Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Summary

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Abstract

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2′-thiodiethanol (TDE), a previously described refractive index matching solution.

Introduction

The ability to image complete neuroanatomical structures is immensely valuable for understanding the brain in health and disease. Traditionally, 3D imaging has required tissue sectioning to provide the axial resolution and to visualize deep anatomical structures. This approach can produce high-resolution data sets, but requires sophisticated image reconstruction techniques and is very labor intensive. As a result, it has been limited to the imaging of small volumes of tissue^1-3. Optical sectioning, on the other hand, is well suited for the creation of high-resolution 3D images of fluorescently labeled tissues. Since optical sectioning is inherently three di....

Protocol

All experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. THY1-YFP, PLP-EGFP and PV-TdTomato mice were used for these experiments, but any mice expressing fluorescent proteins can be successfully cleared and imaged.

1. Tissue Preparation

Caution: Paraformaldehyde (PFA) and acrylamide are toxic and irritants. Perform experiments utilizing these reagents in a fume hood and with appropriate personal protective equipment (lab coat, gloves, and eye protection).

1. Sacrifice the animal in euthanasia chamber with ~1 ml of isoflurane until breathing ceases ....

Results

Visualizing the structural organization of the brain is critical for understanding how morphology and connectivity affect brain function in health and disease. Optical clearing techniques make it possible to image cell populations in 3D in intact tissues, allowing us to study morphology and connectivity cohesively.

Mice that express fluorescent proteins driven by promoters specific for sub-populations of cells permit one to teas.......

Discussion

Passive clearing of hydrogel embedded tissues (passive CLARITY) is a simple and inexpensive method for clearing large pieces of tissue. This approach does not require dedicated equipment and can easily be performed in a temperature-controlled shaker. Over the span of a few weeks, even large, highly myelinated tissues, such as an entire brain or spinal cord, will become transparent and suitable for microscopy. Even though this report has focused on the clearing of CNS tissues, passive CLARITY can be applied to any tissue........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x phosphate buffered saline (PBS)	Fisher	BP399-1	buffers
32% paraformaldehyde (PFA)	Electron Microscopy Sciences	15714-5	perfusion and hydrogel
2,2'-thiodiethanol (TDE)	Sigma-Aldrich	166782-500G	refractive index matching solution
40% acrylamide	Bio-Rad	161-0140	hydrogel
2% bis-acrylamide	Bio-Rad	161-0142	hydrogel
VA-044 initiator	Wako Pure Chemical Industries, Ltd.	VA044	hydrogel
Boric acid	Sigma-Aldrich	B7901	clearing buffer
Sodium dodecyl sulfate (SDS)	Fisher	BP166-5	clearing buffer
Sodium hydroxide	Fisher	55255-1	buffers
Sodium azide	Sigma-Aldrich	52002-100G	preservative
Triton X-100	Sigma-Aldrich	X100-500G	buffers
Heparin	Sigma-Aldrich	H3393-50KU	perfusion
Sodium nitrate	Fisher	BP360-500	perfusion
DAPI	Molecular Probes	D1306	nuclear stain
99.9% isoflurane	Phoenix	57319-559-06	anesthetic
Hydrocholoric acid	Fisher	A1445-500	buffers
Glass bottom dish	Willco	HBSB-5040	Willco dishes
Reusable adhesive	Bostick	371351	Blu-Tack
Silicon elastomer	World Precision Instruments	KWIK-CAST	Kwik-Cast
Lint-free wipe	Kimberly-Clark	34120	KimWipe
Mouse brain matrix	Roboz	SA-2175	sectioning tissue
Matrix blades	Roboz	RS-9887	sectioning tissue
Peristaltic pump	Cole-Parmer	77122-24	pefusion
Laser scanning confocal microscope	Leica	SP5	microscopy
Imaging software	Leica	LAS AF	microscopy