Name: optical clearing of the mouse central nervous system using passive clarity
Uploaded: 2016-06-30T13:00:00.000+00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

Bu çalışma cömertçe Nörolojik Bozukluklar ve İnme (NIH / NINDS) hibe 1R01NS086981 ve Conrad N. Hilton Vakfı Sağlık / Ulusal Enstitüsü Ulusal Sağlık Enstitüleri tarafından desteklenmiştir. Biz konfokal mikroskobu ile onların çok değerli yardım için Dr Laurent Bentolila ve Dr Matthew Schibler teşekkür ederim. Biz AÇIKLIK forumu (http://forum.claritytechniques.org) katkıda bulunmuş olanlar teşekkür ederim. Biz özellikle bu büyüleyici tekniğini öğretmek onun laboratuvarını açılması için Dr. Karl Deisseroth teşekkür ederim. Yazarlar Beyin Haritalama Tıbbi Araştırma Örgütü, Beyin Haritalama Destekleme Vakfı, Pierson-Lovelace Vakfı, Ahmanson Vakfı, Capital Group Şirketleri Hayırsever Vakfı, William M. ve Linda R. Dietel Hayırsever Fonu ve Northstar Fonundan cömert destek için minnettarız . Bu yayında bildirilen araştırma da kısmen Ulusal Araştırma Kaynakları Merkezi tarafından ve Ulusun Başkanlığınca desteklenenÖdül numaraları C06RR012169, C06RR015431 ve S10OD011939 altında Sağlık al Enstitüleri. Içeriği sadece yazarların sorumluluğundadır ve mutlaka Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmemektedir.

Tüm deneyler Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) kurallarına uygun olarak yapıldı. Thy1 YFP, PLP-EGFP PV-tdTomato farenin Bu deneyler için kullanılmıştır, ama floresan proteinleri eksprese bir farenin başarılı bir şekilde temizlenir ve görüntülenebilir. 1. Doku Hazırlanması Dikkat: Paraformaldehyde (PFA) ve akrilamid toksik ve tahriş edicidir. Davlumbaz ve uygun kişisel koruyucu ekipman (laboratuvar önlüğü, eldiven ve göz koruması) bu reaktifleri kullanılarak deneyler. <ol><li> (- 3 dakika ~ 2) solunum sona erene kadar izofluran ~ 1 ml ötenazi odasında hayvan kurban. </li><li> (- 10 dakika 5), kan dokulardan temizlenir kadar ~ 5 ml ayarlanmış bir peristaltik pompa kullanımı / dak intrakardiyak, 4 ° C de perfüzyon çözeltisi (Tablo 1) ile fare perfüze edilmesi için. </li><li> Fiksatif çözüm için perfüzat değiştirin (Tablo 1)(- 10 dakika 5), ​​4 ° C&#39;de, boyun ve kuyruk anlamlı katılaşana dek. </li><li> perfüzyon End ve hayvan başını kesmek. Dikkatle ilk kafatası çevreleyen bağ dokusu çıkarılması ve sonra da kafatasının ventral yüzeyinden kemik kaldırılması ve 20 de açıklandığı gibi, (Şekil 1a) beynini kaldırarak kafatası beyin teşrih. Not: beyincik flocculonodular lobları ve koku ampuller çevreleyen kemiğin çıkarılması için özel bakım verir. <ol><li> Nazikçe omurilik ve vertebral kolon arasında ince bir keskin makas ucu sokarak ve 21 de açıklandığı gibi, uzak bireysel omurga segmentleri keserek vertebra sütundan omurilik parçalara ayır. </li></ol></li><li> plastik bir fare beyin matrisi içine yerleştirerek ve bir matris bıçak ile orta hat boyunca keserek beyin Hemisect. </li><li> santrifüj küvet 50 ml ayırmak için her yarımkürede ve omurilik ekleES ~ hidrojel çözeltisi (Tablo 1) 40 ml. Bu noktada ileri kapaktan alüminyum folyo ile tüpler sonraki tüm adımları sırasında fluorophores korumak için. </li><li> yumuşak 7 gün boyunca (~ 30 rpm) çalkalayarak 4 ° C&#39;de hidrojel örneğini içeren tüp inkübe edin. </li></ol> 2. Polimerizasyon <ol><li> Santrifüj tüpü içinde ~ hidrojelin 25 ml numune ve koruyucu, ~ hidrojelin 25 mi atın. </li><li> kapağı çıkarmadan ve kurutucu kavanoza santrifüj tüpü yerleştirilmesi ve bir vakum kavanozu bağlayarak örnek oksilenlendirilmesi. çözüm ve form kabarcıklar çıkmaya çözünmüş gazlar izin için en az 10 dakika süreyle vakum uygulamak. Yavaşça kabarcıklarını çıkarmak için sallayın. </li><li> 3 dakika - 2 üzerinde% 100 azot gazı ile desiccator kavanoza vakum değiştirin. basıncı (kurutucu kavanozun üst açılabilir kez) eşitleyen sonra, hızlı bir şekilde oksijen reintroduction önlemek için tüp kap. </li><li> PolimerPolimerizasyon işlemi tamamlanana kadar, 3 saat için 37 ° C su banyosu içinde örnek tüpü yerleştirerek hidrojel ize. Polimerize hidrojel jel benzeri bir kıvam (Şekil 1b) alacak. Not: üst polimerleşmemiş hidrojel az miktarda kabul edilebilir. </li><li> Daha sonra, santrifüj tüpüne gelen polimerize örneği kaldırmak örnekten fazla hidrojel kesip bir spatula kullanın. </li><li> silip bir tüy bırakmayan üzerinde örnek yerleştirerek ve yavaşça örnek üzerinde polimerize hidrojel, sadece ince bir tabaka geride bırakarak, sürtünme ve üzerinde doku yuvarlayarak kalan hidrojel çıkarın. </li></ol> 3. Lipit Kaldırma Not: Lipid temizlenir doku kırılgan, dikkatli olun. tüp boşaltırken örnek kaybetmemek için bir hücre süzgeç kullanın. Ayrıca, örnek kırılma indisini uyumlu hale işlemi sırasında ters olabilir şişen, ancak, temizleme işlemi boyunca şişer. Dikkat: Sodium dodesil sülfat (SDS) ve borik asit tahriş edicidir. Davlumbaz ve uygun kişisel koruyucu ekipman (laboratuvar önlüğü, eldiven ve göz koruması) ile onları kullanan deneyler. <ol><li> Temiz bir 50 ml santrifüj tüpüne polimerize örnek aktarın ve tampon (Tablo 1) temizleme 45 ilave edin. hafifçe çalkalanarak 45 ° C (~ 30 rpm) inkübe edilir. kimyasal atık içine kullanılan takas tampon dökülen ve taze takas tampon ~ 45 ml ekleyerek ilk 7 gün boyunca günde tampon değiştirin. florofor korumak için alüminyum folyo ile kaplanmış numunelerin içeren santrifüj tüplerini muhafaza edin. </li><li> 3 gün - ilk 7 gün sonra, her 2 40 ° C ve döviz takas tamponu de örnek kuluçkaya yatmaktadır. Tüm lipidler çözünmüş ve doku şeffaflık (Şekil 1c) ulaşıncaya kadar takas devam edin. Bir fare beyin yarımkürede 6 hafta ve 2 - - Bir omurilik için 4 hafta Bu 4 olacak. </li><li> Doku temizlendikten sonra, aktarmakYeni bir 50 ml santrifüj tüpüne numune ve kalıntı SDS kaldırmak için 24 saat boyunca hafif bir çalkalama (~ 30 rpm) 40 ° C&#39;de PBST (Tablo 1) 4 kez yıkayın. </li></ol> 4. Moleküler Boyama Dikkat: 4 &#39;, 6-Diamidino-2-phenylindole dihidroklorür (DAPI) tahriş edici ve davlumbaz ve uygun kişisel koruyucu ekipman (laboratuvar önlüğü, eldiven ve göz koruması) ile yapılmalıdır kullanan herhangi deneyler olduğunu. <ol><li> Temiz bir 15 ml santrifüj tüpüne örnek transferi ve 1 x PBST içinde 1 ug / ml DAPI, pH 7.5 ile doldurun. 24 saat oda sıcaklığında (RT) enkübe edilir. florofor korumak için alüminyum folyo ile kaplanmış numunelerin içeren santrifüj tüplerini muhafaza edin. </li><li> en az 6 saat / yıkama için oda sıcaklığında PBST içinde 3 kez yıkanır. </li></ol> 5. Refraktif İndeks Eşleştirme Dikkat: sh 2,2&#39;-Thiodiethanol (TDE) tahriş edicidir ve herhangi deneyler onu kullananDavlumbaz ve uygun kişisel koruyucu ekipman (laboratuvar önlüğü, eldiven ve göz koruması) ile yapılabilir ould. <ol><li> Temiz bir 15 ml santrifüj tüpüne örnek aktarın ve% 30 1 x PBS içinde (hac / hac) TDE, pH 7.5 ile doldurun. 24 saat boyunca hafifçe çalkalanarak 40 ° C (~ 30 rpm) inkübe edilir. </li><li> 1 x PBS, pH 7.5 içinde% 63 (h / h) TDE Exchange% 30 TDE. yumuşak, 24 saat boyunca çalkalanarak 40 ° C&#39;de inkübe edin. Not: Örnek, yaklaşık orijinal boyutuna geri çekilmek olacaktır. </li><li> düz ve temiz bir yüzey üzerine numunenin kalınlığından sadece biraz daha düzgün bir çapa sahip bir silindirin içine yeniden bir yapışkan parça döndürün. </li><li> açık uçlu daire yeniden kullanılabilir yapışkan silindiri Lay (~ üst kısmında küçük bir açıklığı olan 25 mm iç çap) 40 mm cam alt çanak. düzeltmeler yapılması gerekiyorsa, cam silindir kaldırmak ve bir jilet ile kesti. </li><li> ile yeniden yapıştırıcı ile cam arasında bir sızdırmazlık oluşturmak için bir P1000 pipet kullanınsilindirin dış kenarı etrafında yuvarlayarak. </li><li> Yeri ~ 100 ul% 63 cam tabak üzerine TDE ve yeniden yapıştırıcı daire içinde cam yüzeyini kapsayacak şekilde yayıldı. </li><li> Bir spatula ile dikkatlice kabarcıkları oluşturmak için özen çemberin ortasında numuneyi. </li><li> hemen ardından tekrar kullanılabilir yapıştırıcı üzerinde 40 mm yuvarlak cam kapak yerleştirin, doğrudan numune üzerine% 63 TDE başka 100 ul pipetle. </li><li> cam kapak örneği ile temas yapmış kadar indeksi, orta ve iki elin yüzük parmağı kullanarak, dikkatli dairenin çevresinde basın. </li><li> Yavaş yavaş bir yüzeye karşı duran dik konuma cam alt yemek getirmek Çözelti en küçük açıklığı ulaşana kadar daha sonra bir pipet yardımıyla% 63 TDE ekleyin. ücretsiz bir çözüm camına damla kalmaması pipet kapalı kuru silin bir tüy bırakmayan kullanın. odasında kabarcıkları önlemek için, pipet tüm yol boş asla. </li><li> içinküçük açma, daire içine ve kapalı bir odasını oluşturmak için silikon elastomer ekleyin. o görüntüleme önce kurumaları için 5 dakika bekleyin. </li></ol> 6. Mikroskopi Dikkat: konfokal, tek düzlem aydınlatma ve ışık levha mikroskopi kullanılan lazerler çok güçlü ve körlüğe neden olabilir. lazerler kullanan herhangi deneyler geniş eğitimden sonra ve büyük bir dikkatle ve uygun göz koruması ile yapılmalıdır. <ol><li> Bir lazer tarama konfokal mikroskop sahnede iç numune ile görüntüleme odasına yerleştirin. </li><li> görüntüleme yazılımı açın (Malzeme Tablo). Argon uyarma lazer açmak için, daha sonra programın lazer simgesinin üstündeki yapılandırma sekmesini tıklatın. lazer güç ve% 30 slayt ölçekli taşımak için argon yanındaki kutuyu işaretleyin. işaretçi yavaşça seçilen pozisyona ısınmak gözlemleyin. <ol><li> üstündeki acquire sekmesine dönün. Büyük kutusuna &quot;Işın Yolu Ayarları &amp;# 34; / Yük gitmek kutusu ayarı kaydetmek ve görüntü YFP (527 nm) için uygun bir dalga boyu kanalı seçmek için açılır menüyü seçin. </li></ol></li><li> kanal ayarları kutusunun altında: &quot;Mevcut nesne objektif&quot; etiketli kırmızı köprü bularak görüntüleme için uygun hedefleri seçin. sekmesini tıklayarak mevcut tüm hedefleri açılır ve bir seçim taret otomatik olarak dönecektir. <ol><li> Kullanım amaçları (örn., 10X), büyük bir çalışma mesafesi ile (doku kalınlığından daha büyük, yansıması için, örneğin 11 mm) ve geniş bir sayısal açıklık (örn., 0.3) düzlem resim çözünürlüğü en üst düzeye çıkarmak ve en aza indirmek için optik kesit kalınlığı. </li></ol></li><li> 1024 x 1,024 &quot;biçim&quot; denilen toplama matriks ayarlamak için pencereyi, ya da objektif lateral çözünürlük sınırı için uygun bir değer: &quot;çözünürlük XY&quot; açık, açılır menüler sol sütun üzerinde. (Mümkün olduğunca düşük yakınlaştırma faktörünü ayarlamak gibi., 10,7). </li><li> Aynı pencerede, buna göre bireysel olarak etiketli menüleri aşağı bırakarak 8 hat ortalama ve 1 çerçeve ortalama parametreleri ayarlayın. hızlı elde edilmesi için yüksek bir sinyal-gürültü oranı ve küçük değerler için büyük değerler kullanın. Not: 8 hat ortalamaları ve 1 kare ortalama yaklaşık 4 saat içinde bir fare beyin yarımkürede yüksek kaliteli görüntüler elde edecektir. </li><li> sahne kontrollerini kullanarak örnek bulun. temsili bir alan görünür sonra, kazanç ve ofset ayarlayın. kazanç ve doku tipi, fluorophores dayalı ölçüde değişecektir ofset ve / veya anatomik özellik görüntülü Not:. YFP için kazanç için 780 760 den ve -1.0 ofset için -1.5 değerleri ayarlayın. </li><li> &quot;Karo tarama&quot; seçin ve ilgi bölgenin sınırlarını belirleyin. z-yığının Set alt ve üst sınırları elde edilecek. Amaç optik bölüm çözünürlük sınırına dayalı optik bölümün kalınlığı ayarlayın. Not: Bir amaç enoptik bölüm çözünürlük sınırı öncelikle sayısal açıklık ile tanımlanır. otomatik olarak objektif bir değer uygun olan hesaplar en mikroskop kontrol yazılımı kullanılmaktadır. 0.3 sayısal diyafram ile 10X objektif için bu yaklaşık 11 mikron olacaktır. </li><li> edinimi başlatın. Not: Bu, birden çok saat sürebilir. </li><li> özel ilgi bölgelerinden daha yüksek büyütme hedefleri kullanarak yüksek çözünürlüklü görüntüler toplayın. </li></ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping.">Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.</a> Nat Protoc. 10, 1860-1896 (2015).</li></ol>

The authors have nothing to disclose.

hidrojel gömülü dokularda (pasif CLARITY) pasif takas dokusunun büyük parçalar temizlenmesi için basit ve ucuz bir yöntemdir. Bu yaklaşım, özel ekipman gerektirmez ve kolay bir şekilde, sıcaklık kontrollü sallayıcı içinde gerçekleştirilebilir. Birkaç haftalık bir sürede içinde, örneğin bütün bir beyin ya da spinal kord bile büyük, çok miyelinli dokular, şeffaf ve mikroskopi için uygun olacaktır. Bu rapor, merkezi sinir sistemi doku temizleme odaklanmış olmasına rağmen, pasif AÇIKLI...

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 tissue1-3. 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 &#181;m in laser scanning confocal microscopy and to less than 800 &#181;m using two-photon excitation microscopy4-8.
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 times15-17. 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 developed17-19. 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.

<table><tbody><tr><td>10x phosphate buffered saline (PBS)</td><td>Fisher</td><td>BP399-1</td><td>buffers</td></tr><tr><td>32% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15714-5</td><td>perfusion and hydrogel</td></tr><tr><td>2,2&#039;-thiodiethanol (TDE)</td><td>Sigma-Aldrich</td><td>166782-500G</td><td>refractive index matching solution</td></tr><tr><td>40% acrylamide</td><td>Bio-Rad</td><td>161-0140</td><td>hydrogel</td></tr><tr><td>2% bis-acrylamide</td><td>Bio-Rad</td><td>161-0142</td><td>hydrogel</td></tr><tr><td>VA-044 initiator</td><td>Wako Pure Chemical Industries, Ltd.</td><td>VA044</td><td>hydrogel</td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B7901</td><td>clearing buffer</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Fisher</td><td>BP166-5</td><td>clearing buffer</td></tr><tr><td>Sodium hydroxide</td><td>Fisher</td><td>55255-1</td><td>buffers</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>52002-100G</td><td>preservative</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500G</td><td>buffers</td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393-50KU</td><td>perfusion</td></tr><tr><td>Sodium nitrate</td><td>Fisher</td><td>BP360-500</td><td>perfusion</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>nuclear stain</td></tr><tr><td>99.9% isoflurane</td><td>Phoenix</td><td>57319-559-06</td><td>anesthetic</td></tr><tr><td>Hydrocholoric acid</td><td>Fisher</td><td>A1445-500</td><td>buffers</td></tr><tr><td>Glass bottom dish</td><td>Willco</td><td>HBSB-5040</td><td>Willco dishes</td></tr><tr><td>Reusable adhesive</td><td>Bostick</td><td>371351</td><td>Blu-Tack</td></tr><tr><td>Silicon elastomer</td><td>World Precision Instruments</td><td>KWIK-CAST</td><td>Kwik-Cast</td></tr><tr><td>Lint-free wipe</td><td>Kimberly-Clark</td><td>34120</td><td>KimWipe</td></tr><tr><td>Mouse brain matrix</td><td>Roboz</td><td>SA-2175</td><td>sectioning tissue</td></tr><tr><td>Matrix blades</td><td>Roboz</td><td>RS-9887</td><td>sectioning tissue</td></tr><tr><td>Peristaltic pump</td><td>Cole-Parmer</td><td>77122-24</td><td>pefusion</td></tr><tr><td>Laser scanning confocal microscope</td><td>Leica</td><td>SP5</td><td>microscopy</td></tr><tr><td>Imaging software</td><td>Leica</td><td>LAS AF</td><td>microscopy</td></tr></tbody></table>

optical clearing of the mouse central nervous system using passive clarity

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&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Beynin yapısal organizasyonu görselleştirme morfolojisi ve bağlantı sağlık ve hastalık beyin fonksiyonlarını nasıl etkilediğini anlamak için kritik öneme sahiptir. Optik takas teknikleri bize cohesively morfolojisi ve bağlantı incelemek için izin sağlam dokularda 3D görüntü hücre popülasyonu için mümkün kılar. Hücreler beyindeki sinir bağlantı karmaşık bir desen ayrı kızdırmak için bir izin ...

Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

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.

Pasif kullanımı Netlik Fare Merkezi Sinir Sistemi Optik Takas

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

optical-clearing-mouse-central-nervous-system-using-passive

Research

JoVE Journal

Neuroscience

13.4K Görüntüleme.  University of California, Los Angeles. 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.

Video: Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

YFP ifade Temizlendi Fare Beyin Multiphoton Mikroskopi

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Nörobilim

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Eşodaklı Işık Sac Mikroskobu ile Tüm Fare Brain mikron çaplı Çözünürlük Optik Tomografi

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Kullanımı Netlik Fare Omurilik içinde Serotonerjik Elyaf Görüntüleme / KÜBİK Tekniği

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Felç ve görsel sistem yaralanma tarafından Nöromyelitis Optica Autoantigen Aquaporin-4 T hücreleri belirli farelerde indüksiyon

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

İmmünoloji ve Enfeksiyon

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

Roman pasif optik şeffaflık bütün sinir dokusunda hızlı üretim için yöntemleri Temizleme

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

CLARITY Doku Temizleme Yöntemi ile Tüm Mount Retinaların İmmünasyonunun İmmünasyon

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

CLARITY Doku Temizleme Yoluyla Bozulmamış Nöronal Dendritlerin Görüntülenmesi ve Nicelleştirilmesi

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

Mezoskopikten Mikroskobik Ölçeklere Nöronal Görüntüleme için Bir Doku Temizleme Yöntemi

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Pasif AÇIKLIĞI temizlenmiş Mouse ovarium vasküler mimarisinin üç boyutlu yeniden yapılandırma

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Biyoloji

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...