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A versatile clearing agent for multi-modal brain imaging

Irene Costantini, Jean-Pierre Ghobril, Antonino Paolo Di Giovanna, Anna Letizia Allegra Mascaro, Ludovico Silvestri, Marie Caroline Müllenbroich, Leonardo Onofri, Valerio Conti, Francesco Vanzi, Leonardo Sacconi, Renzo Guerrini, Henry Markram, Giulio Iannello, Francesco Saverio Pavone

arXiv:1504.03855v2q-bio.NCphysics.med-ph

TL;DR

Mapping brain-wide neuronal connections at microscopic resolution remains challenging. This paper presents TDE as a versatile clearing method, enabling high-resolution imaging that resolves neuronal structures throughout the hippocampus.

  • Problem

    Mapping all brain connections at microscopic resolution remains a tremendous technological challenge.

  • Method

    The paper develops a simple, rapid, inexpensive TDE-based clearing method using a non-viscous aqueous solvent with tunable refractive index.

  • Results

    TDE enabled high-resolution imaging that resolved spines, varicosities, and neuronal processes throughout the mouse hippocampus.

  • Takeaways & Limitations

    The method provides a simple, inexpensive approach for complementary brain-imaging applications.

  • Takeaways & Limitations

    Direct TDE incubation is not suitable for imaging entire organs.

Abstract

from arXiv · show

Extensive mapping of neuronal connections in the central nervous system requires high-throughput um-scale imaging of large volumes. In recent years, different approaches have been developed to overcome the limitations due to tissue light scattering. These methods are generally developed to improve the performance of a specific imaging modality, thus limiting comprehensive neuroanatomical exploration by multimodal optical techniques. Here, we introduce a versatile brain clearing agent (2,2'-thiodiethanol; TDE) suitable for various applications and imaging techniques. TDE is cost-efficient, water-soluble and low-viscous and, more importantly, it preserves fluorescence, is compatible with immunostaining and does not cause deformations at sub-cellular level. We demonstrate the effectiveness of this method in different applications: in fixed samples by imaging a whole mouse hippocampus with serial two-photon tomography; in combination with CLARITY by reconstructing an entire mouse brain with light sheet microscopy and in translational research by imaging immunostained human dysplastic brain tissue.

2. National Institute of Optics, National Research Council, Largo Fermi 6, 50125 Florence, Italy · 3. Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto

This section lists affiliations spanning optics, physics, engineering, neuroscience, and child health institutions in Florence, Lausanne, Rome, and associated clinical settings.

  • 3. Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto: The Laboratory of Neural Microcircuitry is part of EPFL’s Brain Mind Institute in Lausanne, Switzerland.
  • 2. National Institute of Optics, National Research Council, Largo Fermi 6, 50125 Florence, Italy: The National Institute of Optics is part of Italy’s National Research Council in Florence.
  • 3. Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto: The Department of Engineering is located at the University Campus Bio-Medico of Rome.
  • 3. Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto: The Pediatric Neurology and Neurogenetics Unit and Laboratories belong to the Department of Neuroscience.
  • 3. Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto: The Department of Pharmacology and Child Health is affiliated with A. Meyer Children’s Hospital and the University of Florence.
  • 3. Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019 Sesto: The A. Meyer Children’s Hospital address is Viale Pieraccini 24, 50139 Florence, Italy.

7. Department of Biology, University of Florence, Via Romana 17, 50125 Florence, Italy · INTRODUCTION

Mapping brain-wide neuronal connections requires high-throughput, microscopic imaging of large volumes, but existing clearing and sectioning methods trade off transparency, fluorescence, structural preservation, cost, or compatibility. The study introduces water-soluble 2,2'-thiodiethanol (TDE) as a versatile clearing agent for multimodal imaging in mouse and human brain tissue.

  • INTRODUCTION: Existing approaches are limited by sectioning-induced deformation and sample destruction, or by clearing-related fluorescence quenching, shrinkage, long incubations, structural alteration, and incompatibility.These limitations complicate comprehensive mapping across organizational scales and imaging modalities.
  • INTRODUCTION: TDE is presented as a simple, rapid, inexpensive, water-soluble clearing agent for expanding quantitative neuroanatomical imaging.The method is designed to address limitations of existing clearing approaches while supporting large, transparent, fluorescently labeled volumes.
  • INTRODUCTION: TDE applied at 47% to paraformaldehyde-fixed samples increases two-photon penetration depth while preserving protein fluorescence and avoiding structural deformation.This establishes TDE for fixed-sample two-photon imaging without the stated preservation drawbacks of other clearing methods.
  • INTRODUCTION: TDE enables serial two-photon tomography with high-resolution, high-sensitivity 3D reconstruction of the whole hippocampus in a fluorescent transgenic mouse.This demonstrates application to large fixed samples and hippocampal-scale reconstruction.
  • INTRODUCTION: Because TDE’s refractive index can be finely adjusted, it also serves as an optical clearing medium for CLARITY and light sheet microscopy.The study uses this combination to image an entire mouse brain and reconstruct specific cell-type distributions and vascular structures.
  • INTRODUCTION: The approach couples TDE clearing with CLARITY immunohistochemistry to stain large volumes of human dysplastic brain tissue.This extends the method beyond mouse preparations to translational human tissue imaging.
  • INTRODUCTION: The study positions TDE as a multimodal clearing strategy that expands single- and two-photon imaging on large samples for quantitative morphological analysis in mouse and human brain.The stated applications span fixed-sample two-photon tomography, CLARITY-compatible light sheet imaging, and immunostained human tissue.

RESULTS · PFA-fixed brain tissue clearing · Two-photon serial sectioning of whole hippocampus

TDE is a versatile, tunable aqueous clearing agent that improves imaging depth while preserving fluorescence, tissue structure, and anatomical detail. In two-photon serial tomography, it enabled lossless reconstruction and complete neuronal tracing across an entire mouse hippocampus.

  • RESULTS: TDE is presented as a versatile clearing method based on a low-viscosity aqueous solvent whose refractive index can be tuned from 1.33 to 1.52.The method was tested on PFA-fixed mouse brain, CLARITY-processed mouse brain, and formalin-fixed human brain samples.
  • PFA-fixed brain tissue clearing: TDE diffused rapidly and homogeneously through 1-mm mouse brain sections, making them transparent within a few hours.Sections were incubated in phosphate-buffered saline solutions containing increasing TDE percentages.
  • PFA-fixed brain tissue clearing: At 47% TDE/PBS, matched to the objective’s refractive index of 1.42, clearing caused 10% shrinkage without anisotropic distortion.Transmittance increased with wavelength and TDE percentage, reaching SeeDB-comparable levels at 80% TDE/PBS.
  • PFA-fixed brain tissue clearing: TDE increased two-photon imaging penetration depth almost fourfold versus PBS-cleared samples.The comparison used contrast decay as a function of imaging depth in cleared and uncleared tissue.
  • PFA-fixed brain tissue clearing: TDE preserved fluorescence during long-term measurements, without increased bleaching or protein-fluorescence quenching for up to two months.Fluorescence intensity remained constant over time in cleared tissue.
  • PFA-fixed brain tissue clearing: Transmission electron microscopy showed preserved ultrastructure, including mitochondria, synaptic vesicles, and postsynaptic densities, apart from occasional localized myelin swelling.Nuclei, axons, dendrites, vesicles, and organelles remained distinguishable.
  • Two-photon serial sectioning of whole hippocampus: An entire mouse hippocampus was reconstructed by two-photon tomography, enabling complete tracing of single neurons through a large volume without interpolation.Enhanced penetration depth reduced slicing, acquisition time, and cutting artifacts while preserving the whole sample volume.
  • Two-photon serial sectioning of whole hippocampus: High-resolution tomography resolved hippocampal regions, spines, and varicosities, while high sensitivity supported detailed anatomical visualization.Dentate gyrus and Cornu Ammonis areas were recognizable in the reconstructed hippocampus.

Whole mouse brain imaging with light sheet microscopy · Immunostaining and human brain imaging

TDE enabled CLARITY-based light-sheet imaging of whole mouse brains with preserved transparency, fluorescence, and limited deformation. It also supported immunostaining and volumetric imaging of human dysplastic brain tissue, including neuronal tracing and pathology-compatible comparisons.

  • Whole mouse brain imaging with light sheet microscopy: A 63% TDE/PBS medium preserved whole mouse brains, produced uniform transparency, matched FocusClear transmittance, and yielded 16% final tissue expansion after CLARITY.TDE shrank expanded CLARITY tissue similarly to FocusClear while maintaining whole-brain preservation.
  • Whole mouse brain imaging with light sheet microscopy: Whole-brain light-sheet reconstructions achieved sub-cellular resolution, revealing anatomical features, cell-body distributions, axonal bundles, and finer neuronal connections.The method preserved neuronal labeling in PV-cre-tdTomato and GAD2-cre-tdTomato mouse brains for tracing and network analysis.
  • Whole mouse brain imaging with light sheet microscopy: TDE preserved propidium iodide fluorescence throughout whole brains for automated cell counting and revealed smaller capillaries in volumetric FITC-albumin vascular reconstructions.These results extended the approach across nuclear and vascular labeling applications.
  • Immunostaining and human brain imaging: Cleared dysplastic tissue identified giant dysmorphic neurons, with features directly comparable to those obtained by conventional hematoxylin/eosin staining.The comparison used tissue subjected to CLARITY and immunostaining alongside routine anatomopathological characterization.

DISCUSSION

The TDE clearing protocol is a simple, rapid, inexpensive, and versatile method that preserves key tissue properties while supporting diverse brain-imaging applications. Its compatibility with STP, CLARITY-LSM, and immunostaining extends high-resolution imaging from mouse hippocampus and whole brain to human tissue.

  • DISCUSSION: TDE combines rapid, inexpensive clearing with preserved volume, morphology, fluorescence, and compatibility across complementary imaging modalities.The method supports endogenous fluorescence, immunostaining, and multi-modal brain anatomy studies.
  • DISCUSSION: Imaging depth increased fourfold in PFA-fixed samples, while low-viscosity TDE enabled complete STP reconstruction of a mouse hippocampus with spine and varicosity resolution.Automatic 3D stitching supported accurate tracing of single neuronal processes throughout the hippocampus.
  • DISCUSSION: Coupling TDE with CLARITY achieved whole-mouse-brain transparency and provided a lower-cost alternative to FocusClear for affordable large-volume, high-throughput LSM.Adjustable TDE/PBS ratios permit refractive-index optimization for different tissue types and integration of potentially transparency-enhancing compounds.
  • DISCUSSION: Direct TDE incubation is unsuitable for entire organs, and future optimization must balance antibody-limited contrast against sample throughput and maximum thickness.The stated limitation concerns antibody penetration depth and the trade-off between contrast and throughput.
  • DISCUSSION: TDE-compatible immunostaining enabled homogeneous imaging of a 1 mm3 human hemimegalencephaly tissue cube and tracing neuronal fibers throughout its volume.This supports micrometric, sensitive human-brain connectomics and characterization of circuit alterations and aberrant cell morphology.

METHODS … Preparation of fixed mouse brains

The study used defined transgenic mouse lines, an ethically obtained human epilepsy specimen, and standardized fixation protocols for mouse brains. Mouse preparation differed for fluorescence imaging and transmission electron microscopy.

  • Transgenic animal model: Thy1-GFP-M mice enabled sparse GFP labeling of pyramidal neurons, while GAD2-ires-Cre-tdTomato mice visualized GABAergic interneurons.
  • Transgenic animal model: PV-Cre-tdTomato mice were used to image the parvalbumin-positive neuronal subpopulation.
  • Transgenic animal model: Animal experiments were designed and approved in accordance with Italian Ministry of Health laws and requirements.
  • Human brain specimen collection: A human brain sample from a child with drug-resistant epilepsy and HME was collected during surgery after informed consent and ethics approval.
  • Human brain specimen collection: The human specimen was placed in neutral buffered formalin at pH 7.2-7.4 and stored at room temperature until clearing.
  • Preparation of fixed mouse brains: Adult p56 mice were anesthetized, perfused with PBS and 4% PFA, then brains were extracted, fixed overnight at 4 °C, rinsed, and stored in PBS.
  • Preparation of fixed mouse brains: For TEM, mice were perfused with PBS followed by 2.5% glutaraldehyde and 2% PFA, and brains were stored at 4°C in PBS after removal.

Preparation of CLARITY-processed mouse brains … Staining of CLARITY-processed samples

The study prepared mouse brains using hydrogel-based CLARITY or passive CLARITY protocols, then applied graded TDE/PBS clearing. Cleared samples underwent immunostaining or nuclear staining before two-photon or light-sheet imaging.

  • Preparation of CLARITY-processed mouse brains: CLARITY brains were perfused and embedded in a PFA–acrylamide hydrogel, polymerized at 37 °C, then washed in clearing solution.Adult mice were perfused with PBS followed by a solution containing 4% PFA, 4% acrylamide, 0.05% bis-acrylamide, and 0.25% VA044; brains incubated in this solution for 3 days at 4 °C before polymerization.
  • Preparation of passive CLARITY (PC) -processed samples: Passive CLARITY samples were polymerized after two-week fixation in hydrogel precursor, cleared in 4% SDS buffer for two weeks, and washed before staining.Human CLARITY brain samples were cut into approximately 2 mm3 pieces; the protocol was applied to Thy1-GFP-M mouse brain slices and a human brain bioptic sample.
  • Optical clearing with TDE: TDE clearing used serial TDE/PBS incubations tailored to sample type: 20% and 47% for fixed or human samples, and 30% and 63% for CLARITY mouse brains.Fixed samples used 1-hour incubations at 37 °C or 12-hour incubations at room temperature; CLARITY samples used one-day incubations at 37 °C, while human samples used 10-minute incubations.
  • Staining of CLARITY-processed samples: For whole-brain nuclei imaging, CLARITY mouse samples were stained with propidium iodide at 1:50, washed, cleared in 63% TDE/PBS, and imaged by light-sheet microscopy.Propidium iodide incubation lasted 2 days at 37 °C, followed by a one-day PBST0.1 wash at 37 °C.

Measurement of light transmittance and linear deformation · Measurement of fluorescence quenching and bleaching · Evaluation of imaging depth

The study evaluates TDE-based clearing by measuring light transmittance, linear deformation, fluorescence quenching and bleaching, and imaging depth, using cleared and uncleared brain samples or alternative clearing solutions as appropriate.

  • Measurement of light transmittance and linear deformation: CLARITY brain slices were cleared with serial TDE/PBS incubations or FocusClearTM before transmittance and deformation assessment.The TDE/PBS sequence was 20%, 47%, and 63% at 37°C, with one hour per concentration.
  • Measurement of light transmittance and linear deformation: Light transmittance was measured spectrophotometrically in cleared brain slices and whole CLARITY brains treated with TDE/PBS, SeeDB, or FocusClearTM.Transmission images were acquired after one day in TDE/PBS solutions or three days in FocusClearTM.
  • Measurement of light transmittance and linear deformation: Linear deformation was quantified by normalizing cleared-brain area to the PBS area and taking the square root of that quotient.Potential nonlinear distortion was assessed by tracing, resizing, and superimposing brain edges.
  • Measurement of fluorescence quenching and bleaching: Fluorescence quenching was evaluated by repeatedly imaging fixed Thy1-GFP-M slices during incubation in 47% TDE/PBS and measuring mean intensity in homogeneous regions.Fresh TDE solution was used for every measurement.
  • Measurement of fluorescence quenching and bleaching: Bleaching was quantified as temporal decay of mean fluorescence in dendrite-containing regions after subtracting intensity from neighboring background regions.The dendrite ROI measured 20 x 20 µm2, while homogeneous-region measurements used 100 x 100 µm2 ROIs.
  • Evaluation of imaging depth: Imaging depth was evaluated before and after clearing by acquiring 600 µm two-photon stacks with a 2 µm z step and tracking image-contrast decay with depth.The comparison used cleared and uncleared 2-mm brain slices stained with DAPI.

Transmission electron microscopy … Light-sheet microscopy

The section details multimodal imaging workflows spanning electron microscopy, two-photon fluorescence, serial two-photon tomography with TDE, and custom light-sheet microscopy. It also specifies acquisition parameters and notes illumination inhomogeneity caused by tissue heterogeneity.

  • Transmission electron microscopy: Transmission electron microscopy samples consisted of 500-µm vibratome sections from fixed Thy1-GFP-M mouse brain incubated in PBS or 47% TDE/PBS for 4 days at 37°C.Post-fixation, en-bloc staining, resin embedding, sectioning, and imaging followed a published protocol.
  • Serial two-photon tomography with TDE: Serial tomography acquired 0.59 × 0.59 µm2 pixels, 300 × 300 µm2 fields, 1000-µm stack depths, and 200-µm overlap between successive layers.Layers were sliced every 800 µm to support efficient three-dimensional reconstruction along the z axis.
  • Serial two-photon tomography with TDE: Serial two-photon tomography used TDE-cleared mouse hippocampi, acquiring 1000-µm-deep stacks every 4 µm with overlapping fields for 3D reconstruction.Hippocampi were cleared through sequential 20% and 47% TDE/PBS incubations before serial sectioning and imaging.
  • Serial two-photon tomography with TDE: Serial TDE tomography exhibited some illumination inhomogeneity attributed to the inherent heterogeneity of the tissue.This limitation occurred during acquisition according to imaging depth.
  • Light-sheet microscopy: The custom confocal light-sheet microscope was used to image specimens in the light-sheet microscopy workflow.The instrument was described as custom-made and based on the CLSM design reported by Silvestri and colleagues.

29. The light sheet was generated by scanning the excitation beam with a galvanometric mirror (6220H,

The light-sheet microscope used galvo-scanned excitation synchronized with sCMOS rolling-shutter readout for confocal imaging. Terastitcher was extended to reconstruct serially sectioned, multilayered hippocampus data and multichannel human brain images.

  • Light-sheet microscope: Galvo-scanned excitation was synchronized with sCMOS camera line readout to achieve confocality, with custom LabVIEW coordinating the scanners and rolling shutter.
  • Light-sheet microscope: Samples were mounted on a motorized x-, y-, z-, θ-stage in a custom chamber containing 63% TDE/PBS, enabling free three-dimensional motion and rotation.
  • Image reconstruction: Terastitcher was extended to stitch specimens partitioned across overlapping layers for hippocampus reconstruction and to process images containing multiple channels for human brain tomography.
  • Image reconstruction: The multilayer workflow separately stitched input layers, imported them using instrument coordinates, aligned adjacent layers with multi MIP-NCC, and blended overlaps.
  • Image reconstruction: For multichannel registration, MIP-NCC could use fused or individual channels to prioritize informative signals or discard noisy channels while preserving the raw channel composition.

FIGURE LEGENDS

The figure set characterizes TDE as a low-deformation, fluorescence-preserving clearing medium and demonstrates its use for mouse hippocampus, whole-brain CLARITY imaging, and human brain immunostaining.

  • TDE characterization: 47% TDE/PBS caused no fluorescence quenching for up to two months and enabled two-photon imaging of 2 mm brain slices to 1 mm depth.Fluorescence intensity remained stable during incubation, while DAPI-stained slices were reconstructed along the z axis.
  • Hippocampus tomography: Whole mouse hippocampus was reconstructed after fixation and clearing with 47% TDE/PBS using two-photon excitation and serial sectioning.Six 1 mm layers were sampled every 4 µm, with serial sectioning at 800 µm depth.
  • TDE refractive index matching for CLARITY: In CLARITY brains, transmittance increased with refractive index, and 63% TDE/PBS achieved transmittance comparable to FocusClearTM.After ETC-induced expansion, tissue shrank back toward its original size during clearing.
  • Whole mouse brain tomography: CLARITY-treated whole mouse brains cleared with 63% TDE/PBS were imaged by light-sheet microscopy across multiple fluorescent and stained preparations.The figure includes parvalbumin-, GAD-, PI-, and FITC-albumin-labeled brains with high-resolution inserts.
  • Human brain immunostaining: A formalin-fixed dysplastic human brain block was immunostained with PV and GFAP antibodies, cleared with 47% TDE/PBS, and imaged by two-photon excitation.Nuclei were counterstained with DAPI in the illustrated preparations.
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