The authors acknowledge Howard Berg for the gift of the bead-tracking microscope/photomultipliers and the Texas A&amp;M Engineering Experiment Station for funds.

1. Hücre Hazırlanması <ol><li> 10 ml taze TB 100 seyreltme: 1 ile inokülasyon ardından Tryptone Broth (TB,% 1 pepton,% 0.5 NaCl) alleli 15,22 yapışkan Flic taşıyan istenen gerginlik gecede kültürleri büyütün. OD 600 = 0.5 kadar bir çalkalama inkübatöründe 33 ° C&#39;de kültür büyütün. </li><li> 1,500 xg&#39;de hücreleri Pelet 5-7 dk yeniden disperse pelet kuvvetli bir şekilde süzülerek sterilize motilite tamponu 10 mL (MB; 10 mM fosfat tampon maddesi ile: 0,05-0,06 M NaCl, 10 4 M EDTA, 1 uM metionin pH 7.0). </li><li> Adımı yineleyin 1.2 iki kez daha ve 1 mL MB son pelet yeniden dağıtmak. </li><li> (- 12 cm uzunluğunda, 0.58 mm iç çapı 7) polietilen boru ile bağlı 21 ila 23 ölçü adaptörleri ile iki şırınga arasında ileri ve geri ~ 75 kez geçerek süspansiyon kayma. 45 s - 30 kesme için toplam süreyi sınırlayın. </li><li> 5-7 dakika boyunca 1.500 xg&#39;de makaslanmış hücreleri Santrifüj ve yeniden dağıtmakMB 500 ul - 100 pelet. </li></ol> 2. Slayt Hazırlama <ol><li> Bir kapak kayma ve bir mikroskop lamı arasında iki çift taraflı yapışkan bantlar sandviç ile bir görüntüleme odası hazırlayın. kemotaksis deneyleri için, MB ve kimyasal uyarıcılar alışverişini sağlayan herhangi bir mikroakışkan odasını kullanır. </li><li> odası içinde% 0.01 poli-L-lisin solüsyonu ilave edin ve 5 dakika sonra yavaşça MB yüzeyleri yıkayın (80-100 ul). </li><li> odasına hücre süspansiyonu 40 ul ekleyin ve cam yüzeyine bağlanması için yeterli bir süre sağlar (7-8 dakika). Diğer taraftan bir filtre kağıdı ile çözelti emme ise, odacığın bir tarafında 100 uL MB eklenerek yapışmamış hücreleri dışarı akar. </li><li> odasına lateks boncuk 15 mcL ve boncuk yerleşmek ve hücrelere takmak için yeterli zaman tanımak - 10 Ekle (7-8 dk). boncuk ayrılmak kaldırmak için, adım 2.3 anlatıldığı gibi yavaşça, MB 100 uL ile yıkayın. boncuk-si bir dizi kullanıniyi bir kontrast mevcut sürece deneyler için zes. </li></ol> 3. Boncuk İzleme <ol><li> Bir mikroskop sahnede örnek yerleştirin ve motorlara bağlı boncuklar için yüzey taraması. Faz mikroskopi gerekli değildir, ancak gözlem yapmaya 40X fazlı amacı kullanın. Alternatif olarak, bu kadar uzun yeterli kontrast açıkça karanlık bir arka plan üzerinde parlak boncuk ayırt etmek korunur olarak parlak alan görüntüleme kullanır. </li><li> Bir kordon seçildikten sonra, Şekil 1B &#39;de gösterildiği gibi önceden belirlenmiş bir köşesinde boncuk konumlandırmak için yanal olarak sahne hareket ettirin. aynı köşede Pozisyon boncuk tanenin dönme yönü doğru bilinen emin olmak için. İdeal boncuk yörünge yaklaşık dairesel ama eliptik yörüngeleri kabul edilebilir. </li><li> aliasing ile ilişkili hataları önlemek için motorun iki dönme frekansı daha örnekleme frekansı yüksek koruyun. Bu çalışmada, dönen bir motor kullanmak10 kat daha yüksektir (500 Hz) olan frekanslarda 50 Hz ve örnek, düzgün bir sinyal elde edildi. </li></ol> 4. Veri Analizi <ol><li> 23 Gerekirse Merkezi afin dönüşümler yörüngelerde PMT çıkış gerilimleri ve doğru ekliptiğinde ölçek ve. Rotasyon oranlarını 17 belirlemek için bir güç spektrum analizi kullanın. </li><li> polar açıları, θ (t) = Atan (y (t) / x (t)) belirler. motor devir değişimleri belirlemek ve omega hesaplayarak zamanla geçiş <img alt="denklem 1" src="/files/ftp_upload/55240/55240eq1.jpg" /> 14. </li><li> Motor hız verilerini düzeltmek için bir medyan filtresi kullanır. Iki tam rotasyonlar üzerinde bir filtre penceresi 23,24 tavsiye edilir. </li></ol>

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The authors have nothing to disclose.

gergin boncuk izleme ve motor tork doğru tahmin kolaylaştırmak amacıyla, aşağıdaki bilgiler gözden geçirilmelidir. flagellated hücrelerle bu ölçümleri yaparken, kesme kritik bir adımdır. Kesme ve böylece motordaki viskoz yük boncuk ağırlıklı nedeniyle ve% 10 hata 16 içinde tahmin edilebilir olmasını sağlamak, sadece saplama flagellar filamanın azaltır. Kesme de sıkı dağıtılan dışmerkezliliğinden (&lt;boncuk çapı 14) ile dairesel yörüngeleri bulma şansını artırır. izleme ve yapışkan drags hesaplanmasında yanı sıra zayıf sinyal-gürültü oranı ile sonuçlanan hataları birleştirmeler şımarık yörüngeleri, uygunsuz kesme sonuçları. Bir osiloskop kullanımı gibi verilerin hızlı eleme sağlar. kamçılı ipliklerin biyomekanik özellikleri türleri ile değiştirilmesi için beklendiği için, kesme yöntemleri olasılıkla t yeterli kesme sağlanması için uygun olması gerekirO ilgi bakteriler. makaslama ile ilişkili hataları azaltmanın etkili bir yoludur lif proteinlerini kodlayan genlerden yoksun hücreler ile çalışmaktır. Prob boncuklar daha sonra anti-kancalı antikorları ile kanca doğrudan bağlı olabilir. uygun zor olabilir gergin bir boncuk bulma. görüş alanında en boncuklar ya hücre gövdeleri veya cam yüzeyine yapışmış olacak olmasıdır. Bu tür boncuklar kolayca keskin odak haline getirilebilir. Diğer boncuklar titremesini görünür ya da büyük genlikleri veya büyük dışmerkezliliğinden ile gözle görülür döner (&gt; 1.5 - 2x boncuk çapı). Bunlar tipik olarak tam olarak kesilmiş veya fokal düzleme eğimli bir düzlemde döner edilmemiştir filamanlar flagella gergin bulunmaktadır. Bu tür boncukların örnekleme tipik olarak daha yüksek gürültü ile sonuçlanır, ve yapışkan yükleri zaman varyasyonlar belirli bir tane boyutunda için motorlar torkların düşük hesaplanmasına sebep olabilir. gergin boncuk küçük bir kısmı rastgele uğrayacaktırhareket; Bu sadece Brown rotasyon geçiyor. boncuk bir kısmı bulanık görünecektir ve kolayca odak getirdi olamaz. Bunlar uygun gergin ve ilgi motorlardır edilmiş motorlar olması muhtemeldir. Burada açıklanan gibi izleme tek motor sınırlamalar arasında yüksek verimlilik deneyler için yetersizliğidir. Bu görüntüler yüksek hızlı bir kamerası, ilgili daha geniş bir bölgesi bu konuda avantajlı olabilir. Diğer sınırlamalar birden fazla sinyal Proje Yönetim Ekipleri görüş alanında, birbirine yakın döner boncuk kaynaklanan ilişkili hata içerir. Son olarak, iki foto çoklayıcı yarıkları ile ilgili olarak kaydedilen boncuk doğru konumunun belirlenmesinde hatalar sviçleme dinamikleri kesin tahmin neden olur. Burada tarif edilen kurulum avantajları, uzun süreler boyunca gerçek zamanlı olarak boncuk dönüşünü izlemek için yeteneğini içerir. Buhata eğilimli yörüngeleri hızlı eliminasyonu, belki zor ultra hızlı kamera ile elde şey sağlar. Buna ek olarak, bir kaç değişiklik ile ayar çeşitli uyaranlara hücrelerin maruz için tasarlanmış tahliller ile entegre edilebilir. Termoelektrik 26 Soğutma ile birlikte, teknik termal uyaranlara tek tek motorlar tepkilerini ölçmek için kullanılabilir. Son zamanlarda 6 yapıldığı gibi optik cımbız ile entegrasyon, mekanik uyaranlara tepki olarak bireysel motorların yeniden ölçümlerini etkinleştirebilirsiniz. Son olarak, kimyasal uyarıcıların motorun adaptasyon uygun bir perfüzyon odasının kullanımı ile ölçülür ve 11 pompa olabilir. Bilinen bakteri türlerinin büyük bir kısmı hareketli ve flagellar aracılı motilite doğada baskındır. Burada gösterilen yöntemler yapısal-yeniden içine anlayışlar geliştirilmesi ve uyum o yardımcı devam etmesi bekleniyorflagellar motora ön.

Kamçılı motorlar sarmal dışı filamentler çevirerek yüzmeye hücreleri sağlar. Motor flagellum&#39;un (yani, viskoz yük) belirli bir uzunluk için oluşturabilir tork miktarı, yüzme hızlarını belirler. Öte yandan, dönüş yönünü değiştirmek için yeteneğidir kimyasallar, kemotaksis olarak bilinen bir işleme karşılık olarak hücre göçü kontrol eder. Kemotaksis ve hareketlilik olan virülans 1-3, kamçılı motorlar yıllar 4 üzerinde iyi karakterize edilmiştir faktörleri. Montaj kanıtlar motor mekanosensör olarak işlev gördüğünü düşündürmektedir - mekanik katı yüzeylerde 5,6 varlığını algılar. Bu yetenek muhtemelen yüzey kolonizasyonu ve enfeksiyonlara 5,7 tetikleme yardımcı olur. Bunun bir sonucu olarak, motorlu yüzeyleri ve başlatır sinyal algılar ve böylece mekanizmaları önemli 8,9 sahiptir. Flagellar motoru kolayca flagell tethering ele alınabilirum bir alt tabakaya ve hücre dönme gözlemleyerek. Bu tür bağlama birinci anti-kancalı antikorlar 10 cam alt tabakalar için polyhook E. coli içinde mutant ve başarılı bir şekilde bağlanmış kanca ile çalışmış Silverman ve Simon, elde edildi. gergin hücre deneyi kimyasal uyaranlara çeşitli motor anahtarı yanıtları çalışma etmesini sağlamıştır. Örneğin, Segall ve arkadaşları kimyasal iyontoforetik pipetler yardımıyla gergin hücreleri uyarılır. CW önyargı karşılık gelen değişiklikler kemotaksis ağı 11,12 adaptasyon kinetiği ölçmek için onları etkin (zaman motorların fraksiyonu CW, saat yönünde döndürmek). Gergin hücre deneyi anahtarı yanıtları okuyan etkili iken, sadece viskoz yükleri 13 sınırlı bir aralıkta motorlu mekaniği içgörü sunmayı başardı. Bu sorunu aşmak için, Ryu ve arkadaşları yüzeylere yapışmış hücreler üzerinde koçanları filament için küresel, lateks boncuklar gergin. boncuklar vardıDaha sonra zayıf optik tuzakları 14 ile geri odak interferometrik kullanılarak izlenir. farklı boyutlarda boncuk ile birlikte çalışarak, araştırmacılar yükler çok daha geniş bir aralıkta motoru çalışma olabilir. Bu tahlil, daha sonra lazer karanlık alan aydınlatma ile birlikte photomultiplier tabanlı boncuk izleme tekniğini geliştirdi Yuan ve Berg tarafından geliştirildi. Dış viskoz dirençleri rotasyon 15,16 iç viskoz dirençleri kıyasla düşük olduğunu bu nedenle küçücük gergin altın nanobeads (~ 60 nm) Onların yöntemi etkin izleme. Bu, E. coli içinde elde edilebilen maksimum hızdan (~ 300 Hz) ölçümlerine yol açtı. V. alginolyticus&#39;un olarak, benzer boncuk deneyleri ara viskoz yükler (~ 700 Hz) 17 yaşında iplik oranları ölçümleri sağladı. (Sıfır yükten yakın durak) viskoz yüklerin tüm olası aralığında motor yanıtların ölçümleri sağlayarak, boncuk deneyleri t anlamak için önemli bir biyofiziksel araç sağladıorque nesil süreci 18,19. Son zamanlarda, bireysel motorlar 6 hassas mekanik uyaranlara uygulamak için etkin optik cımbız içerecek şekilde Yuan-Berg tahlil güncellenmiştir. Onlar viskoz yüklerde değişikliklere tepki olarak pişmanlık - Bu tekniği kullanarak, biz motoru döndürmek kuvvet jeneratörler dinamik mechanosensors olduğunu göstermiştir. Mekanizmaları tam olarak bilinmemektedir, ancak böyle bir yük algılama, kaynıyordu bakterilerin içine hücre farklılaşmasını tetikler mümkündür. Doğrudan bir kanıt yoktu, ancak diğer türlerdeki flagellar motorlar da 20 mechanosensitive olması da muhtemeldir. Burada, kamçılı filamentler 15 gergin lateks boncuk dönüşünü izlemek için photomultiplier tabanlı (PMT) yaklaşımları tartışıldı. gerçek zamanlı olarak ve uzun dura üzerinde tek boncuk izlemek için nispeten basittir, çünkü çok hızlı kameralarla izlemeye karşılaştırıldığında, photomultiplier-Kur avantajlıleri. Nedeniyle çevresel uyaranlara 21 Flagellar motoru komplekslerinde biçimlenme uzun süre eğitim zaman özellikle yararlıdır. Özel olarak, E. coli için ayrıntılarıyla protokolleri rağmen, hali hazırda diğer türlerdeki flagellar motorlar çalışmak için adapte edilebilir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-L-lysine Solution (0.1%)</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Polybead Microspheres</td>
			<td>Polysciences, Inc.</td>
			<td>7307</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>1 ml Luer Slip Tip Syringe</td>
			<td>Exel Int.</td>
			<td>26048</td>
			<td>http://www.exelint.com/tuberculin_syringes.php</td>
		</tr>
		<tr>
			<td>Clay Adams Intramedic Luer-Stub Adapter 23-gauge</td>
			<td>Becton, Dickinson and Company</td>
			<td>427565</td>
			<td>http://www.bd.com/ds/productCenter/ES-LuerStubAdaptors.asp</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Harvard Apparatus</td>
			<td>59-8325</td>
			<td>http://www.harvardapparatus.com/laboratory-polye-polyethylene-non-sterile-tubing.html</td>
		</tr>
		<tr>
			<td>Photomultiplier Tubes</td>
			<td>Hamamatsu</td>
			<td>R7400U-20</td>
			<td>Spectral response range of 300 to 920 nm, Peak wavelength 630 nm,&#160; 0.78 ns response time&#160; 
			http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html</td>
		</tr>
		<tr>
			<td>3x1 mm precision slits</td>
			<td>Edmund Optics</td>
			<td>NT39-908</td>
			<td>2 slits mounted at right angles to one another on photomultiplier tubes</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TBS 1032B</td>
			<td>Alternative brands are acceptable. Digital Oscilloscope, TBS 1000B Series, 2 Analogue, 30 MHz, 500 MSPS, 2.5 kpts&#160; 
			http://www.tek.com/oscilloscope/tbs1000b-digital-storage-oscilloscope</td>
		</tr>
		<tr>
			<td>8 Pole LP/HP Filter</td>
			<td>Krohn-Hite</td>
			<td>3384</td>
			<td>Alternative brands are acceptable. A frequency range from 0.1 Hz to 200 kHz is recommended.&#160;&#160;&#160; 
			http://www.krohn-hite.com/htm/filters/PDF/3384Data.pdf</td>
		</tr>
		<tr>
			<td>Optiphot microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>Any upright or inverted phase microscope can be used. 
			https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=754</td>
		</tr>
		<tr>
			<td>50:50 (R:T) Cube Beamsplitter</td>
			<td>ThorLabs</td>
			<td>BS013</td>
			<td></td>
		</tr>
	</tbody>
</table>

biophysical characterization of flagellar motor functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Photomultiplier kurulumu Şekil 1A&#39;da gösterilmiştir. Proje Yönetim Ekipleri ilgi boncuk tarafından dağınık dalga boyları aralığında yüksek hassasiyetleri sahip olması önemlidir. Burada kullanılan Proje Yönetim Ekipleri görünür ve yakın kızılötesi aralıkları faaliyet ve halojen ışık kaynağı tarafından aydınlatılan boncuk tarafından saçılan ışık tespit başardık. Optimum aydınlatma koşulları ve stok gerilimleri bir kurulum diğerine değişecektir. Bu çalışmada kullanılan kurulum, bir PMT kazanç ~ için 10 Nisan-10 Mayıs yeterli olduğunu kanıtladı. Her photomultiplier foto çoğaltıcı önüne yerleştirilmiş bir 3 x 1 mm yarık dışında kaplandı. Yarıklar ışık fotoçoğaltıcılar girebilir olan hücre numunesinde bölge sınırlar ve iki yarık, birbirine ortogonal olan. Dönen boncuk doğru konuma (Şekil 1B) yerleştirilmiş olduğunda, photomultiplier artar giren ışık miktarı boncuk geliyordairesel yol olarak görünümü ve azalır uzak görünümden alır. sinüzoidal PMT gerilim çıkışlarının frekansları dönme hızını gösterir ve iki sinyal arasındaki faz farkları dönme yönünü gösterir. PMT çıkışları görüntülemek için bir osiloskop kullanımı gerçek zamanlı olarak boncuk yörüngeleri görselleştirme sağlar. Temsili bir motordan zamanla değişen PMT sinyalleri, y (t) X (t), Şekil 2A&#39;da gösterilmiştir. İki yarıkların diklik iki sinyal arasında bir faz boşluğu getirmektedir. sinyal amplitüdleri, sinyal-gürültü oranı hem de dönme eksantrikliği bağlıdır. Boncuk mukabil yörüngeleri Şekil 2B&#39;de gösterilmiştir. Bir Chey bir Örnek motorundan ölçülen hızları histogram - silinmiş streyn Şekil 3A&#39;da gösterilmiştir </strong&gt;. silme Flagellar motorlar geçiş ve münhasıran saat yönünün tersine (sola) döndüremezsiniz sağlar. Bu özel motordan ortalama hızı 60 Hz ve ölçülen hızlar 1 mikron boncuk 25 ile gergin motorlar için önceki raporlarda ile tutarlıdır. Şekil 1B şematik olarak görüldüğü gibi kordon İlk olarak, sağ alt köşesinde yerleştirildi. Karşılık gelen açısal hızı Şekil 3B&#39;de (üst panel) &#39;de gösterilmiştir. bitişik sol alt köşesine boncuk konumlandırma motor hızının işareti (alt panel) ters sonuçlandı. Bu durumda, bitişik bir köşe boncuk hareket motorunu dönme gözlenen yönünü değiştirecektir. Bu bağlamda, çapraz köşe aynıdır. Doğru anahtarlama dinamikleri belirlemek için ölçümler sırasında boncuk yerini bilmek çok önemlidir. Şekil 3C, dönme iki yönde doğal tipte bir motor tekrarlanan geçişleri gösterir. <html> Veri toplama yazılımı için özel kodlar bir bilgisayarda 15 veri kaydetmek için önce işten adapte edilmiştir. PMT çıkış AC ile birleştirilmiş ve alçak geçiren 100 Hz kesme frekansı ile filtre edilmiştir. Gerçek zamanlı izleme bir osiloskop süzülmüş çıkışlarını bağlayarak sağlanmıştır. <img alt="Şekil 1" src="/files/ftp_upload/55240/55240fig1.jpg" /> Şekil 1: Boncuk izci Kur. PMT tabanlı izleme kurulumu A) şematik. B) iki ortogonal yarık boncuk (siyah küre) akrabası ideal konumu. Yörünge noktalı çizgilerle gösterilir. Dışmerkezlik e noktalı dairenin yarıçapı. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> ove_content &quot;fo: keep-together.within sayfa =&quot; 1 &quot;&gt; <img alt="şekil 2" src="/files/ftp_upload/55240/55240fig2.jpg" /> Şekil 2: PMT Çıkışlar. A) Alçak geçiren merkezleme / ölçeklendirme sonra, iki fotoçoğaltıcılar çıkışlarını süzülür. B) 3 saniye boyunca örneklenmiş PMT verileri, elde edilen boncuk yörüngeleri. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/55240/55240fig3.jpg" /> Şekil 3: Boncuk Yörüngeler. A) temsili bir motor CCW sadece hız Histogram. B) sağ alt köşesinde (üst panel) görüntülü bir CCW sadece motorun dönme hızı. Aynı motorun dönme hızları sol alt köşesinde (alt panel) konumlandırılmış zaman. C) </strong&gt; temsili vahşi tip motorda geçiyorum. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Biophysical Characterization of Flagellar Motor Functions at JoVE.com

Biophysical Characterization of Flagellar Motor Functions

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

Kuyruktaki Motor fonksiyonların Biyofiziksel Karakterizasyonu

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

biophysical-characterization-of-flagellar-motor-functions

Nanyang Technological University

Research

JoVE Journal

Biology

8.2K Görüntüleme.  Texas A&M University. Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

Video: Biophysical Characterization of Flagellar Motor Functions

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

Yakın Kızılötesi Spektroskopisi ile bebekler, Çocuk ve Yetişkin Bilişsel Fonksiyonları keşfetmek

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Biyoloji

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Hazırlanması Aplysia Duyusal-motor Nöronal Hücre Kültürleri

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Parafin ve Dondurulmuş Bölüm Drosophila Yetişkin Kaslar

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

İzolasyon ve Meyve cuticles biyofiziksel Çalışması

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Genomik DNA 5-hydroxymethylcytosine seçici Yakalama

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Gen Yönetmelik Tek molekül Görüntüleme<em&gt; In vivo</em&gt; Dilinim tarafından Cotranslational Aktivasyon (CoTrAC) kullanma

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bakteriyel Bölümü Makine Super çözünürlüklü Görüntüleme

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

Elektrofizyoloji Tabanlı Katı Desteklenen Membran Giriş

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Verimli Üretimi ve Saflaştırılması Rekombinant murin kindlin-3 Biophysical Çalışmaları Böcek hücrelerinden

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.