Name: visualizing stromule frequency with fluorescence microscopy
Uploaded: 2016-11-23T14:00:00
Description: Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

J.O.B. and A.M.R. were supported by predoctoral fellowships from the National Science Foundation.

NOTE: For this protocol, we have focused on assaying stromule frequency in the epidermis of N. benthamiana leaves. Several stable transgenic lines have been generated that can be used for this purpose, including 35SPRO:FNRtp:EGFP13 and NRIP1:Cerulean6. Both of these lines show robust expression of fluorophores in the chloroplast stroma of leaves grown under a wide range of conditions. Alternatively, chloroplast-targeted fluorophores may be transiently expressed ...

<ol>
	<li>Koussevitzky, S., 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=Signals+from+chloroplasts+converge+to+regulate+nuclear+gene+expression.">Signals from chloroplasts converge to regulate nuclear gene expression.</a> Science. 316 (5825), 715-719 (2007).</li><li>Burch-Smith, T. M., Brunkard, J. O., Choi, Y. G., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PNAS+Plus:+Organelle-nucleus+cross-talk+regulates+plant+intercellular+communication+via+plasmodesmata.">PNAS Plus: Organelle-nucleus cross-talk regulates plant intercellular communication via plasmodesmata.</a> Proc. Natl. Acad. Sci. USA. 108 (51), E1451-E1460 (2011).</li><li>Stonebloom, S., Brunkard, J. O., Cheung, A. C., Jiang, K., Feldman, L., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+states+of+plastids+and+mitochondria+differentially+regulate+intercellular+transport+via+plasmodesmata.">Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata.</a> Plant Physiol. 158 (1), 190-199 (2012).</li><li>Nomura, H., 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=Chloroplast-mediated+activation+of+plant+immune+signalling+in+Arabidopsis.">Chloroplast-mediated activation of plant immune signalling in Arabidopsis.</a> Nat. Commun. 3, 926 (2012).</li><li>Avenda&#241;o-V&#225;zquez, A. -. O., 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=An+uncharacterized+apocarotenoid-derived+signal+generated+in+&#950;-carotene+desaturase+mutants+regulates+leaf+development+and+the+expression+of+chloroplast+and+nuclear+genes+in+Arabidopsis.">An uncharacterized apocarotenoid-derived signal generated in &#950;-carotene desaturase mutants regulates leaf development and the expression of chloroplast and nuclear genes in Arabidopsis.</a> Plant Cell. 26 (June), 2524-2537 (2014).</li><li>Caplan, J. L., 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=Chloroplast+stromules+runction+during+innate+immunity.">Chloroplast stromules runction during innate immunity.</a> Dev. Cell. 34 (1), 45-57 (2015).</li><li>Hanson, M. R., Sattarzadeh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromules:+recent+insights+into+a+long+neglected+feature+of+plastid+morphology+and+function.">Stromules: recent insights into a long neglected feature of plastid morphology and function.</a> Plant Physiol. 155 (4), 1486-1492 (2011).</li><li>Gray, J. C., 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=Plastid+stromules+are+induced+by+stress+treatments+acting+through+abscisic+acid.">Plastid stromules are induced by stress treatments acting through abscisic acid.</a> Plant J. 69 (3), 387-398 (2012).</li><li>Brunkard, J., Runkel, A., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplasts+extend+stromules+independently+and+in+response+to+internal+redox+signals.">Chloroplasts extend stromules independently and in response to internal redox signals.</a> Proc. Natl. Acad. Sci. USA. 112 (32), 10044-10049 (2015).</li><li>Dupree, P., Pwee, K. -. H., Gray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+photosynthesis+gene-promoter+fusions+in+leaf+epidermal+cells+of+transgenic+tobacco+plants.">Expression of photosynthesis gene-promoter fusions in leaf epidermal cells of transgenic tobacco plants.</a> Plant J. 1 (1), 115-120 (1991).</li><li>Charuvi, D., Kiss, V., Nevo, R., Shimoni, E., Adam, Z., Reich, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+and+loss+of+photosynthetic+membranes+during+plastid+differentiation+in+the+shoot+apex+of+Arabidopsis.">Gain and loss of photosynthetic membranes during plastid differentiation in the shoot apex of Arabidopsis.</a> Plant Cell. 24 (3), 1143-1157 (2012).</li><li>Chiang, Y. -. H., 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=Functional+characterization+of+the+GATA+transcription+factors+GNC+and+CGA1+reveals+their+key+role+in+chloroplast+development,+growth,+and+division+in+Arabidopsis.">Functional characterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development, growth, and division in Arabidopsis.</a> Plant Physiol. 160 (1), 332-348 (2012).</li><li>Schattat, M., Barton, K., Baudisch, B., Kl&#246;sgen, R. B., Mathur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastid+stromule+branching+coincides+with+contiguous+endoplasmic+reticulum+dynamics.">Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics.</a> Plant Physiol. 155 (4), 1667-1677 (2011).</li><li>Erickson, J. L., 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=Agrobacterium-derived+cytokinin+influences+plastid+morphology+and+starch+accumulation+in+Nicotiana+benthamiana+during+transient+assays.">Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays.</a> BMC Plant Biol. 14 (1), 127 (2014).</li><li>Ho, J., Theg, S. 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+formation+of+stromules+in+vitro+from+chloroplasts+isolated+from+Nicotiana+benthamiana.">The formation of stromules in vitro from chloroplasts isolated from Nicotiana benthamiana.</a> PLoS One. 11 (2), e0146489 (2016).</li><li>Brunkard, J. O., Burch-Smith, T. M., Runkel, A. M., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+plasmodesmata+genetics+with+virus-induced+gene+silencing+and+an+Agrobacterium-mediated+GFP+movement+assay.">Investigating plasmodesmata genetics with virus-induced gene silencing and an Agrobacterium-mediated GFP movement assay.</a> Method. Mol. Biol. , 185-198 (2015).</li><li>Schattat, M. H., Kl&#246;sgen, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+stromule+formation+by+extracellular+sucrose+and+glucose+in+epidermal+leaf+tissue+of+Arabidopsis+thaliana.">Induction of stromule formation by extracellular sucrose and glucose in epidermal leaf tissue of Arabidopsis thaliana.</a> BMC Plant Biol. 11, 115 (2011).</li><li>Joly, D., Carpentier, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+intact+chloroplasts+from+spinach+leaves.">Rapid isolation of intact chloroplasts from spinach leaves.</a> Method. Mol. Biol. 684, 321-325 (2011).</li><li>Waters, M. T., Fray, R. G., Pyke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromule+formation+is+dependent+upon+plastid+size,+plastid+differentiation+status+and+the+density+of+plastids+within+the+cell.">Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell.</a> Plant J. 39 (4), 655-667 (2004).</li></ol>

When investigating stromules, three important factors must be considered throughout: (i) manipulation of the plant tissue must be kept to an absolute minimum, (ii) the experimental system must be kept consistent, and (iii) sampling strategies must be carefully planned to ensure robust, reproducible data are analyzed.
Stromules are remarkably dynamic: they can extend and retract rapidly before an observer&#39;s eyes under the microscope. Moreover, stromule frequency varies significantly in resp...

Chloroplasts are dynamic organelles in plant cells responsible for photosynthesis and a host of other metabolic processes. Signaling pathways from the chloroplast also exert significant influence on plant physiology and development, coordinating plant responses to environmental stress, pathogens, and even leaf shape1-6. Recently, biologists have gained interest in a poorly understood aspect of chloroplast structure: stromules, very thin stroma-filled tubules that extend from the surface of the chloroplast7.
The biological functions of stromules remain unknown, although stromule frequency is known to vary in response to environmental stimuli7-9, and stromules may be capable of transmitting signaling molecules between organelles6. All types of plastids (not only the green, photosynthetic chloroplasts, but also clear leucoplasts, starch-filled amyloplasts, and pigmented chromoplasts, to name a few types of plastids) make stromules, and stromules are found in all land plant species that have been examined to date. Stromules can extend and retract dynamically, appearing or disappearing within seconds, or they can remain relatively stationary for long times. One of the major hurdles facing stromule biologists is that stromules are often studied using dramatically different methods, tissues, and species, making comparisons across the stromule biology literature difficult. Going forward, standard practices and thorough descriptions of the experimental systems used to study stromules will be critical to discovering the function of these ubiquitous features of chloroplast morphology.
Here we describe methods for visualizing stromule formation in the epidermal chloroplasts of Nicotiana benthamiana leaves. In the mesophyll, chloroplasts are densely packed into large, three-dimensional cells, which makes it difficult to accurately and rapidly visualize stromules by confocal microscopy. By contrast, epidermal cells are relatively flat, contain fewer chloroplasts, and are at the surface of the leaf, allowing for easy and rapid visualization of stromules. N. benthamiana is an ideal model system for these experiments because, unlike many plant species, all cells in the epidermis of N. benthamiana make chloroplasts10. In the epidermis of most plants, including Arabidopsis thaliana, only the stomatal guard cells have chloroplasts, while other epidermal cells have "leucoplasts", plastids that are clear, relatively amorphous, and nonphotosynthetic9,11,12. Thus, whereas a single field of view of an A. thaliana epidermis might show only a handful of chloroplasts in a pair of guard cells, a field of view of an N. benthamiana epidermis will include dozens or even hundreds of chloroplasts. All of the methods described here, however, can be modified to investigate other questions in stromule biology; for example, we have used the same approach to study leucoplast stromules of A. thaliana9.

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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.

This protocol was used to visualize stromule frequency at day and at night in the cotyledons of young N. benthamiana seedlings. Slices from a z-stack were merged into a single image (Figure 1A). For visual purposes, that image was then desaturated and inverted so that stroma appears black (Figure 1B). The chloroplasts were labeled either as having no stromules (green asterisk) or having at least one stromule (magenta asterisk). Of the 87...

Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

Visualizing Stromule Frequency with Fluorescence Microscopy

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

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

The overall goal of this procedure is to visualize and determine stromule frequency using live cell confocal fluorescence microscopy within leaves, and for visualizing stromules in vitro using chloroplast extracted from leaves. These experimental methods can help answer key questions in plant cell biology and chloroplast research by discovering how stromules function. The main advantage of these techniques is that they produce reproducible and robust data, even of these highly dynamic chloroplast structures.We had the idea for the chloroplast isolation study because some research suggested that stromule formation required cytosolic structures, like the cytoskeleton, to form. So we decided to blend up the cell, and see if stromules could still form. To prepare leaf samples, begin by using a very sharp razor blade to cut a small section of an Nicotiana benthamiana leaf.Immediately after cutting the leaf section, submerge it in a five milliliter syringe filled with water. Then, remove the air from the syringe, and apply a vacuum by using a finger to cover the syringe orifice, before pulling the plunger. Gently release the plunger to avoid damaging the leaf section.Then repeat the air removal two or three times, or until the air has been removed, and the leaf appears to be deep green. Add a drop of water to a slide, and place the leaf section on the drop. Then add another drop of water to the top of the leaf, and add a cover slip.If there are any air bubbles, gently tap the cover slip until they are removed. With transmitted light, and a 20x objective, focus on a field of view near the center of the leaf section, visualizing multiple chloroplasts simultaneously. Save an image with transmitted light, so that cell types can be distinguished later if necessary.Then switch to laser illumination with the appropriate excitation and emission filters for the fluorophore being used, and save another image. Using the microscope software, select the GFP channel, and click to adjust the pinhole aperture to one airy unit. Select laser scanning to start visualizing the sample, and then click the GFP channel and detector gain to minimize the laser power while still clearly visualizing stromules.Next, prepare a Z stack experiment that will collect the series of images through the leaf epidermis in the field of view. Adjust the scanning speed and image resolution as necessary to make sure that the Z stack is collected quickly. Then save the Z stack for later analysis.Using image J, merge the Z stack into a single image using the maximum intensity in the software. Then identify and manually count all chloroplasts in the image. For each chloroplast, visually determine whether one or more stromules are seen extending from the chloroplast in the merged Z stack image.To extract intact chloroplasts, prepare cold extraction buffer using the following reagents. Then, with NaOH and HCl, adjust the pH to 6.9, and refrigerate before use. Prepare isolation buffer, and adjust the pH to 7.6.If the leaves are not expressing a genetically encoded stromofluorophore, prepare Carboxyfluorescein diacetate, or CFDA solution. To isolate chloroplasts, remove approximately 5 to 10 grams of leaves from several plants and briefly rinse in cold water. Then immediately transfer the leaves to 50 milliliters of cold extraction buffer.Using a blender with several short pulses, grind the leaves, then filter the mixture through two to three layers of cheese cloth to remove leaf debris. Divide the extracted chloroplast into two 50 milliliter centrifuge tubes, and centrifuge for one minute at 750 x g. Discard the supernatant, and use 10 milliters of isolation buffer to re-suspend the green chloroplasts.Then centrifuge again for one minute at 750 x g, discard the supernatant, and use isolation buffer to re-suspend the chloroplasts up to a final volume of five milliliters. If the chloroplasts are from transgenic plants expressing plastid-targeted fluorescent proteins, transfer 20 microliters of the chloroplasts to a slide and add a cover slip. Then carry out microscopy.To stain chloroplasts from plants that are not expressing plastid-targeted fluorescent proteins, add five microliters of 50 millimolar CFDA stock to the five milliliters of chloroplasts in isolation buffer. After allowing the chloroplasts to incubate for five minutes transfer a small aliquot to a slide, add a cover slip and carry out microscopy. Finally, use a FITC or GFP filter set and a 20x objective to visualize multiple chloroplasts simultaneously.Or a higher objective to visualize a single isolated chloroplast. A merged stack showing GFP stomule frequency in the codoledence of young N.Benthamiana seedlings is shown here. In this panel, the image was de-saturated and inverted so that the stroma appears black.The chloroplasts were labelled either as having no stromules, or having at least one stromule. Of the 87 epidermal chloroplasts visualized, 33 have stromules, for a frequency of 37.9 percent in this leaf. This graph represents the analysis of over 23, 000 chloroplasts, and several hundred cells from a single leaf from 21 different plants.The average stromule frequency during the day was 20.8 plus or minus 1.8 percent, and the average nighttime stromule frequency was 12.8 plus or minus 0.9 percent, indicating a significantly higher daytime frequency, as determined by the Welch's T test. In this figure, chloroplast stromules were observed in vitro after isolation from N.benthamiana expressing plastid-targeted GFP, or after using CFDA to stain chloroplasts from N.benthamiana, or Spinachia oleracea. While conducting live cell imaging of stromules, it is important to work quickly, and to manipulate the plant tissue as little as possible.Following this procedure, other methods like gene silencing or chemical treatments can be used to discover additional molecular players or pathways involved in stromule formation and function. This technique has been used by researchers in plant cell biology and plant immunity to study the role of stromules in cellular signalling pathways and plant responses to pathogens. After watching this video, you should have a good understanding of how to calculate stromule frequency in a leaf, and how to observe stromules in extracted chloroplasts.And remember that the lasers and the electrical voltage supplying a scanning electron confocal microscope can be extremely hazardous, and it's important to understand the system requirements for using this equipment.

visualizing-stromule-frequency-with-fluorescence-microscopy

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Biology

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

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.

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 ...

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.

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

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.

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.

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

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.

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

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 ...

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.

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

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...