful, tie off the major vessels supplying that organ, excise the organ for PACT clearing, and continue to perform PARS clearing with the remaining body. Starting over with a new PARS preparation should only be used as the last resort. Poor flow to specific If PACT is not a desirable organ due to anatomic option and the organ is sizable reasons (poorly with accessible vasculature vascularized) consider PARS clearing the single organ, akin to published decellularization methods.
  • High crosslinking High crosslink density in density A4P1-4 -hybridized tissues will slow antibody diffusion - thus antibody incubations should be extended.
  • fixative-induced autofluorescence such as autofluorescence, tissue bleaching, performing elastin, collagen wash steps in PBST containing 100 mM glycine to quench aldehydes, and treating tissue with histology stains that quench or mask autofluorescence, may be adapted to thick-sectioned cleared tissues - typically by performing longer wash steps after the appropriate countermeasure;
  • photobleaching tissue prior to IHC at wavelengths that exhibit the highest autofluorescence may also help.
  • Tissue mounting Poor image quality Tissue is of insufficient Extend the tissue incubation and imaging and/or poor imaging transparency for light time in RIMS to several days
  • Steps 12-13 depth to penetrate before imaging; for bone, incubate for an additional 1 day in RIMS- 1.48 or RIMS- 1.49 before imaging.
  • TerraStitcher or option C Vaa3D TerraFly; consider upgrading computer workstation and/or adding RAM and/or new graphics card; down sample the data set (of note, compression cannot be used with Imaris); process the images in tiles (i.e., analyze each tile individually).
  • mice (AAV9:CAG-GFP) were administered systemically (via retro-orbital injection) to 6-week-old female C57B1/6 mice. Three weeks later, mice were perfused and cleared via PARS, and individual organs were harvested and equilibrated in RIMS until clear (up to 7 days) before mounting in fresh RIMS and imaging.
  • mice and rats must conform to all relevant governmental and institutional regulations. Animal husbandry and all experimental procedures involving mice and rats were approved by the Institutional Animal Care and Use Committee (IACUC) and by the Office of Laboratory Animal Resources at the California Institute of Technology.
  • IACUC Institutional Animal Care and Use Committee
  • Quantitative image analysis was carried out using a custom MATLAB script. To factor out attenuation loss along the z-axis and account for varying cell density, the antibody fluorescence signal was scaled by the average DAPI intensity and computed perpendicular to the tissue surface to estimate labelling intensity as a function of depth. To fit this signal, we first identified the location of the surface as the point of maximum staining level and the center of the section as the point of minimum staining level. Since the surface is not perfectly flat, we excluded the top 20 ⁇ above the maximum point of the signal from further analysis. Data points plotted in Figure 4D show the staining level starting 20 ⁇ below the maximum down to the minimum staining level.
  • staining level between 20% and 80% depth to fit the exponential model (this range is indicated by the extent of the straight lines for each sample shown in Fig. 4D).
  • lipid-extracted tissues make poor subjects for high-resolution EM studies.
  • a fundamental component of tissue clearing, delipidation compromises the structural integrity of remaining subcellular constituents and eliminates a primary source of ultrastructural contrast in EM (i.e., osmium tetroxide based fixation-staining of lipids, and thus of all cell membranes and membrane-enclosed organelles). Without this contrast, the process of identifying fine structures or tracing cellular elements between images, assuming they survive clearing, becomes problematic.
  • tissue that is slated for ultrastructural analysis undergoes a highly regimented fixation, freeze-substitution, and/or immunostaining process to ensure that the subcellular architecture is well-preserved for high-resolution visualization and reliable immunolocalization.
  • the tissue clearing procedure departs from standard EM methods even at its onset: tissues are initially fixed in 4% PFA with no inclusion of glutaraldehyde, a staple in EM fixatives.
  • extended incubations at RT-to-37 °C in solutions where pH and osmolarity are imprecisely controlled are suboptimal conditions for the preservation of fine structures.
  • Brain tissue samples from each of the clearing conditions were processed simultaneously for subcellular examination via electron tomography. Each sample was placed in a petri dish containing 0.1 M sodium cacodylate trihydrate + 5% sucrose. Similar regions were extracted from each sample and cut into 0.50-0.75 mm cubes. These pieces were placed into a brass planchette (Ted Pella, Inc.) prefilled with cacodylate buffer supplemented with 10% Ficoll (70kD; Sigma-Aldrich) which serves as an extracellular cryoprotectant. The sample was covered with second
  • Planchettes containing vitrified samples were transferred under liquid nitrogen to cryotubes (Nunc) filled with 2.5% osmium tetroxide, 0.05% uranyl acetate in acetone. Tubes were placed into an AFS-2 freeze-substitution machine (Leica Microsystems) and processed at -90 °C for 48 hours, warmed slowly over 12 hours to -20 °C and further processed at that temperature for 10 hours. The tubes were then warmed to 4 °C for 1 hour and the samples rinsed 4 ⁇ with cold acetone. Samples were removed from the planchettes, infiltrated into Epon-Araldite resin (Electron Microscopy Sciences, Port Washington PA) and flat-embedded between two Teflon-coated glass microscope slides.
  • Epon-Araldite resin Electrode
  • Embedded samples were observed with a phase-contrast light microscope and similar portions from each condition was extracted and glued to plastic sectioning stubs.
  • Semi-thick sections 350 nm were cut with a UC6 ultramicrotome (Leica Microsystems) and a diamond knife (Diatome-US, Port Washington PA). Sections were placed on Formvar-coated copper/rhodium slot grids (Electron Microscopy Sciences) and stained with 3% uranyl acetate and lead citrate.
  • Colloidal gold particles (10 nm) were placed on both sides of the grid to serve as fiducial markers for tomographic image alignment. Grids were placed in a 2040 dual-axis tomography holder (E.A.
  • control sample of this experiment was fixed only by 4% PFA perfusion, akin to cleared samples, and then processed for TEM. Its retention of fine structure was readily apparent (see Fig. 5B, Control), and the level of gross tissue damage was minimal considering the nontraditional method of preparation. Significantly less fine structure was discernable in the cleared samples, as was predicted, and the lipid-containing membranes were fully solubilized or highly ruptured. Of note, the addition of paraformaldehyde to hydrogel formulations served to attenuate lipid extraction and led to greater preservation of neuronal processes (see Fig. 5B, A4P4).
  • Sudan Black B lipofuscin treatment (SB): Prepare 0.2% Sudan Black B in 70% ethanol in a sealable container or jar; protect from light. Alternatively, if tissue possesses a high level of lipofuscin, or if tissue will undergo extensive immunolabeling, which can wash off SB staining, prepare 1% SB in 70% ethanol. With the container sealed and wrapped in tin foil, stir the SB solution on a stir-plate at high-speed for 2 hours (or up to overnight).
  • Thyl-YFP mice were perfusi on-fixed with 4% PFA, and the excised brains were cut into 0.5 mm and 1 mm coronal sections. Upon brief post-fixation, sections were rinsed in l x PBS and then dd H20, and then incubated in either l x PBS (control), 10 mM CuS04, 0.2% SB, or 1% SB for 2-3 hours at RT with shaking. Sections were then dipped in dd H20 to remove excess stain, briefly rinsed in l x PBS, and incubated in A4P1 for 48 hours at 4 °C before hydrogel polymerization according to standard PACT procedures.
  • Collagenase crude from Clostridium histolyticum (Sigma-Aldrich, cat. no. CO 130) 16 Calcium chloride (Sigma-Aldrich, cat. no. C1016)
  • silicone rubber sheet any, such as: Press-to-SealTM Silicone Sheet with Adhesive, Fischer Scientific, cat. no. P-24745)
  • 6 ePACT sample washing and clearing wells such as 6-well tissue culture plates and/or petri dish
  • ePACT acrylate-acrylamide copolymer (AcAm): Combine 2.5% acrylamide, 8.625%) sodium acrylate, 0.15%. bis-acrylamide in 1 * PBS with 2 M NaCl; store at -20 °C prior to use. For tissue embedding, thaw AcAm on ice, and add the following (w/w): 0.01%> 4- hydroxy TEMPO, 0.2% TEMED, and 0.2% APS (see Fig. 13D)
  • PBS Phosphate-buffered Saline
  • Boric acid buffer (BB) Prepare a 1 M boric acid buffer stock solution through stirring 61.83 g boric acid and 10 g NaOH in 900 ml water with gentle heating. Once sodium hydroxide pellets and boric acid are fully dissolved, adjust the pH to 8.5 with NaOH and add distilled and deionized water (dd H20) to a total volume of 1 L. Dissolve this stock 5-fold for 0.2 M boric acid buffer (BB).
  • BBT boric acid wash buffer
  • 0.2 M boric acid buffer with 0.1% Triton X-100 (vol/vol), pH 8.5 dilute the 1 M boric acid stock to 0.2 M boric acid in dd H20, adding 1 ml of Triton X-100 per liter of BBT.
  • ePACT Clearing solution For borate-buffered 10% SDS in 0.2 M BB at pH 8.5 (10% SDS-BB), dilute 500 ml 20% SDS and 200 ml 1 M boric acid buffer stock to 1 L with dd H20; adjust the pH to 8.5, if necessary.
  • TESCA buffer Prepare 50 mM TES, 0.36 mM Calcium chloride solution, pH 7.4; sterile filter and store at RT.
  • Mounting media 2% low-melt agarose. Expanded samples may be stored at RT for ⁇ 72 hours prior to imaging if 0.01% sodium azide is added to the melted agarose solution immediately prior to pouring, and if the cover-slipped sample is sealed with Entellan.
  • the sample may be stored at -80 °C for up to a few months.
  • Sections may be stored at 4 °C in 1 x PBS with 0.01% sodium azide for ⁇ 1 day.
  • Sections may be stored at 4 °C in lx PBS with 0.01% sodium azide for ⁇ 1 day.
  • the section may be mounted on a non-treated glass slide in PB in order to visualize gross tissue morphology and, if applicable fluorescent signal strength. For finer visualization, incubate the section in RIMS for 1-2 hours prior to imaging. After imaging, wash RIMS-infused samples in lx PBS (3 x 15 minutes). ePACT Hydrogel Embedding
  • samples may be digested within a larger sample well cut from a silicon matte and adhered onto a slide (see Fig. 13D); coverslip and digest within a petri dish humidifier at 37 °C.
  • the AcAm-embedded section must be mounted in, for example agarose, to prevent sample drift during imaging. Also, the mounted sample must be sealed between the coverslip and glass slide so that the water content of the agarose and of the expanded AcAM tissue- hydrogel remains at a steady-state. Otherwise, the sample will shrink from dehydration and/or expand when it absorbs water from the agarose (i.e. if placed in a humidity chamber).
  • vacuum grease may be used to reinforce the seal.
  • Tissue size fluctuations can place undue stress upon cellular architecture, causing concern about utilizing tissue clearing procedures like ePACT. Indeed, we provide conclusive evidence that fine processes are compromised with unchecked expansion (see Fig. 13A-B, item 3). However, we have also found that tissue swelling may be used to great advantage in certain applications. As an example, certain cell populations are difficult to study due to their high density. To determine virus infectivity for AAV vector engineering or the expression level and coverage of an optogenetic construct, accurate and time- intensive cell-counting must often be performed in discrete tissue regions.
  • NPS neuronal positioning system
  • the ePACT procedure by clearing tissue through both SDS-based lipid removal and RI homogenization, can greatly assist in these circumstances.
  • enzymatically digesting and inflating the tissue with water serves to optically clear the tissue as well as to dilute the fluorescent, nonspecific and/or background signal.
  • fluorescently labeled targets come into view (see Fig. 13A-B, items 1 and 2), and automated cell counting becomes possible.
  • FIG. 13 demonstrates that the ePACT technique is applicable to the "microscale" study of cellular morphology and cell populations, we believe that an additional future value lays in the nanoscale measurement of fluorescent probes in smFISH experiments.
  • Our previous research revealed that, even with the 1.5 ⁇ expansion conveyed by standard PACT, single labeled transcripts were more readily discernable in PACT tissue than in the customary thin sections due to both lower background and their expansion-based separation. Instead, by utilizing the 4x tissue expansion conferred by ePACT to further enlarge the optical space within a cell (see Fig. 13C), quantitative analysis of multiple transcripts isolated to their subcellular locations can be more easily performed.
  • the sections begins to Insufficient clearing, Transfer the section crack during such that back to 10% SDS- expansion collagenase cannot BB (pH 8.5) and access all tissue clear for several during digestion hours as in steps
  • tissue begins to expand evenly during washing - proceed
  • Sodium borohydride is highly flammable when in contact with moisture and is very toxic to the skin. Do not leave the flask uncapped. Prepare dilutions fresh, on ice, in fume or chemical hood. Close it tightly after weighing, seal with parafilm, and return to its containment canister (if applicable to institutional laboratory practices).
  • FISH probes 20mer oligo probes, 1 nM each per hybridization reaction and labeled with Alexa 594
  • Microscope Nikon Ti Eclipse microscope with an Andor Ikon-M camera and an Plan- Apo 60x 1.4 N.A. 1 oil objective (w.d 0.13 mm) with an additional 1.5x magnification.
  • Excitation lasers (589 nm (SDL-589-XXXT), 532 nm (SDL-532-200TG) and 405nm(SDL-405-LM-030), all manufactured by Shanghai Dream Laser
  • Aminosilane-treated coverslips Coverslips were sequentially transferred between and sonicated in three solutions: first 1M NaOH, then 100% EtOH, and finally acetone. The cleaned coverslips were immediately submerged into a 2% solution of (3-Aminopropyl) triethoxysilane (Sigma 440140) in acetone for two minutes. Amine-modified coverslips were rinsed and stored in ultrapure water at RT. 189
  • Ethanol dilutions Prepare graded dilutions of 100%, 95%, 70% ethanol in RNase-free sterile H20.
  • Permeabilization Buffer Prepare a solution of 0.5% sodium borohydride (wt/vol) in 70% ethanol.
  • 2x SSC For 2x SSC, combine 100 ml 20x SSC with 850 ml RNase-free sterile H20, pH to 7.0, then add H20 to a total volume of 1 L.
  • Hybridization buffer Prepare 10% dextran sulfate (wt/vol, Sigma D8906), 10% formamide (vol/vol) in 2x SSC.
  • (A) Determine the average background of the sample. For example, the images were median filtered using a 50 ⁇ 50 pixel kernel and the average pixel intensity of the center 200 ⁇ 200 pixel sub-image was used as the average background value of the image.
  • Tube lens Thorlabs 2" Achromatic e.g. AC508-400-A- doublet, various focal ML

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Abstract

La présente invention porte, dans divers modes de réalisation, sur des procédés de clarification de tissu, les tissus étant rendus perméables à des macro-molécules et optiquement transparents, ce qui permet d'exposer leur structure cellulaire avec une connectivité intacte. Selon certains modes de réalisation, la présente invention concerne le protocole ePACT, qui est un protocole pour une meilleure clarification de tissu par le biais d'un développement. Selon certains modes de réalisation, la présente invention concerne la visualisation d'un tissu qui a été développé par le biais du protocole ePACT.
PCT/US2016/047430 2015-08-17 2016-08-17 Stabilisation de tissu de tout le corps et extractions sélectives par le biais d'hybrides hydrogel-tissu pour un phénotypage et un appariement de circuit intact à haute résolution Ceased WO2017031249A1 (fr)

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CN108693151A (zh) * 2017-04-11 2018-10-23 中国科学技术大学 Storm/palm显微成像方法及装置
WO2018155925A3 (fr) * 2017-02-27 2019-03-14 고려대학교 산학협력단 Procédé de production de tissu décellularisé à l'aide d'un polymère de type hydrogel et tissu décellularisé ainsi produit
WO2019055558A1 (fr) * 2017-09-12 2019-03-21 Worcester Polytechnic Institute Encapsulation d'hydrogel d'organismes vivants pour microscopie à long terme
US20200041514A1 (en) * 2017-02-24 2020-02-06 Massachusetts Institute Of Technology Methods for Diagnosing Neoplastic Lesions
CN111487130A (zh) * 2020-04-16 2020-08-04 河海大学 一种水凝胶拉伸测试的新型免夹持测试设备
US10794802B2 (en) 2013-09-20 2020-10-06 California Institute Of Technology Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high resolution intact circuit mapping and phenotyping
US10955322B2 (en) 2017-01-18 2021-03-23 California Institute Of Technology Methods and devices for soft and osseous tissue clearing and fluorescent imaging
WO2021188704A1 (fr) * 2020-03-18 2021-09-23 Genentech, Inc. Analyse spatiale de matériel biologique provenant d'échantillons de tissu intacts
EP3778920A4 (fr) * 2018-05-18 2022-02-16 Binaree, Inc. Procédé de diagnostic par imagerie tridimensionnelle d'acides nucléiques d'un bio-tissu, utilisant une amplification isotherme d'acides nucléiques
US11333588B1 (en) 2020-12-07 2022-05-17 Nebulum Technologies Co., Ltd. Matrix-assisted methods and compositions to prepare biological samples for super-resolution imaging
KR20220121178A (ko) * 2021-02-24 2022-08-31 한국과학기술원 팽창된 전-유기체의 생물학적 구조 정보를 초고해상도로 관찰하기 위한 이미징 방법
CN116718450A (zh) * 2017-06-07 2023-09-08 深图有限责任公司 用于标记、透明化和成像大型组织的方法
EP4249889A1 (fr) 2022-03-23 2023-09-27 Nebulum Technologies Co., Ltd. Procédés de préparation et d'analyse de biopsies et d'échantillons biologiques
EP4253941A1 (fr) * 2022-03-31 2023-10-04 Abberior GmbH Procédé et appareil d'imagerie optique avec un nombre élevé d'échantillons d'imagerie
EP4253942A1 (fr) * 2022-03-31 2023-10-04 Abberior GmbH Procédé et appareil d'imagerie optique avec un nombre élevé d'échantillons d'imagerie
US11802872B2 (en) 2017-02-24 2023-10-31 Massachusetts Institute Of Technology Methods for examining podocyte foot processes in human renal samples using conventional optical microscopy
US11802822B2 (en) 2019-12-05 2023-10-31 Massachusetts Institute Of Technology Multiplexed expansion (MultiExM) pathology
US11873374B2 (en) 2018-02-06 2024-01-16 Massachusetts Institute Of Technology Swellable and structurally homogenous hydrogels and methods of use thereof
CN117571436A (zh) * 2023-11-16 2024-02-20 中国人民解放军海军军医大学 棕色脂肪透射电子显微镜样品的新固定方法
WO2024094851A1 (fr) * 2022-11-03 2024-05-10 Deep Piction Gmbh Système robotisé de micro-biopsie et procédé robotisé de micro-biopsie
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US12265004B2 (en) 2019-11-05 2025-04-01 Massachusetts Institute Of Technology Membrane probes for expansion microscopy
US12405193B2 (en) 2019-02-22 2025-09-02 Massachusetts Institute Of Technology Iterative direct expansion microscopy
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US10794802B2 (en) 2013-09-20 2020-10-06 California Institute Of Technology Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high resolution intact circuit mapping and phenotyping
US10955322B2 (en) 2017-01-18 2021-03-23 California Institute Of Technology Methods and devices for soft and osseous tissue clearing and fluorescent imaging
US20200041514A1 (en) * 2017-02-24 2020-02-06 Massachusetts Institute Of Technology Methods for Diagnosing Neoplastic Lesions
US11802872B2 (en) 2017-02-24 2023-10-31 Massachusetts Institute Of Technology Methods for examining podocyte foot processes in human renal samples using conventional optical microscopy
US12061199B2 (en) * 2017-02-24 2024-08-13 Massachusetts Institute Of Technology Methods for diagnosing neoplastic lesions
WO2018155925A3 (fr) * 2017-02-27 2019-03-14 고려대학교 산학협력단 Procédé de production de tissu décellularisé à l'aide d'un polymère de type hydrogel et tissu décellularisé ainsi produit
CN108693151A (zh) * 2017-04-11 2018-10-23 中国科学技术大学 Storm/palm显微成像方法及装置
CN116718450A (zh) * 2017-06-07 2023-09-08 深图有限责任公司 用于标记、透明化和成像大型组织的方法
WO2019055558A1 (fr) * 2017-09-12 2019-03-21 Worcester Polytechnic Institute Encapsulation d'hydrogel d'organismes vivants pour microscopie à long terme
US11248995B2 (en) 2017-09-12 2022-02-15 Worcester Polytechnic Institute Hydrogel encapsulation of living organisms for long-term microscopy
US11873374B2 (en) 2018-02-06 2024-01-16 Massachusetts Institute Of Technology Swellable and structurally homogenous hydrogels and methods of use thereof
US12258454B2 (en) 2018-02-06 2025-03-25 Massachusetts Institute Of Technology Swellable and structurally homogenous hydrogels and methods of use thereof
EP3778920A4 (fr) * 2018-05-18 2022-02-16 Binaree, Inc. Procédé de diagnostic par imagerie tridimensionnelle d'acides nucléiques d'un bio-tissu, utilisant une amplification isotherme d'acides nucléiques
US12233184B2 (en) 2018-07-13 2025-02-25 Massachusetts Institute Of Technology Dimethylacrylamide (DMAA) hydrogel for expansion microscopy (ExM)
US12405193B2 (en) 2019-02-22 2025-09-02 Massachusetts Institute Of Technology Iterative direct expansion microscopy
US12265004B2 (en) 2019-11-05 2025-04-01 Massachusetts Institute Of Technology Membrane probes for expansion microscopy
US11802822B2 (en) 2019-12-05 2023-10-31 Massachusetts Institute Of Technology Multiplexed expansion (MultiExM) pathology
WO2021188704A1 (fr) * 2020-03-18 2021-09-23 Genentech, Inc. Analyse spatiale de matériel biologique provenant d'échantillons de tissu intacts
CN111487130A (zh) * 2020-04-16 2020-08-04 河海大学 一种水凝胶拉伸测试的新型免夹持测试设备
US11333588B1 (en) 2020-12-07 2022-05-17 Nebulum Technologies Co., Ltd. Matrix-assisted methods and compositions to prepare biological samples for super-resolution imaging
KR20220121178A (ko) * 2021-02-24 2022-08-31 한국과학기술원 팽창된 전-유기체의 생물학적 구조 정보를 초고해상도로 관찰하기 위한 이미징 방법
KR102728714B1 (ko) 2021-02-24 2024-11-13 한국과학기술원 팽창된 전-유기체의 생물학적 구조 정보를 초고해상도로 관찰하기 위한 이미징 방법
EP4249889A1 (fr) 2022-03-23 2023-09-27 Nebulum Technologies Co., Ltd. Procédés de préparation et d'analyse de biopsies et d'échantillons biologiques
EP4253941A1 (fr) * 2022-03-31 2023-10-04 Abberior GmbH Procédé et appareil d'imagerie optique avec un nombre élevé d'échantillons d'imagerie
WO2023187190A1 (fr) * 2022-03-31 2023-10-05 Abberior GmbH Procédé et appareil d'imagerie optique avec un nombre élevé d'échantillons d'imagerie
WO2023187189A1 (fr) * 2022-03-31 2023-10-05 Abberior GmbH Procédé et appareil d'imagerie optique avec un nombre élevé d'échantillons d'imagerie
EP4253942A1 (fr) * 2022-03-31 2023-10-04 Abberior GmbH Procédé et appareil d'imagerie optique avec un nombre élevé d'échantillons d'imagerie
WO2024094851A1 (fr) * 2022-11-03 2024-05-10 Deep Piction Gmbh Système robotisé de micro-biopsie et procédé robotisé de micro-biopsie
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WO2025233421A1 (fr) * 2024-05-08 2025-11-13 Fundación Para La Investigación Biomédica Del Hospital Gregorio Marañón (Fibhgm) Système de clarification de tissu et procédé associé

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