Yazarlar bu yöntem geliştirme konusundaki yorumlar yararlı ve teknik destek için Laura Diaz, Kevin Phillips, Willa A. Hsueh ve Kral C. Li teşekkür etmek istiyorum.

1. Hayvan Hazırlama ve Soğuk Tedavi <ol><li> Laboratuvar fareleri accomodating için onaylanmıştır 4 ° C soğuk oda bulun ve inceleyin. </li><li> Soğuk oda gecede Pre-chill hayvan kafesleri. Kafesler yem ve yatak olmadan ama bir şişe su ile monte edilir. </li><li> Deney gününün sabahında, yer fareler 30 dakika aralıklarla önceden soğutulmuş kafesleri her birine göre bir. Bu görüntüleme laboratuara taşınan önce her tek tek kafesli fare yaklaşık 4 saat boyunca soğuk bir odada kalmalı. Fareler ama su erişimi olan oruç olun. </li><li> Görüntüleme Lab için bir defada 4 saat sonrası soğuk tedavi taşımacılık az bir hayvanın her 30 dk. Bu, buz ile plastik bir kap doldurma ve kutu içine buz üzerine bir ön-soğutulmuş bir konut kafes yerleştirilmesi ile elde edilebilir. Gevşekçe strafor kutusunda kapağı yerleştirin. </li></ol> 2. Kur Micro-PET/CT Görüntüleme Akışı <p class=Bu protokol micro-PET/CT görüntülemede &quot;jove_content&quot;&gt; Siemens Inveon Dedicated PET (dPET) Sistemi ve Inveon modelli (MM) demirledi modunda Sistemi (BT / SPECT) ile elde edilir. Hayvan ilk olarak anatomik referanslar için CT taraması ile MM giriş, ikinci yerleştirilir ve daha sonra bir statik F18 PET elde edilmesi için dPET merkezine doğru kaydırılır. Ana bilgisayar otomatik olarak bu sıralı görevleri yürütmek için etkinleştirmek için, aşağıdaki &quot;iş akışı&quot; önce gerçek görüntüleme oturumuna Inveon Acquisition İşyeri (IAW) yazılımı ile programlanır. <ol><li> CT Toplama: tüm vücut BT tarama için 500 cari uA, 80kV, 200 msn de pozlama süresi ve 240 ° dönüş için 240 adımları gerilim ayarlanır. X-ray dedektörü için, 78 mm aksiyel görüntüleme alanı ve tek kişilik yatak modu ile &quot;düşük sistem büyütme&quot; çözünürlük seçin. Böylece &quot;Ortak Cone-Beam İmar&quot; yöntemi ile &quot;gerçek zamanlı rekonstrüksiyon&quot; seçeneğini ile ana bilgisayar görüşmelergörevi başlatmak için özel bir gerçek zamanlı rekonstrüksiyon bilgisayar (Kobra). </li><li> PET Emisyon Toplama: &quot;zaman kazanmak&quot; seçeneğinde &quot;sabit tarama zamanı&quot; için Set 600 saniye (10 dakika). &quot;Çalışma izotop&quot; F-18 seçin ve &quot;enerji seviyesi&quot; olarak 350-650 keV kullanın. </li><li> PET Emisyon Histogram: statik tarama elde etmek için, sürenin tamamı için bir çerçeve olarak işlem verileri için &quot;siyah&quot; olarak ayarla &quot;dinamik çerçeve&quot;. Histogram türü olarak &quot;3D&quot; seçin ve &quot;hayır dağılım düzeltme&quot; seçin. </li><li> PET İmar: rekonstrüksiyon algoritması olarak 2D Z&#39;ye Altküme Beklenti Maksimizasyonu (OSEM2D) kullanın. </li></ol> 3. FDG enjeksiyonu <ol><li> Görüntüleme laboratuara onun varış ~ 30 dk zamanlanmış ilk enjeksiyon öncesi için bölgesel bir satıcıdan 18F-FDG (10 mCi) bir klinik paket sipariş. Radyoaktif malzemeler içeren paketi almak ve incelemektir enstitünün güvenlik prosedürleri takipls (RAM). </li><li> L-blok masa üstü kalkan kısım FDG ve steril serum fizyolojik ile dilüsyon yapmak tarafından sağlanan koruma ile. FDG nispi aktivite konsantrasyonu her bir enjeksiyon için 200-300 μCi/100 ul de mevcut olmalıdır. 26G 1/2 inç iğne ile bir 1 ml şırınga içine FDG çözelti çekmek ve bir doz kalibratör ile bütün şırınga radyoaktivitesi ölçülür. </li><li> Sadece intraperitoneal (ip) yoldan FDG çözeltisinden 100 ul soğuk oda (Adım 1.4 &#39;e bakınız) taşınır hayvan enjekte. Enjeksiyon zamanı kaydedin. Dozunu kalibratör ile tekrar şırınga kalıntı radyoaktivite ölçülür. </li><li> Buz ile tutulan bir köpük soğutucu içinde soğuk kafesine geri hayvan koyun. FDG alımı için, 1 saat boyunca soğuk (~ 4 ° C) içinde hayvan inkübe edin. </li><li> Aşağıdaki formül ile her bir fare için enjeksiyon FDG etkinlik hesaplanır: Enjeksiyondan önce enjektör içerisinde Injected aktivite (μCi) = aktivitesi- Enjeksiyondan sonra şırınga faaliyet </li></ol> 4. Micro-PET/CT Görüntüleme <ol><li> Micro-PET/CT görüntüleme soğuk tedaviden sonra FDG enjeksiyon ya da 5 saat sonra 1 saat başlar. Oksijen İzofluran% 3 ile anestezi indüksiyon odasına hayvan koyun. </li><li> Bir kez anestezi meydana gelir, hayvanın kafa sürekli olarak 2 L / dak &#39;lık bir akış hızında (% 2) Isoflurane sağlayan bir koni yüz maskesi içinde oturtulmuş bir mikro-pelet CT (hayvan yatağı) üzerine hareket eder. Bir elektrikli ısıtma yastığı (BioVet, m2m Imaging Corp) vücut ısısını korumaya yardımcı olmak için hayvanın altına yerleştirilir. </li><li> MM tarayıcının giriş hayvan kaydırın, araç çubuğundan &quot;lazer&quot; simgesini etkinleştirmek ve hayvanın göğüs yatay ve dikey lazer çizgileri arasında çapraz üstünde olacak şekilde yatakta hareket ettirmek için dokunmatik kontrolü kullanın. &quot;Lazer hizalama&quot; penceresinde Optio olarak &quot;PET edinimi iş akışı dahil&quot; CT tarama gibi &quot;ilk tarama tipi&quot; seçin ven. </li><li> &quot;Scout Görünüm&quot; penceresini açın ve x-ray grafisi bir izci görünüm kazanır. CT bakış merkez alan fare gövde (karaciğer) merkezinde yer alır, böylece hayvan yatağın pozisyonunu ayarlamak için IAW kullanın. Bu adımı gerekirse tekrarlayın. </li><li> Adım 2 yılında kurulan &quot;iş akışı&quot; başlayın. Seçenekleri açılır, CT yeniden kullanılmak üzere uygun bir 3D PET-BT dönüşüm matrisi dosyayı seçin ve zayflama düzeltme ile PET rekonstrüksiyon için yeni oluşturulmuş normalleşme dosyası seçin. IAW sonra BT ve PET taramaları sıralı olarak programlanmıştır başlayacaktır. </li><li> Tüm iş akışı tamamlandıktan sonra, hangi 20-25 dakika sürebilir, kısaca edinilen CT ve ASIPro VM, bir mikro-PET analiz yazılımı IAW ile birlikte yüklü PET görüntülerinin kalitesini değerlendirmek. Arşiv görüntüleme bir veri depolama aygıtına veri veya daha fazla analiz için bir post görüntü analiz istasyonu (Adım 5) ağ üzerinden veri aktarımı. </li><li> Release görüntüleme sistemlerinden hayvan ve oda sıcaklığında kurtarma için normal gıda ve su ile temiz bir konut kafesine yerleştirin. Sistemleri artık kuyruğunda sonraki hayvan için hazırız. Sonrası görüntüleme hayvanların bakımı ve kullanımı &quot;RAM ile enjekte laboratuar hayvanlarının kullanımı&quot; ile ilgili enstitünün düzenlemelere uymak gerekir unutmayın. Ayrıca enstitünün ilgili RAM atık bertaraf yönetmeliklerine göre iğne / şırınga, emici pedler, eldiven, ve tüm yatak kullanılan ve dışkı, radyoaktif atık olarak kabul edilmeli ve ele unutmayın. </li></ol> 5. Post-görüntüleme Analizi <ol><li> El Açık Inveon Araştırma İşyeri (IRW) yazılım (Siemens) ve BT ve PET veri setleri hem içe. &quot;Kayıt&quot; &quot;Genel analizi&quot; fonksiyonu ile araçlarını kullanarak pencere ve &quot;Yorum&quot; penceresi altında Co-kayıt BT ve PET görüntüleri BT ve tüm 3 boyutlu PET görüntüleri arasında mükemmel bir uyum emin olun. </li><li>Co-kayıtlı BT görüntüleri tarafından sağlanan referansları ile &quot;Region of Interest (ROI) Niceleme&quot; penceresinde, Nereden, boyun, erişkin farelerde en baskın soğuk indüklenebilir BAT interskapular bölgede BAT belirlemek ve ilgi hacmi çekmek (VOI) PET veri seti üzerinde BAT. &quot;Niceleme Türü&quot; olarak &quot;Voksel Yoğunluğu&quot; seçin ve Bq / ml olarak VOI içinde ortalama radyoaktivite kaydedin. Bq / ml sayım / sn dönüştüren bir kalibrasyon faktörü önceden bilinen radyoaktivite ile bir hayalet tarama tarafından kurulmuştur. </li><li> BAT FDG tutulumu çürüme düzeltme gram doku başına yüzde enjekte doz (% ID / g) olarak ölçülür. Enjekte edilen doz Adım 3.5 sonucu olan, ancak, becquerel (Bq) bloğu (1 μCi = 37,000 Bq) dönüştürülür, bu doku, 1 ml 1 g eşit olduğunu varsaymaktadır. </li></ol>

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Çıkar çatışması ilan etti.

Bu çalışmada, bir micro-PET/CT-based görüntüleme yöntemi sadece bir soğuk işleme ve 18 F-FDG ticari olarak mevcut bir enjeksiyon gerektirir yetişkin farelerde BAT aktiviteleri tespit etmek için geliştirilmiştir. Tüm prosedürü bir tedavi ve bütün hayvanların tedavi ve görüntülü kadar her 30 dakikada başlar görüntüleme sırasını izleyerek bir gün içinde yapılabilir. Anlatılan deney koşulları altında, 10 fareler (5 fare ya da 2 grup) toplam tek bir görüntüleme sistemi ile a...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Micro-PET/CT Görüntüleme Sistemi </td><td> Siemens Tıp Çözümleri USA, Inc </td><td> Inveon Dedicated PET Sistemi ve Inveon modelli BT / SPECT Sistemi (yerleştirilmiş) </td><td></td></tr><tr><td> Propranolol </td><td> Sigma </td><td> P0884 </td><td></td></tr><tr><td> BRL 37344 </td><td> Sigma </td><td> B169 </td><td></td></tr><tr><td> 18F-FDG </td><td> Cyclope A.Ş. </td><td></td><td></td></tr><tr><td> C57BL/6J Erkek Fare </td><td> Jackson Laboratuvarı </td><td> 000664 </td><td> 3-4 aylık </td></tr></tbody></table>

functional imaging of brown fat in mice with 18f-fdg micro-pet/ct

Kahverengi yağ dokusu (BAT) ayrık konumu ve zengin vaskülarizasyon ve mitokondri yüksek yoğunluk nedeniyle kahverengi-kırmızı renkli beyaz yağ dokusu (WAT) farklıdır. BAT enerji tüketimi ve non-titreme termojenezdeki yeni doğmuş, memelilerde hem de yetişkin 1 önemli bir rol oynar. BAT-aracılı thermogenesis yüksek β adrenerjik reseptör 2, 3 ile ağırlıklı olarak, sempatik sinir sistemi tarafından düzenlenir. Son çalışmalar yetişkin insan BAT faaliyetlerini olumsuz vücut kitle indeksi (VKİ) ve diğer diyabetik parametreleri 4-6 ile ilişkili olduğunu göstermiştir. BAT böylelikle anti-obesity/anti-diabetes terapisi enerji dengesi 6-8 modülasyonu odaklanarak için potansiyel bir hedef olarak öne sürülmüştür. Birkaç soğuk sorgulama tabanlı pozitron emisyon tomografisi (PET) yöntemleri insan BAT 9-13 algılamak için kurulmuş olsa da, görüntüleme ve quantif için standardize protokol aslında yokturfare gibi küçük hayvan modellerinde BAT ication. Burada farelerde BAT fonksiyonel değerlendirmesi için sağlam bir PET / BT görüntüleme yöntemi açıklanmaktadır. Kısaca, yetişkin C57BL/6J fareler, 18 F-Florodeoksiglukoz (FDG) bir doz almış önce 4 saat süreyle açlık koşullarında tedavi soğuk vardı. Fareler bir ilave saat sonrası FDG enjeksiyon için soğuk olarak kalmış ve daha sonra küçük bir hayvan adanmış micro-PET/CT sistemi ile tarandı. Kazanılmış PET görüntülerinde anatomik başvuruları için BT görüntüleri ile birlikte tescil ve BAT faaliyet sunmak interskapular BAT alanda FDG tutulumu için analiz edildi. Bu soğuk muamele standardize ve görüntüleme protokol farmakolojik faaliyetler sırasında test BAT ile onaylanmıştır, örneğin, β-adrenoseptör 14, 15 propranolol antagonist, agonist ya da β3 BRL37344 16 tarafından geliştirilmiş BAT aktivasyon işlemi ile bastırılmış BAT aktivasyonu. Yöntem desburada açıklanan şekilde değiştirin BAT aktivitesini düzenleyen, ya da gen / çeşitli klinik öncesi ve temel araştırmalar BAT geliştirilmesi ve düzenlenmesi dahil olan yollar belirlemek için ilaç / bileşiklerin taranması için kullanılabilir.

Fare BAT micro-PET/CT görüntüleme bir örneği Şekil 1&#39;de gösterilmiştir. BT görüntüleme anatomik bilgi sağlar iken, PET görüntüleme bütün vücutta 18F-FDG tutulumunun dağılımı ve miktarı kodlar. Bu görüntüleme verileri (1C) erimiş, ya da maksimal intensite projeksiyon (MIP, 1D) gibi bir 3D özelliği ile gösterdi (1A ve 1B) ayrı ayrı izlenebilir. 3 boyutlu bir görüntüleme aracı, ilgilenilen bir hacim (VOI) yardımı ile, burada interskapular BAT bölgesi <s...

Watch this Scientific Journal Video about Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT at JoVE.com

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Fare kahverengi adipoz doku (BAT) işlevsel görüntüleme için bir yöntem olup BAT 18F-Florodeoksiglukoz soğuk-uyarılmış alımı (FDG) invaziv olmayan standart bir protokol micro-PET/CT ile değerlendirilmiştir olduğu tarif edilmektedir. Bu yöntem, fare modellerinde BAT faaliyetlerinde farklılıkları tespit etmek için sağlam ve hassastır.

FDG micro-PET/CT ile Fare Brown Fat Fonksiyonel Görüntüleme

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

functional-imaging-of-brown-fat-in-mice-with-18f-fdg-micro-petct

Research

JoVE Journal

Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

19.0K Görüntüleme.  The Methodist Hospital Research Institute, Houston. Fare kahverengi adipoz doku (BAT) işlevsel görüntüleme için bir yöntem olup BAT 18F-Florodeoksiglukoz soğuk-uyarılmış alımı (FDG) invaziv olmayan standart bir protokol micro-PET/CT ile değerlendirilmiştir olduğu tarif edilmektedir. Bu yöntem, fare modellerinde BAT faaliyetlerinde farklılıkları tespit etmek için sağlam ve hassastır.

Video: Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Fareler için açık / koyu Geçiş Testi

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Biyoloji

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Demir Oksit hESC ve hMSCs Etiketleme MR Görüntüleme in vivo Takip Non-Invasive Nanopartiküller

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Takviye, Eyetracking ve Fizyolojik İzleme Fonksiyonel Görüntüleme

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Fareler için Yükseltilmiş Artı Labirent

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Kurul, Yükleme ve Analitik ultrasantrifüjdeki Örnek Hücre Hizalama

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Görüntüleme Ca Teknikleri 2 + İnsan Sperm sinyal

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

Fare Geleneksel NASA Hindlimb Boşaltma Model bir Alternant Yöntemi

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Theta sonrası etkileri Haritalama Fonksiyonel Görüntüleme ile İnsan İşitsel korteks üzerine Stimülasyon Burst

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Transgenik Olmayan Omurgasızlarda Tek Nöron Çözünürlüğü ile Ağ Dinamiklerini Kaydetmek için Fotodiyot Tabanlı Optik Görüntüleme

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...