المقاصة البصري للنظام العصبي المركزي الماوس عن طريق وضوح السلبي

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 dimensional, it does not require extensive computation to produce a 3D image volume. However, light scattering and tissue opacity limit the depth at which tissues can be optically sectioned. The depth of imaging is limited to about 150 µm in laser scanning confocal microscopy and to less than 800 µm using two-photon excitation microscopy^4-8.

In order to overcome these limitations, several optical clearing techniques have been recently developed and then further refined to permit the deep microscopic imaging of intact tissues. The use of benzyl alcohol and benzyl benzoate (BABB) to render fixed tissues transparent was among one of the earliest approaches⁹. However, this approach was limited by the quenching of fluorescence in the samples and incomplete clearing of highly myelinated structures¹⁰. Refinements of this technique, such as 3D Imaging of Solvent Cleared Organs (3DISCO), have led to very rapid and complete tissue clearing, but still suffer from rapid loss of fluorescent signals, especially yellow fluorescent protein (YFP)¹⁰. Water-based clearing solutions, such as Sca/e¹¹ and SeeDB¹², preserve fluorescent signals, but do not completely clear highly myelinated tissues. Clear, Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) is a promising new optical clearing technology that appears to overcome the limitations of previously developed water-based clearing solutions¹³. In contrast with other optical clearing techniques, Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY)¹⁴ embeds the brain in a porous matrix that provides structural integrity to proteins, nucleic acids and small molecules, while leaving lipids unbound. The lipids are then removed electrophoretically, culminating in an optically cleared brain that can be easily visualized and equally easily probed using commonly available techniques.

Electrophoretic tissue clearing (ETC) is used to remove lipids from hydrogel embedded tissues in CLARITY, however, ETC can be difficult to implement consistently and tissues subject to electrophoresis can exhibit tissue distortion, browning, loss of fluorescence and antibody reactivity. Passive lipid clearing avoids these limitations, but requires increased clearing times^15-17. Passive clearing also permits the clearing of large numbers of samples in parallel, since it is not limited by the number of ETC apparatuses available. Decreasing the concentration of paraformaldehyde and excluding bis-acrylamide from the hydrogel have led to great increases in the speed of clearing at the expense of greater tissue expansion¹⁶. Less expensive alternatives to the refractive index matching solution used in the original CLARITY technique have been developed^17-19. 2,2′-thiodiethanol (TDE) is an inexpensive and rapid brain clearing agent that reverses some of the tissue expansion that occurs during lipid removal¹⁸. This report incorporates passive clearing of hydrogel embedded tissue and the use of TDE as a refractive index matching solution to the original CLARITY technique to produce a highly reproducible and inexpensive protocol for the optical clearing of the mouse central nervous system.

Protocol

أجريت جميع التجارب وفقا للرعاية الحيوان المؤسسية والمبادئ التوجيهية للجنة الاستخدام (IACUC). استخدمت THY1-YFP، حزب العمال التقدمي-EGFP وPV-TdTomato الفئران لهذه التجارب، ولكن أي الفئران معربا عن البروتينات الفلورية يمكن مسح بنجاح وتصويرها. 1. …

Representative Results

تصور التنظيم الهيكلي للدماغ أمر بالغ الأهمية لفهم كيفية التشكل والاتصال تؤثر على وظيفة الدماغ في الصحة والمرض. تقنيات المقاصة البصرية تجعل من الممكن لسكان الخلية الصورة في 3D في الأنسجة سليمة، مما يسمح لنا لدراسة مورفولوجية والربط متماسك. <p class="jov…

Discussion

المقاصة سلبية من هيدروجيل جزءا لا يتجزأ من الأنسجة (وضوح السلبي) هي طريقة بسيطة وغير مكلفة لإزالة أجزاء كبيرة من الأنسجة. لا يتطلب هذا النهج معدات مخصصة، ويمكن بسهولة أن يؤديها في شاكر التحكم في درجة حرارته. على مدى غضون بضعة أسابيع، حتى الكبيرة، والأنسجة العصبي للغا?…

Materials

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