سمير حمدان ساعد في تطوير هذه التقنية. E. البروتينات القولونية هي من مختبر البروفيسور نيك ديكسون ، جامعة ولونغونغ ، والبروتينات هي من T7 البروفيسور تشارلز ريتشاردسون ، كلية هارفارد الطبية. وتدعم العمل الذي قامت به المعاهد الوطنية للصحة (GM077248 لAMvO) والتابوت جين تشايلدز مؤسسة (JJL).

<ol>
	<li>Tanner, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+single-molecule+observation+of+rolling-circle+DNA+replication.">Real-time single-molecule observation of rolling-circle DNA replication.</a> Nucleic Acids Res. 37, e27 (2009).</li><li>Tabor, S., Huber, H. E., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+thioredoxin+confers+processivity+on+the+DNA+polymerase+activity+of+the+gene+5+protein+of+bacteriophage+t7.">Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage t7.</a> J Biol Chem. 262, 16212-16223 (1987).</li><li>Sofia, S. J., Premnath, V. V., Merrill, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+oxide)+grafted+to+silicon+surfaces:+Grafting+density+and+protein+adsorption.">Poly(ethylene oxide) grafted to silicon surfaces: Grafting density and protein adsorption.</a> Macromolecules. 31, 5059-5070 (1998).</li><li>Tanner, N. A., van Oijen, A. M., Vengrova, S., Dalgaard, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+observation+of+prokaryotic+DNA+replication.">Single-molecule observation of prokaryotic DNA replication.</a> DNA replication: Methods and protocols. 521, 397-410 (2009).</li><li>Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+DNA+replication+loops+reveal+temporal+control+of+lagging-strand+synthesis.">Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.</a> Nature. 457, 336-339 (2009).</li><li>Heller, R. C., Marians, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+reactivation+downstream+of+a+blocked+nascent+leading+strand.">Replication fork reactivation downstream of a blocked nascent leading strand.</a> Nature. 439, 557-562 (2006).</li></ol>

المراقبة الحرجة واحد هو دراسة تأثير أورانج SYTOX صمة عار ، على البروتينات تكرار الفائدة. وهناك طريقة بسيطة للقيام بذلك هي لتنفيذ تكرار التجربة في الخلية التدفق كما هو موضح ولكن مع إغفال SYTOX. بعد خليط التفاعل قد تدفقت عبر الغرفة ، إضافة إلى SYTOX العازلة مع وصمة عار الحمض النووي ودراسة توزيع طول جزيئات منسوخة. بدلا من ذلك ، يمكن استخدام معظم ردود الفعل القياسية لفحص أي تأثير على معدل تكرار SYTOX والكفاءة. التجربة الموصوفة هنا فقط يستخدم تدفق قناة واحدة. يمكن تغيير هذا بسهولة من خلال خلق قنوات متعددة الغرف تدفق أو استخدام أو الأجهزة ميكروفلويديك PDMS مماثلة. زيادة عدد القنوات ييسر فحص تركيزات البروتين ، والمسوخ ، أو الجزيئات المانع ويزيد من سرعة جمع البيانات تكرارها. كما ذكرنا ، فإننا أداء E. تكرار التجارب القولونية عند 37 درجة مئوية باستخدام تدفق الالومنيوم تدفئة المنازل بنيت الخلية ، عنصر التدفئة مقاوم (خرطوشة سخان) والمتغير امدادات الطاقة. وهذا يعطي درجة حرارة جيدة الاستقرار والابتعاد عن شراء سخان الهدف. لمعايرة جهاز التدفئة ، ونحن ببساطة حفر حفرة في وسط تدفق رأس خلية الكوارتز ، وإدراج الحرارية في قناة تدفق وتدفق العازلة كالمعتاد. قياس درجة حرارة التدفق العازلة خلية في زيادة الفولتية يسمح التدفئة دقيقة.

واضاف تريس 40 ملم درجة الحموضة 7.5 ، الغلوتامات البوتاسيوم 50 ملي و 10 ملي مول كلوريد المغنيسيوم ، و 100 ميكروغرام / مل جيش صرب البوسنة ، مع dithiothreitol 5 مم ، 600 ميكرومتر dNTPs ، و 300 ميكرومتر اعبي التنس المحترفين ، و 300 ميكرومتر CTP ، و 15 نانومتر SYTOX أورانج على الفور من قبل : T7 المتماثل الاستخدام. وأضاف البروتينات على النحو التالي : 5 نانومتر gp4 (مسدوس) ، 40 نانومتر بوليميريز (1:1 gp5 : thioredoxin) ، 360 نانومتر gp2.5 1،5. إي كولاي المتماثل : HEPES 50 ملم 7.9 درجة الحموضة ، 12 المغنيسيوم خلات ملم ، 80 ملم كلوريد البوتاسيوم و 100 ميكروغرام / مل مع جيش صرب البوسنة dithiothreitol 10 ملم ، و 40 ميكرومتر dNTPs ، 200 rNTPs ميكرومتر ، و 15 نانومتر SYTOX أورانج واضاف على الفور قبل استخدامه. وأضاف البروتينات على النحو التالي : 30 نانومتر DnaB (مسدوس) ، 180 نانومتر DnaC (مونومر) ، 30 نانومتر αεθ ، 15 نانومتر τ 2 1 γ δδ&#39;χψ أو τ δδ&#39;χψ 3 ، 30 β نانومتر (ديمر) ، 300 نانومتر DnaG ، 250 نانومتر SSB (tetramer) ، 20 نانومتر بريا و 40 نانومتر PriB ، 320 نانومتر PRIC ، 480 نانومتر DNAT 1،6

visualizing single-molecule dna replication with fluorescence microscopy

وصفنا بسيطة مضان المجهري المستندة في الوقت الحقيقي لمراقبة طريقة تكرار الحمض النووي على مستوى واحد جزيء. ومرفق بهذا التعميم ، قالب الحمض النووي متشعب إلى ساترة functionalized الزجاج على نطاق واسع وتكرارها بعد إدخال البروتينات النسخ والنيوكليوتيدات (الشكل 1). يتم توسيع المتزايد الناتج المزدوج حبلا الحمض النووي (dsDNA) مع تدفق الصفحي وتصور باستخدام صبغة الإقحام. قياس موقف نهاية الحمض النووي المتنامي في الوقت الحقيقي يسمح التحديد الدقيق لمعدل التكرار (الشكل 2). وعلاوة على ذلك ، وطول الحمض النووي الانتهاء من المنتجات تقارير عن processivity من تكرارها. يمكن إجراء هذه التجربة بسهولة وبسرعة جدا ويتطلب سوى مضان المجهر مع كاميرا حساسة إلى حد معقول.

Watch this Scientific Journal Video about Visualizing Single-molecule DNA Replication with Fluorescence Microscopy at JoVE.com

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

يوضح هذا البروتوكول بسيطة المجهري مضان المفرد جزيء تقنية لتصور تكرار الحمض النووي التي replisomes الفردية في الوقت الحقيقي.

تصور تكرار الحمض النووي جزيء واحدة مع مضان المجهري

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

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22.3K Views.  Harvard Medical School. يوضح هذا البروتوكول بسيطة المجهري مضان المفرد جزيء تقنية لتصور تكرار الحمض النووي التي replisomes الفردية في الوقت الحقيقي.

Video: Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

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 level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

تصور المجمعات الجزيئية واحدة في فيفو استخدام الميكروسكوب الإسفار متقدم

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 ...

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.

النظام الآلي للقياسات واحدة الإسفار جزيء من الجزيئات الحيوية سطح يجمد

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

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

الملاحظة المباشرة من الحمض النووي الانزيمات تكرار استخدام الحمض النووي جزيء واحدة تمتد الفحص

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

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.

الوقت الفاصل بين التصوير من الانقسام الميتوزي بعد ترنسفكأيشن سيرنا

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

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

متصور الكهربائي زنزانة مع مضان المجهري

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

رصد النسخ المتماثل البلازميد في خلايا الثدييات يعيش أكثر من أجيال متعددة من مضان المجهري

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

الجمع بين التلاعب جزيء واحدة والتصوير لدراسة التفاعلات-DNA البروتين

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

دراسة الحمض النووي عن طريق حلقات جزيء واحد الحنق

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

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.

تصور التردد Stromule مع مضان المجهر

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

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.