The authors thank Drs. Steven Chu and Joe W. Gray for helpful discussions, Henry Marr for his initial work on the BiFC-PALM project, and Alexis Shoemaker for her technical assistance. This work is supported by startup funds to X.N. from OHSU. Research in the Nan laboratory was also supported by NIH 5U54CA143836-05, the Damon Runyon Cancer Research Foundation, the M. J. Murdock Charitable Trust, and the FEI company.

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The authors have nothing to disclose.

PALM sağlam biyolojik örneklerin nano ölçekli sokulmuşlardır son tek bir molekül superresolution mikroskopi tekniği ise BiFC, tespit ve hücrelerde PPIs görselleştirmek için yaygın olarak kullanılan bir teknik, olmuştur. PALM ile BiFC kombinasyonu nanometre mekansal çözünürlükte ve tek bir molekülün duyarlılığı ile bir hücrenin içinde PPİ seçici görüntüleme elde etti. BiFC PALM her iki tekniğin yararını uzanır ve bu çalışmada gösterildiği gibi, kendi ana hücresel bağlamda PPİ ...

Protein-protein etkileşimleri (PPİ) biyoloji 1 için temel olan ve sıkı birçok zaman ve uzunluk ölçeklerinde genelinde zamanmekansal mekanizmalar üzerinden düzenlenir. Hücre zarı üzerinde hücre sinyal ile ilgili çalışmalar, örneğin, belirli bir PPIs ve hücresel süreçlerin 2 kolaylaştıran dinamik nano ölçekli mekansal bölmeleri göstermektedir. Bu nedenle, yeterli mekansal ve zamansal çözünürlükte, yüksek özgüllük ve yüksek hassasiyet ile biyolojik sistemlerde PPIs prob yeteneği biyoloji mekanistik anlayış ulaşmanın anahtarıdır. 5 - Bimoleküler floresan tamamlama (BiFC) hücre içi çözünürlük ve canlı hücre uyumluluğu 3 bir hücreye PPIs görselleştirmek için birkaç mevcut araçlardan biridir. Tekniği oldukça basit ve iki floresan olmayan parçalara bir floresan proteininin yarma içerir; genetik iki etkileşen proteinlere etiketlendi zaman veyakınına getirildiğinde, fragmanları, flüoresan sinyali verecek şekilde tam bir floresan protein oluşturmak üzere sulandırmak olabilir. Doğru tasarlandığında, BiFC probları kendiliğinden PPİ yokluğunda sulandırmak gerekir. Bunun gibi, bir BiFC tahlilinde floresan sinyali sadece yüksek özgüllük ile PPİ doğrudan görüntülenmesine olanak tanıyan PPİ varlığında ortaya çıkacaktır. Okuma olarak floresan kullanmanın ek yararlar diğerleri arasında yüksek verimlilik ve yüksek içerik tarama deneyleri, yüksek hassasiyet, subsellüler çözünürlük ve uyumluluk. Bu faydalar, farklı üst floresan proteinleri göre BiFC probları bir dizi geliştirilmiştir. Geleneksel ışık mikroskopisi göre tüm diğer algılama teknikleri gibi, ancak, BiFC uzaysal çözünürlüğü ışık difraksiyonu ile ~ 250 nm ile sınırlıdır. Bu lipit sallar 6 a göre daha önce değindiğim ve örneklendiği gibi, nano, at PPİ düzenlenmesini incelemek için bunu bir meydan okuma yaparnd Ras nanokümeler 7, örneğin sinyalizasyon gibi birçok hücresel süreçleri anlamak için kritik bir uzunluk ölçektir. BiFC PPIs 10 görüntülenmesi için mekansal çözünürlükte bu sınırı aşmak için fotoaktive yerelleştirme mikroskobu (PALM) 8,9 ile kombine edilmiştir. PALM stokastik aktivasyonu ve tek flüoresan moleküllerinin subdiffractive lokalizasyonu ile floresan görüntüleme kırınım sınırı atlatır son superresolution mikroskopi tekniğidir. Her bir aktivasyon çevriminde, bir flüoresan molekül birkaç bin fotonlara birkaç yüz verir ve dedektör üzerinde tek bir molekülün bir görüntüye yol açmaktadır. Görüntü kırınım sınırlı olsa da (~ genişliği 250 nm) olarak, ağırlık merkezi, tipik olarak tespit edilen fotonların sayısına bağlı olarak 10-50 nm mertebesinde, daha yüksek hassasiyet ile tespit edilebilir. Aktive ve numunedeki her floresan molekülü lokalize ederek, yüksek çözünürlüklü bir görüntü yeniden inşa edilebilir. Performecanlı hücreler üzerinde d tek bir hücreden 11 protein difüzyon yörüngeleri binlerce tek bir molekül izleme (smt-) PALM ileri izin edinimi. Önemlisi, PALM stokastik aktivasyonunu elde etmek için bu tür ışıkla aktive floresan proteinleri (PA-FP) gibi özel flüoresan millerini kullanır. BiFC ve PALM kullanım floresan proteinleri hem beri onlar amino asitler 159 ve 160 arasında iki parça halinde, PALM için PAmCherry1, yaygın olarak kullanılan bir PA-FP bölerek kombine edilmiştir. Bölünmüş PAmCherry1 dayalı BiFC sistemi iki parçalarının kendiliğinden sulandırıldıktan düşük arka plan sinyalini gösterir. Genetik olarak etkileşen proteinlerin bir çift etiketli, iki fragman (RN = 1-159 kalıntıları RC = Met + kalıntıları 160-236) da 37 ° C &#39;de ve daha düşük sıcaklıklarda kuluçkaya olmadan PAmCherry1 proteinleri oluşturmak için etkin bir şekilde yeniden, burada Böyle ebeveyn mCherry 13 gibi diğer BiFC çiftleri 12 için durum böyle değil. Furthermoyeniden yeniden PAmCherry1 proteini gibi doğru tek bir molekül lokalizasyonu ve yüksek çözünürlüklü PALM görüntüleme için kritik olan diğerleri arasında yüksek kontrast oranı, orta foton verimi ve hızlı fotoaktivasyon gibi ana PAmCherry1 ait Fotofiziksel özelliklerini korudu. Bu protokolde, split PAmCherry1 (Şekil 1A) kullanarak U2OS hücrelerinde Ras-Raf etkileşimlerini görüntüleme için BiFC PALM kullanılması tarif edilmektedir. İlk adım PAmCherry1 fragmanları (örneğin, RN ve Re) ve ilgili proteinler arasındaki füzyon proteinleri ifade etmek için yapıları tasarlamaktır. Teorik olarak, aday protein (A ve B) her çifti için, füzyon proteinlerinin sekiz çifti, test edilecek vardır: RN-A / RC-B; RN-A / B-RC; RC-A / RN-B; RC-A / B-RN; A-RN / RC-B; A-RN / B-RC; A-RC / RN-B; ve A-RC / B-RN. Bu süreç genellikle dikkate aday proteinlerin yapısal veya biyokimyasal özellikleri alarak basitleştirilmiş olabilir. Ras&#39;ın bir durumda, proteintranslasyon sonrası C-terminal CAAX kutusunun modifiye (C = Cys; A = alifatik, X = herhangi bir), ve bundan sonra AAX motifi kesilir. Bu nedenle, RN RC yalnızca Ras&#39;ın N-terminine kaynaşmış olabilir; Bu dört (Şekil 1B), füzyon proteini çiftlerinin sayısını azaltır. Raf için domain Ras bağlayıcı (RBD; artıkları 51-131) kullanılır ve her iki uçta etiketlenmiş olabilir. Bu dört füzyon konfigürasyonları elde edilmiştir: RN-Kras / RC-Raf RBD; RN-Kras / Raf RBD-RC; RC-Kras / RN-Raf RBD; ve RC-KRAS / Raf RBD-RN. Buna ek olarak, fragmanlar ve ilgi proteinleri arasındaki bağlayıcı olarak kabul edilmesi gerekir. Yaklaşık on amino asit esnek bir bağlayıcı genellikle tamamlama oluşması için yeterli özgürlüğü sağlar olarak kullanılır. Çoklu bir klonlama bölgesinin (MCS) 14 elde edilen rasgele diziler dahil olmak üzere, başarılı bir şekilde uygulanmıştır diğerleri, olmasına rağmen böyle bir bağlayıcı (GGGGS) x2. Bağlayıcıların uzunluğu dependin optimize edilmesi gerekebilirilgi proteinlerin ve yönelimleri etkileşim boyutuna g. PAmCherry1 fragmanları MCSS yan küçük bir klonlama omurgası içerdiği (Malzeme listesi). Söz konusu genler sınırlama siteleri yoluyla, ya da bir ligasyon bağımsız yöntemi ile yerleştirilebilir. Klonlama ve Sekans doğrulama sonrasında, ekspresyon kaseti, bir yeniden bağlayıcı reaksiyonlar, yüksek aslına uygunluk ve yüksek verimlilik ile bir klonlama işlemi kullanılarak bir ekspresyon vektörüne transfer edilir. Daha sonra, elde edilen sentezleme konstruktları hedef hücre hattı enfekte etmek için sabit bir hücre serisi isteniyorsa, lentivirüs içine paketlenmiş, hedef hücre hattına transfekte veya edilir. Geçici transfeksiyon BiFC yapılandırmasının hızlı doğrulama için sağlar, ancak potansiyel problemler dikkat edilmelidir. Genellikle kimyasallar kullanarak Transfeksiyon yüksek otofloresans sonuçlanan hücreleri vurgular; ise toplam iç yansıma floresan (TIRF) mikroskobik kullanımıpy PPİ hücre zarındaki yer alırken aydınlatma hacmi, TIRF idealini sınırlayarak bu arka plan sinyalini azaltmak olabilir. Buna ek olarak, geçici nakiller genellikle çok PPIs saptanmasında eserler neden endojen proteinlerin aşan, protein ekspresyonunun yüksek seviyeleri yol açar. Bu nedenle, uygun bir BiFC yapılandırması ilk test ile belirlenmiştir sonra kararlı hücre soyları kurulmuştur önerilir. Stabil hücre hatları da doksisiklin indüksiyonu 15 vasıtasıyla ayarlanabilir ekspresyon için potansiyele sahiptir. Enfeksiyondan sonra, hücreler tipik haliyle puromisin ve her bir yapı için neomisin, bir antibiyotik kullanılarak seçilir. Numune hazırlama, görüntü elde etme ve veri analizi için bir sonraki adım protokoller detaylı olarak tarif edilecektir. Bu yaklaşımla, Ras-Raf RBD BiFC PALM görüntülerde nano kümelerin oluşumu rutin görülmektedir (Şekil 2A &lt;/ strong&gt;). Sürekli, Ras-Raf RBD smt-PALM yörüngeleri difüzyon devletler (Şekil 2B-D) heterojen bir dağılım gösterir. Bu sonuçlar, Ras-Raf kompleksleri potansiyel biyolojik etkileri olan, muhtemelen monomerlerin ve kümeleri gibi, hücre zarı üzerinde birden fazla eyalette mevcut olduğunu göstermektedir. Bu çalışma, geleneksel BiFC, floresan co-lokalizasyon veya floresan rezonans enerji transferi (FRET) ile elde etmek zor olurdu nanometre uzamsal çözünürlük ve tek bir molekülün duyarlılık, hücrelerin içinde PPİ seçici görüntülenmesinde BiFC PALM gücünü gösteriyor. BiFC deneyler tasarlama ve sonuçları yorumlama, bu BiFC süreci bölünmüş PAmCherry1 dahil çoğu durumda, geri dönüşümsüz akılda tutmak önemlidir. Iki parça bir araya ve tam bir PAmCherry1 protein, iki parça arasındaki bağlantı oluşturabilir ve sonra bu şekilde PPI kalıcı olur. Bu BiFC ve BiFC-PAL kullanımını sınırlamaktadırPPI dinamiklerini (örneğin, bağlayıcı bir kinetik olup oluşturulduğu bir kez protein kompleksinin difüzyon dinamikleri) izlenmesi için ve zaman zaman M da PPI komplekslerinin mislocalization yol açabilir. BiFC PALM deney tasarlama floresan proteinleri doğasında vardır kromofor olgunlaşmasında gecikme olduğunda ek bir faktör dikkate. İki protein etkileşim ve FP fragmanları bir araya gelip, onlar genellikle saniye (EYFP 3 60 saniyelik yarı zamanlı) mertebesinde yeniden katlayın. Ancak, sonraki kromofor olgunlaşma ve flüoresan sinyal dakika mertebesinde gelişir. Floresan bölünmüş Venüs, hızlı bir katlama YFP varyant sulandırılmasından sonra 10 dakika içinde tespit edilebilir iken, genel olarak olgunlaşması için yarı-zamanı genellikle yaklaşık 60 dakika 16. Split-PAmCherry1 benzer fiyat bilgisi gözlendi. Dolayısıyla, BiFC ve BiFC PALM gerçek zamanlı ÜFE kinetiği izlemek için şu anda kötü seçimlerdir; Diğer meörneğin FRET ve yeni geliştirilmiş bir dimerizasyon bağlı floresan proteini 17 gibi thods, bu amaç için daha uygun olabilir.

<table><tbody><tr><td>Microscope frame</td><td>Nikon</td><td>Ti-U</td><td></td></tr><tr><td>60X oil immersion TIRF objective with 1.49 NA</td><td>Nikon</td><td>CFI Apo TIRF 60x H</td><td></td></tr><tr><td>EMCCD camera</td><td>Andor</td><td>iXon Ultra 897</td><td></td></tr><tr><td>561 nm laser</td><td>Opto Engine</td><td>MGL-FN-561</td><td></td></tr><tr><td>405 nm laser</td><td>Coherent</td><td>CUBE-405 50mW</td><td></td></tr><tr><td>561 nm dichroic mirror</td><td>Semrock</td><td>&amp;nbsp;Di01-R405/488/561/635-25x36</td><td></td></tr><tr><td>561 nm filter</td><td>Semrock</td><td>FF01-525/45-25</td><td></td></tr><tr><td>405/561 nm notch filter</td><td>Semrock</td><td>NF01-405/488/568-25</td><td></td></tr><tr><td>Temperature and CO&lt;sub&gt;2&lt;/sub&gt; controlled stage</td><td>Tokai Hit</td><td>INUBG2ATW-PLAM</td><td></td></tr><tr><td>pENTR-D-TOPO-PAmCherry1_1-159-MCS</td><td>Addgene</td><td>60545</td><td></td></tr><tr><td>pENTR-D-TOPO-PAmCherry160-236-MCS</td><td>Addgene</td><td>60546</td><td></td></tr><tr><td>pcDNA3.2-DEST</td><td>Life Technologies</td><td>12489-019</td><td></td></tr><tr><td>pLenti-DEST</td><td>Addgene</td><td></td><td>http://www.addgene.org/Eric_Campeau/</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Scientific</td><td>F-531</td><td></td></tr><tr><td>In-Fusion HD Cloning</td><td>Clontech</td><td>639649</td><td></td></tr><tr><td>LR Clonase</td><td>Life Technologies</td><td>11791</td><td></td></tr><tr><td>Vira Power Lentivirus Packaging</td><td>Life Technologies</td><td>K497500</td><td></td></tr><tr><td>X-tremeGENE Transfection Reagent</td><td>Roche</td><td>13873800</td><td></td></tr><tr><td>Lab-Tek II Chambered Coverglass</td><td>Thermo Scientific</td><td>155409</td><td>#1.5 glass bottom dishes</td></tr><tr><td>U2OS cells</td><td>ATCC</td><td>HTB-96</td><td></td></tr><tr><td>293T/17 cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>DMEM with phenol red</td><td>Life Technologies</td><td>11995</td><td></td></tr><tr><td>DMEM no phenol red</td><td>Life Technologies</td><td>21063</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>10082</td><td></td></tr><tr><td>Leibovit&#039;s L-15, no phenol red</td><td>Life Technologies</td><td>21083-027</td><td></td></tr><tr><td>Reduced serum medium</td><td>Life Technologies</td><td>31985</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Life Technologies</td><td>14040</td><td></td></tr><tr><td>Syringe</td><td>BD Biosciences</td><td>309604</td><td></td></tr><tr><td>Syringe filter</td><td>Millipore</td><td>SLHV033RB</td><td></td></tr><tr><td>Lentiviral concentrator</td><td>Clontech</td><td>631231</td><td></td></tr><tr><td>Retroviral concentrator</td><td>Clontech</td><td>631455</td><td></td></tr><tr><td>10 cm culture dish</td><td>BD Biosciences</td><td>353003</td><td></td></tr><tr><td>6-well culture plate</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>Polybrene</td><td>Sigma</td><td>107689</td><td></td></tr><tr><td>Puromycin</td><td>Life Technologies</td><td>A11138</td><td></td></tr><tr><td>G-418</td><td>Calbiochem</td><td>345812</td><td>Neomycin</td></tr><tr><td>Doxycyline</td><td>Fisher</td><td>BP2653</td><td></td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP152</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>EDS</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G6257</td><td></td></tr><tr><td>PIPES</td><td>Sigma</td><td>P6757</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>3777</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma</td><td>M2643</td><td></td></tr><tr><td>Potassium Hydroxide</td><td>Sigma</td><td>221473</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>BP358</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>M33</td><td></td></tr><tr><td>100 nm gold particles</td><td>BBI Solutions</td><td>EM.GC100</td><td></td></tr><tr><td>Molecular grade water</td><td>Life Technologies</td><td>10977</td><td></td></tr><tr><td>Dpn1</td><td>New England Biolabs</td><td>R0176</td><td></td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Miniprep kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>Midiprep kit</td><td>Macherey-Nagel</td><td>740410</td><td></td></tr><tr><td>0.6 ml microcentrifuge tubes</td><td>Fisher</td><td>05-408-120</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Fisher</td><td>05-408-137</td><td></td></tr><tr><td>15 ml tubes</td><td>Fisher</td><td>05-539-12</td><td></td></tr><tr><td>5 ml polypropylene round-bottom tubes</td><td>BD Biosciences</td><td>352063</td><td></td></tr><tr><td>14 ml polypropylene round-bottom tubes</td><td>BD Biosciences</td><td>352059</td><td></td></tr><tr><td>50 ml tube</td><td>BD Biosciences</td><td>352070</td><td></td></tr><tr><td>PCR tube</td><td>GeneMate</td><td>C-3328-1</td><td></td></tr><tr><td>SOC medium</td><td>Life Technologies</td><td>15544</td><td></td></tr><tr><td>LB broth</td><td>BD Biosciences</td><td>244610</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Fisher</td><td>BP906</td><td></td></tr><tr><td>Competent cells</td><td>Life Technologies</td><td>C4040</td><td></td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td></td></tr></tbody></table>

photoactivated localization microscopy with bimolecular fluorescence complementation (bifc-palm)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Gösterilen BiFC PALM örnek Craf etki (RBD) (Şekil 1A) bağlayıcı Ras ile etkileşim Kras G12D mutant olduğunu. Tartışıldığı gibi Ras zar lokalizasyonu ve dolayısıyla biyolojik aktivitesini bozacak, çünkü, RN veya RC fragmanları Ras&#39;ın C-terminaline etiketlenmemiş. Bu dört (Şekil 1B) sekizden olası kombinasyonları azalır. Bu dört kombinasyon her lentiviral enfeksiyonu ile U2OS hücrelerine dahil edilmiştir. Protokolde ayrıntılı olarak hücreler sabitlend...

Watch this Scientific Journal Video about Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM at JoVE.com

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Bimoleküler Floresan tamamlama ile fotoaktive Yerelleştirme Mikroskobu (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

photoactivated-localization-microscopy-with-bimolecular-fluorescence

Research

JoVE Journal

Bioengineering

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

9.8K Görüntüleme.  Oregon Health and Science University. Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes. 

Video: Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).

Değişken Açılı Toplam İç Yansıma Floresan Mikroskopi (VA-TIRFM) üzerine Hücre yapışma Nanotopology

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

Biyomühendislik

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Fotoaktive Tek molekül Takip Kullanma Canlı Bakteriyel Hücrelerde Protein-DNA Etkileşimleri görselleştirme

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

İmmünoloji ve Enfeksiyon

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

Canlı Hücresüper çözünürlüklü mikroskopi ve Tek Moleküllü İzleme için Konvansiyonel BODIPY Konjugeleri

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

Biyokimya

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Planar on Tek Molekül Floresans Mikroskopi bilayers Desteklenen

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Yüzey immobilize Biyomoleküller Tek Molekül Floresans Ölçümler için Otomatik Sistem

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Biyoloji

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Bimoleküler Floresan tamamlama

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

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 ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Çok renkli yerelleştirme mikroskobu canlı memeli hücre organelleri tek membran proteinlerin

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

To prepare a sample for imaging, plate about 5.5 x 104 stable expression cells per well of an 8-well glass-bottomed chamber slide, in 350 microliters of phenol red-free DMEM. Use fresh paraformaldehyde with glutaraldehyde to fix the cells, and after replacing the fixative with PBS or imaging buffer, vortex 100-nanometer gold particles, and add 35 microliters per well for tracking stage drift during imaging.
Approach the microscope and power on the 405 and 561-nanometer lasers. Keeping the shutters closed at this point, ensure the 561-nanometer dichroic mirror and notch filter are in place. Open the image acquisition software.
Turn on the EMCCD camera and allow it to cool down, and set the exposure time to 100 milliseconds and the EMCCD gain to an appropriate value. Add immersion oil to the objective, and secure the sample on the microscope stage. Next, with either brightfield or the 561-nanometer laser, bring the sample into focus.
For imaging Ras and other membrane proteins, use a 60X apochromat TIRF objective with a 1.49 numerical aperture, and bring the microscope into TIRF configuration.
Adjust the excitation laser so that it is off-centered when hitting the back aperture of the TIRF objective, which will cause the laser to deflect upon reaching the coverslip buffer interface. Keep adjusting the laser incident position until the critical angle is reached and the laser is being reflected back.
Search for a cell to image with several gold particles in view. Then, set a region of interest that encloses the cell or a region of it and the gold particles.
In case sufficient activation is already occurring because of high expression levels, begin acquisition with the 405-nanometer laser off and the 561-laser on. Otherwise, turn on the 405-nanometer laser at the lowest factory power setting, and increase it gradually until there are tens of molecules per frame or so that single molecules are well-separated.
As data acquisition continues, gradually increase the 405-nanometer laser power to keep the spot density roughly constant. Continue image acquisition until high 405 power does not initiate any more activation events.