Biz Prof. Judith Armitage (Oxford Üniversitesi, İngiltere) ve Prof. Conrad Mullineaux (Londra Queen Mary Üniversitesi, UK) grupları bakteri suşlarının tür bağışlar kabul etmiş oluyorsunuz. IMD Biyokimya Bölümü (Oxford Üniversitesi) ve OCISB tarafından ortaklaşa finanse edilen AR Mühendislik ve Fiziksel Bilimler Araştırma Konseyi (EPSRC) DTC öğrencilikle tarafından finanse edilmektedir; ND Biyoteknoloji ve Biyolojik Bilimler Araştırma Konseyi (BBSRC) finanse edilmektedir; MCL Royal Society Üniversitesi Araştırma Bursu tarafından finanse edilmektedir.

<ol><li> Bu yordamı başlamak için, Escherichia coli bakteri hücrelerinin ifade floresan protein dondurulmuş stoklarının 50 ul ilk çözülmüş ve C. sabah 37 derece gecede 5 ml LB büyüme ortamı sallayarak aerobik yetiştirilen, bu doymuş kültür 50 ul ayıklanır 4 ila 6 saat boyunca 30 ° C&#39;de inkübe minimal M63 glikoz kültür ortamı, alt-kültür. Burada, biri bir elektron taşıma ifade iki farklı hücre suşları, açıklamak GFP, GFP için erimiş bakteriyel flagellar motor yer alan bir protein ifade diğer erimiş sitokrom. </li><li> Hücreler, büyüyen alt-kültür immobilize örnekleri olarak varsa da doğrudan hasat, ya da &quot;gergin&quot; koşullar altında görüntülerken, bakteri kamçısı kesecek makaslanmış olabilir. </li><li> Kırpma, steril boru ile birleştirilmiş iki steril şırıngalar oluşan bir cihazın içine alt-kültür genellikle 1-5ml yerleştirerek içerir. Kesme dar bir boru aracılığıyla kültür itmek için her şırınga pompası iterek alternatif ile yapılır. Bu gerekli kesme ölçüde bağlı olarak, 50-100 kez yapılır. Kültür kamçı parçalarını kaldırmak için asgari medya yeniden süspanse pelet hücreler, daha sonra santrifüj edilir. </li><li> Daha sonra 20 dakika için etanol KOH doymuş bir çözüm çeker, sonra de-iyonize su ve etanol iyice durulama ve en az 1 saat boyunca kurumaya bırakarak BK7 cam lamelleri temizlenir hazırlamak </li><li> Biz hücrelerin mikroskop evine basit bir akış hücresi oluşturmak. Bu parafin yağ hatları BK7 cam mikroskop lamı üzerine çizim ve daha sonra bir tünel sandviç oluşturarak üstüne temizlenir coverlips birini koyarak ve forseps bir çift ile hafifçe aşağı doğru bastırarak, 5-10 ul bir akış hücresi hacmi veren içerir . </li><li> Immobilize hücreler gözlemlemek için% 0.1 w / v poli-L-lisin bir çözüm akış hücre enjeksiyonu ile doldurmak ve en az 1 dakika süreyle oda sıcaklığında inkübe izin. Daha sonra 100 ul minimal medya akışını hücrenin bir ucundan, medya enjekte ve diğer kağıt mendil ile esneklik kullanarak dışarı floş. </li><li> Daha sonra lamel yüzey işaretlemek için minimum medya, 200 nm çaplı lateks mikroküreler (Polysciences) bir 1:500 dilüsyon 20 ul fitil. Akış hücre lamel aşağıya doğru, basit bir nem odası yüzey açık bir platform üzerine yerleştirilen karşı karşıya olduğu gibi ters ve oda sıcaklığında 5 dakika inkübe edilir. Unbound boncuk sonra 100 ul minimal medya aracılığıyla esneklik yıkanır. </li><li> Gergin hücreleri gözlemlemek isterseniz, poli-L-lisin adım ihmal ve 5 mg / ml anti-flagellin antikor yerine akış hücre doldurun. Akış hücresi, 10 dakika nem odası yer ve sonra esneklik tarafından temizlendi. </li><li> Hücre kültürü 20 ul immobilize hücreleri inceleyen gergin hücreleri veya unsheared numune gözlemleyerek, ya makaslanmış örnek kullanarak, akış hücresi ile sonra kötülerin. </li><li> Akış hücre ters ve 20 dakika boyunca nem odasında yerleştirilir. Unbound hücreler daha sonra 100 ul minimal medya aracılığıyla esneklik tarafından yıkanır. </li><li> Bir damla immersiyon lamel üst yüzey merkezinde yerleştirilir ve akış-hücreli yüksek sayısal diyafram objektif lens ile optik temas eden, özel inşa edilmiş floresan mikroskop numune tutucu hafifçe yerleştirilir. </li><li> Elektron mikroskobu çarparak kamera açık ve kamera -70 ° C ye soğutulmalıdır. Ayarlamak, yazılım, başlangıçta kazanç kontrolü disenabling çerçeve transfer modunda 25 Hz tipik bir kare hızında görüntüler elde etmek için ayarlanır kamera. </li><li> Aydınlık aydınlatma açık ve görüntü lamel yüzey uzun eksenine paralel çakılı olmanın temelinde görüntülü hücreleri uygun bir hücre veya grup seçerek, odak noktası haline getirdi. Odak ince lamel yüzeyine yapışmış 200 nm lateks boncuk sadece odak noktası olmasını sağlamak için ayarlanabilir. </li><li> Hücre gövdesi taslağını kaydetmek için aydınlık bir görüntü dizisi elde edilir. Aydınlık aydınlatma kapalı ve kamera kazanç maksimum etkindir. </li><li> Toplam iç yansıma floresan (veya TIRF) uygun dalga boyu üzerinde bir lazer ışını (Burada, yeşil flüoresan protein uyarma, 473 nm lazer) kullanarak, standart bir satın alma için arka odak düzlemi odaklı olacak şekilde önceden ayarlanmıştır. objektif lens ama hücre örneği içine floresans uyarma için kaybolan bir alan oluşturmak üzere optik ekseni yanal yerinden. </li><li> Kamera alımına başlanması ve lazer deklanşör bakteri içinde floresan proteinleri heyecanlandırmak için açıldı. Lazer yoğunluk ve hız için satın alma parametreleri farklı değerler ile denemeler tarafından soruşturma altında belirli bir biyolojik sistem için optimize edilmiş olması gerekir, ancak tipik bir aralığı r5-40 ms kare başına bir pozlama süresi ile, hücre zarı mobil protein kompleksleri okuyan elevant çapı ~ 30 mikron dairesel bir uyarım alana 1-10 mW lazer güç vardır. Örnekleri photobleached, genellikle ~ 10 sn kadar aydınlatılmış </li><li> Bu protokol hem vücut hücre lamel ve flagellar saplama arasında ek bir nokta etrafında dönen gergin hücreleri ve katı lamel yüzey sabit hareketsiz hücreler için kullanılabilir. </li><li> Kompleksleri yüksek bir kopya sayısı bazı hücre suşları TIRF görüntüleme önce hücre tek kutuplu bir başlangıç ​​kırınım sınırlı odaklı lazer çamaşır suyu yarar. Bu çamaşır suyu (ya da sıkı bağlamak) photobleaching sonra floresan kurtarma kullanılan eşdeğerdir. Bizim özel mikroskop kullanarak ikaz lazer ışığı, sıkı bağlamak-tipi beyazlatma için kullanılan ikinci bir bağımsız yoluna beslenebilir. Genellikle 1-10mW lazer güç aralığı 10-300 ms tipik bir çamaşır suyu zaman kullanılır. Bu sonuçlar, daha sonra bu alana daha kolay görüntülenebilmekte diffüz bireysel kompleksleri sağlayan hücre ağartılmış bölge çok daha yüksek görüntüleme kontrast. </li><li> &quot;Slimfield&quot; olarak adlandırılan farklı bir aydınlatma modu istihdam hücre zarının dışında çok daha hızlı diffüz hücre sitoplazma kompleksleri görselleştirme. Burada odaklanmış bir lazer yanal sadece tek bir hücre kapsayacak şekilde genişletilmiştir. Bu tipik bir milisaniyeden daha hızlı pozlama alınacak izin çok yoğun bir alan üretir. </li><li> Veri toplama, görüntüler, özel yazılmış yazılım (LabVIEW 8.5 kodlu) beslenir. Bu hücreleri genellikle birkaç nanometre hassas bir floresan noktalar pozisyonları otomatik olarak algılar ve kendi büyüklüğü ve parlaklığı ayıklar. Brightnessof, paletli bir moleküler kompleks zaman saygı ile photobleaching iz sonra stokiyometri tahmin etmek için kullanılan tek bir moleküler kompleks oluşturan kaç bireysel floresan proteinleri yani. </li></ol> Temsilcisi Sonuçlar: Protokol yapılır aydınlık içinde görüntülenen hücrelerin düzgün görüntüleri koyu bir beyaz / gri hücre gövdesi (Şekil 1a) karşı hücre gövdeleri çevrelerinin, çok farklı. Floresan immobilize hücreler kullanarak, farklı noktalar genellikle genişliği 250-300 nm (Şekil 1b) yoğunluğu, görebilirsiniz. Sağlıklı, gergin hücreleri aydınlık görüntüler halata eki nokta etrafında döndürmek için görülecektir. Floresans uyarma altında bizim durumumuzda bazı moleküler kompleksler flagellar motor etiketli protein lokalizasyonu gösteren eki noktada olabilir. Bu lekeler, bireysel moleküler kompleksler ve sayıda görülen onları aydınlatma modu ve komplekslerin çoğu aslında herhangi bir zamanda hücrede bulunur bağlıdır. Noktalar hareketlilik çalışma altında belirli bir biyolojik sistem bağlıdır. Lekelerin yoğunluğu başlangıçta çok yüksek ise, sonra bir ilk sıkı bağlamak çamaşır suyu görüntü kontrastı artırabilir gerçekleştirerek, burada kullanılan etiketli sitokromlar durumda. <img src="/files/ftp_upload/1508/1508Figure1tmb.jpg" border="0" alt="Şekil 1" /> Şekil 1. (A) aydınlık ve (B) hareketsiz bir Escherichia coli hücre için TIRF (sahte renk) bakteri flagellar motorlar yer olarak bilinen yeşil floresan protein (GFP) erimiş bir protein ifade. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1508/1508Figure1.jpg">buraya tıklayınız</a> .

<ol>
	<li>Leake, M. C., Chandler, J. H., Wadhams, G. H., Bai, F., Berry, R. M., Armitage, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+and+turnover+in+single,+functioning+membrane+protein+complexes.">Stoichiometry and turnover in single, functioning membrane protein complexes.</a> Nature. 443, 355-358 (2006).</li><li>Leake, M. C., Greene, N. P., Godun, R. M., Granjon, T., Buchanan, G., Chen, S., Berry, R. M., Palmer, T., Berks, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+stoichiometry+of+the+TatA+component+of+the+twin-arginine+protein+transport+system+observed+by+in+vivo+single-molecule+imaging.">Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.</a> Proc Natl Acad Sci U S A. 105, 15376-15381 (2008).</li><li>Leake, M. C., Wilson, D., Gautel, M., Simmons, R. M., M, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+titin+molecules+using+a+two-bead+optical+tweezers+assay.">The elasticity of single titin molecules using a two-bead optical tweezers assay.</a> Biophys. J. 87, 1112-1135 (2004).</li><li>Leake, M. C., Wilson, D., Bullard, B., Simmons, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+single+kettin+molecules+using+a+two-bead+laser-tweezers+assay.">The elasticity of single kettin molecules using a two-bead laser-tweezers assay.</a> FEBS Lett. , 535-555 (2003).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+clustering+and+dynamics+of+cytochrome+bd+complexes+in+the+Escherichia+coli+plasma+membrane.">In vivo clustering and dynamics of cytochrome bd complexes in the Escherichia coli plasma membrane.</a> Mol. Microbiol. 70, 1397-1407 (2008).</li><li>Lenn, T., Leake, M. C., Mullineaux, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Escherichia+coli+OXPHOS+complexes+concentrated+in+specialised+zones+within+the+plasma+membrane.">Are Escherichia coli OXPHOS complexes concentrated in specialised zones within the plasma membrane.</a> Biochem. Soc. Trans. 36, 1032-1036 (2008).</li><li>Lo, C. J., Leake, M. C., Pilizota, T., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+measurements+of+Membrane+Potential,+Sodium-Motive+Force+and+Flagellar+Motor+Speed+in+Escherichia+coli.">Single-cell measurements of Membrane Potential, Sodium-Motive Force and Flagellar Motor Speed in Escherichia coli.</a> Biophys. 93, 294-302 (2007).</li><li>Lo, C. J., Leake, M. C., Berry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+measurement+of+intracellular+sodium+concentration+in+single+Escherichia+coli+cells.">Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells.</a> Biophys. J. 90, 357-3565 (2006).</li></ol>

The authors have nothing to disclose.

Bakım bu yana gergin bakteri bakarak &quot;flagellar motorlar üzerine kesme&quot; hücre işlevlerini bozabilir değil alınmalıdır. Oksijen tükenmiş olabilir hücreleri mikroskop lamı üzerine bir kez bir saat daha uzun kullanmak için önemlidir. Önemli bir optimizasyon en iyi mikroskop görüntüleme koşulları soruşturma altında belirli bir biyolojik sistem yiyecek ve içecek bulmak için gerekli olabilir. Özellikle mikroskop sistemi için gerekli olan doğru yoğunluklu lazer uyarma tespit etmek için t...

visualizing single molecular complexes in vivo using advanced fluorescence microscopy

Sadece hücresel düzeyde olayları ortaya çıkarmak ve doğrudan temel süreçleri araştıran, canlı hücrelerin mekanizmalarını içine tam fikir elde edilebilir. Yerine nispeten ham dökme topluluk ortalamanın ölçümler kullanılarak yapılan çalışmalarda tek sistemleri odaklanarak, biyolojik sistemlerin kayma karmaşıklığı Bugüne kadar çok talep olması kesin tek molekül deneyler neden oldu. Ancak, çok önemli süreçler, sadece bir düzeyinde yaşayan hücre ya da birkaç molekül oluşur; topluluk ölçümleri genellikle bu olayların stokastik ve heterojen doğası maske. Burada, gelişmiş optik mikroskop ve analitik görüntü analiz araçları kullanarak, tek bir canlı bakteri tek moleküllerin hassas bir hücre ve biyolojik makineleri işleyen moleküler kompleksler içinde dinamiklerini izleyebilirsiniz içinde proteinler izlemek için nasıl gösterilmektedir. Teknikleri fizyolojik doğrudan ilgili. Minimal pertürbatif ve biyolojik örnek çalışma altında non-invaziv ve yaşam malzemesi araştırmalar için tamamen uydurulmuş, biyofizik diğer tek-molekül yaklaşımlara hazır özellikleri. Buna ek olarak, biyolojik örneklerin hepsi doğal olarak ortaya daha belirgin olarak daha fazla protein üretmek için, daha sık ama daha az ideal bir yaklaşım karşı değiştirilmemiş hücre suşları (&quot;genomik kodlama&quot;), hemen hemen aynı seviyelerde floresan etiketli proteini üretmek okudu (&quot;plazmid ifade&quot;). Böylece, gerçek biyolojik örnekler ele alınacaktır doğal organizmalar için önemli ölçüde yakın ve gerçek fizyolojik süreçleri nedenle gözlemler daha uygun.

Watch this Scientific Journal Video about Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy at JoVE.com

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Burada izlenir ve sayısal, fonksiyonel moleküler kompleksler tespit sağlamak için canlı bakteri hücreleri tek-molekül floresan mikroskopi gerçekleştirmek için protokolleri göstermektedir.

Tek Moleküler Kompleksleri görselleştirme In Vivo Gelişmiş Floresan Mikroskopi

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

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Biology

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

9.2K Görüntüleme.  University of Oxford. Burada izlenir ve sayısal, fonksiyonel moleküler kompleksler tespit sağlamak için canlı bakteri hücreleri tek-molekül floresan mikroskopi gerçekleştirmek için protokolleri göstermektedir.

Video: Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Floresan Mikroskop ile görselleştirme Tek molekül DNA replikasyonu

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Biyoloji

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Floresan Mikroskobu kullanılarak Nematodlar Bakteriler görselleştirme

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Gelişmiş 3D Floresan Mikroskobu kullanılarak Otofaji Kantitatif Analiz

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Tek hücreli Yara Şifa sırasında Sitoplazmik Akışı görselleştirme<em&gt; Stentor coeruleus</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Hızlı Analiz ve Floresans Mikroskopi Görüntüler Exploration

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Floresan Mikroskopi ile Stromule Frekansı görselleştirme

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

VirusMapper Süper çözünürlüklü Mikroskopi Açık kaynak Tek parçacık Analizi

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

3D Floresans mikroskobu kullanılarak Viral transkripsiyon fabrikaların fonksiyonel görüntüleme

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.