We are grateful to National doctoral scholarships (ANID 21211333; 21191389) for the financing. Grant Fondecyt 1161232.
This work was supported by de Advanced Microscopy Facility UMA UC.

1. Considerations and selection of the cellular model
NOTE: Cyanobacteria have a strong autofluorescence due to the presence of photosynthetic pigments. This signal is in the red part of the spectrum, therefore, the fluorescent proteins suitable for imaging in cyanobacteria are the ones that far from the red emission. For example, GFP, YFP, Venus, Turquoise and BFP.
<ol>
	<li>Select a divisome component present in the target filamentous cyanobacterial strain. For the experiments, it is necessary to construct a filamentous cyanobacterial mutant that expresses the selected divisome component fused to a fluorescent protein. In this protocol, an Anabaena sp. mutant that expresses FstZ-sfGFP, as an example. This strain was obtained by triparental mating with E. coli11.</li>
	<li>Check the segregation level of the mutant that will be used. In the example, the segregation was determined by PCR amplification of the target gene. The FtsZ-sfGFP mutant used in this protocol is a fully segregated strain that lacks the wild-type ftsZ gene.</li>
</ol>
2. Growth conditions
<ol>
	<li>Make a fresh subculture of the mutant using 10 mL culture in stationary phase (grown for approximately 14 days in constant light, at 25&#176;C , for Anabaena sp.) and adding to 90 mL of BG11 medium supplemented with antibiotics (Spectinomycin and Streptomycin 10 &#181;l ml-1 for the FtsZ-sfGFP mutant).</li>
	<li>Grow the new cell culture in constant light and at the optimum temperature until reaching the exponential phase. The FtsZ-sfGFP mutant of Anabaena sp. is grown at 25 &#176;C for 7 days before the sample preparation.</li>
</ol>
3. Sample preparation in an agarose matrix
<ol>
	<li>Take 2 mL of the homogenized growth culture.</li>
	<li>Centrifuge at 2,500 x g for 10 min at room temperature.</li>
	<li>In the meantime, in a 50 mL flask, heat 30 mL of 3% low melting point agarose in BG11 liquid medium. Use a microwave set to the medium power level and check the solution every 15 s until completely dissolved. Set aside to cool down to 37 &#176;C. 
	NOTE: Autoclave the solution of agarose to avoid contamination.</li>
	<li>Once centrifugation is done, discard 1.9 mL of the supernatant and resuspend the cells in the remaining volume by pipetting at least three times up and down.</li>
	<li>Mix the resuspended cells with 900 &#181;L of the 3% agarose solution by pipetting up and down at least three times. 
	NOTE: The agarose solution must be liquid but not too hot to avoid cell damage. The next step must be performed fast and before the agarose solution forms a gel like consistency =.</li>
	<li>Aspirate the agarose matrix in a 1 mL syringe carefully. It is important to avoid the generation of bubbles while the sample is being aspirated with the syringe. 
	NOTE: Before this step, make sure that the tip of the syringe is cut to eliminate the narrow entry hole.</li>
	<li>Let the sample-agarose mixture solidify by placing the syringe in the horizontal position at room temperature for 2 h.</li>
	<li>Remove the agarose matrix carefully using the plunger and put it on a clean and flat surface.</li>
	<li>Cut the solidified sample into slices, in a thickness range of 0.5 - 1 mm, maintaining the integrity of the matrix. 
	NOTE: For better results, cut the agarose matrix using a scalpel or thin razor blades.</li>
	<li>Place the slices side by side on a coverslip. The number of disks on a coverslip will depend on its dimensions. 
	NOTE: For time-lapse microscopy, it is recommended to use a cell chamber made for microscopy analysis (e.g., Attofluor or Chamlide Magnetic chambers). These chambers allow the acquisition of the sample avoiding dehydration. In this example the samples are prepared on rounded coverslips (25 mm) using the Attofluor chamber.</li>
	<li>Add 2 mL of melted 3% agarose solution on the sample placed in the chamber to prevent dehydration.</li>
	<li>Wait until the agarose solution is completely solidified to form a gel.</li>
</ol>
4. Image acquisition
<ol>
	<li>Standardize the parameters to visualize the fluorescent protein of interest in the mutant strain in a confocal microscope. Make sure that the microscope can detect both the autofluorescence and the selected fluorescent marker.</li>
	<li>Visualize the sample in the bright field conformation with maximum magnification, and search a vertically oriented filament as shown in Figure 1.</li>
	<li>Find the division site of interest with an initial scanning using the autofluorescence signal along the Z plane.</li>
	<li>Use the parameters of the fluorescent protein marker to visualize the signal of the protein of interest at the division site.</li>
	<li>Perform time-lapse experiments according to the biological model and the protein of interest. For example, in this case time-lapse experiments of 10 s in 50 s were performed to record the dynamics of FtsZ along the Z-ring in Anabaena sp. (Figure 2).</li>
</ol>

<ol>
	<li>L&#246;we, J., Amos, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+bacterial+cell-division+protein+FtsZ.">Crystal structure of the bacterial cell-division protein FtsZ.</a> Nature. 391 (6663), 203-206 (1998).</li><li>Erickson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FtsZ,+a+prokaryotic+homolog+of+tubulin.">FtsZ, a prokaryotic homolog of tubulin.</a> Cell. 80 (3), 367-370 (1995).</li><li>Huang, K., Mychack, A., Tchorzewski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+FtsZ+C-terminal+variable+(+CTV+)+region+in+Z-ring+assembly+and+interaction+with+the+Z-ring+stabilizer+ZapD+in+E.">Characterization of the FtsZ C-terminal variable ( CTV ) region in Z-ring assembly and interaction with the Z-ring stabilizer ZapD in E.</a> coli cytokinesis. , 1-24 (2016).</li><li>Perez, A. J., 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=Movement+dynamics+of+divisome+proteins+and+PBP2x:FtsW+in+cells+of+Streptococcus+pneumoniae.">Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae.</a> Proceedings of the National Academy of Sciences. 116 (8), 3211-3220 (2019).</li><li>Bisson-Filho, A. W., 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=Treadmilling+by+FtsZ+filaments+drives+peptidoglycan+synthesis+and+bacterial+cell+division.">Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division.</a> Science. 355 (6326), 739-743 (2017).</li><li>Yang, X., 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=GTPase+activity-coupled+treadmilling+of+the+bacterial+tubulin+FtsZ+organizes+septal+cell+wall+synthesis.">GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis.</a> Science. 355 (6326), 744-747 (2017).</li><li>Whitley, K. D., 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=FtsZ+treadmilling+is+essential+for+Z-ring+condensation+and+septal+constriction+initiation+in+Bacillus+subtilis+cell+division.">FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division.</a> Nature Communications. 12 (1), (2021).</li><li>Maz&#243;n, G., 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=LexA-binding+sequences+in+Gram-positive+and+cyanobacteria+are+closely+related.">LexA-binding sequences in Gram-positive and cyanobacteria are closely related.</a> Molecular Genetics and Genomics. 271 (1), 40-49 (2004).</li><li>Mandakovic, D., 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=CyDiv,+a+conserved+and+novel+filamentous+cyanobacterial+cell+division+protein+involved+in+septum+localization.">CyDiv, a conserved and novel filamentous cyanobacterial cell division protein involved in septum localization.</a> Frontiers in Microbiology. 7 (FEB), 1-11 (2016).</li><li>Camargo, 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=ZipN+is+an+essential+FtsZ+membrane+tether+and+contributes+to+the+septal+localization+of+SepJ+in+the+filamentous+cyanobacterium+Anabaena.">ZipN is an essential FtsZ membrane tether and contributes to the septal localization of SepJ in the filamentous cyanobacterium Anabaena.</a> Scientific Reports. 9 (1), 1-15 (2019).</li><li>Thiel, T., Peter Wolk, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugal+Transfer+of+Plasmids+to+Cyanobacteria.">Conjugal Transfer of Plasmids to Cyanobacteria.</a> Methods in Enzymology. 153 (C), 232-243 (1987).</li><li>Moffitt, J. R., Lee, J. B., Cluzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-cell+chemostat:+An+agarose-based,+microfluidic+device+for+high-throughput,+single-cell+studies+of+bacteria+and+bacterial+communities.">The single-cell chemostat: An agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities.</a> Lab on a Chip. 12 (8), 1487-1494 (2012).</li></ol>

There is no conflict of interest.

The study of the dynamics of divisome proteins is without doubt a challenge. Particularly, in filamentous cyanobacteria, one the challenges is the visualization the Z-ring in the horizontal plane, for which the cells must be vertically oriented. The method we described here allows the positioning of the Z-ring in the XY plane to perform the different analyzes. This is the first simple method for filamentous cyanobacteria that allows the complete visualization of the Z-ring. 
Although this prot...

Bacterial cell division is the process in which a mother cell generates two daughter cells, in most cases by the mechanism known as binary fission. One of the earliest events in the septation process is the localization of FtsZ in the middle of the cell1. This protein, which is structurally homologous to tubulin2, is conserved and widely distributed in most bacteria, and its polymerization generates a contractile structure known as the Z-ring3. This ring serves as a scaffold for other division proteins and together they form a molecular machinery called a divisome. Several studies have shown that the Z-ring is highly dynamic and that FtsZ protofilaments move by treadmilling4,5,6. To study the Z-ring in time-lapse experiments, it is advisable to record the division site in the XY plane for better resolution and rapid sampling. In order to achieve this, it is necessary to develop vertical cell immobilization methods that commonly include the nanofabrication of microhole cell traps and complex microfluidic devices7.
Cyanobacteria are photosynthetic microorganisms that are classified as gram-negative by their cellular morphology. However, phylogenetically they are closer to gram-positive bacteria8. These organisms have cell division genes that are common within gram-positive and gram-negative bacteria, but their divisome also contains unique elements9. Anabaena sp. PCC 7120 (hereafter Anabaena sp.) is filamentous cyanobacteria with one division plane and is a model for the study of cell division in multicellular cyanobacteria. In this strain, it has been possible to determine the positioning of FtsZ in the middle of the cells10. Nevertheless, there are no studies showing the in vivo dynamics of FtsZ in this model. In our laboratory, through triparental mating and homologous recombination, we obtained a totally segregated mutant of Anabaena sp. that expresses the FtsZ protein fused to sfGFP, which replaced the complete endogenous ftsZ gene. We developed a rapid and simple cell immobilization method that favors the vertical orientation of the mutant strain filaments for time-lapse experiments to visualize the division proteins in filamentous cyanobacteria. This method does not need microfluidic devices that can be expensive and difficult to develop. As an example, we used this protocol to visualize the Z-ring in the FtsZ-sfGFP mutant by confocal microscopy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 ml syringe</td>
			<td>Qingdao Agna Medical Technology Co., Ltd.</td>
			<td>-</td>
			<td>The disposable medical plastic syringe with 1 ml needle generally used to pump liquid or injection liquid, in this experiment it is used to suck the sample and make it polymerize.</td>
		</tr>
		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>ThermoFischer Scientific</td>
			<td>A7816</td>
			<td>The Attofluor Cell Camera is a durable and practical coverslip holder designed for viewing live cell samples in upright or inverted microscopes.</td>
		</tr>
		<tr>
			<td>Axygen MaxyGene II Thermal Cycler with 96 well block</td>
			<td>CORNING</td>
			<td>THERM-1001</td>
			<td>The new MaxyGene II Thermal Cycler increased speed and advanced features, providing the premium performance you have come to expect from Axygen brand products. Unique flexible programming. Rapid run times. Improved workflow over traditional gradient cyclers. Ramping rates up to 5&#176;C/sec. Adjustable heated lid accommodates strips, tubes and microplates.</td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses: Circles</td>
			<td>ThermoFischer Scientific</td>
			<td>12-546-2P</td>
			<td>Made of finest optical borosilicate glass, with uniform thickness and size. Circular shape. Corrosion-resistant.&#160;</td>
		</tr>
		<tr>
			<td>Low Melting Point agarose</td>
			<td>Promega</td>
			<td>V3841</td>
			<td>Agarose, Low Melting Point, Analytical Grade, is ideal for applications that require recovery of intact DNA fragments after gel electrophoresis.</td>
		</tr>
		<tr>
			<td>LSM 880 microscope from Zeiss Airyscan</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>The Zeiss Airyscan LSM 880 microscope is a laser scanning focal microscope that can acquire images under the resolution limit (lateral resolution ~ 120nm and axial resolution ~ 350nm). The detectors are highly sensitive making it possible to acquire super-resolution images at high speed.</td>
		</tr>
		<tr>
			<td>Microcentr&#237;fuga Fresco 17</td>
			<td>ThermoFischer Scientific</td>
			<td>75002402</td>
			<td>Speed up routine sample preparation processes up to 17,000 &#215; g with our standard microcentrifuge, available with refrigeration. These microcentrifuges offer productivity, versatility, safety and convenience in an easy-to-use, compact design laboratory instrument.</td>
		</tr>
		<tr>
			<td>Nikon Timelapse Microscope</td>
			<td>Nikon</td>
			<td>-</td>
			<td>The Nikon C2 laser scanning confocal microscope is an ideal microscope for long-term timelapse, because thanks to its incubation chamber it is possible to keep samples in optimal conditions for more than 8 hours.</td>
		</tr>
		<tr>
			<td>Thin razor blades Schick Super Chromium</td>
			<td>Farmazon</td>
			<td>SKU: 401146</td>
			<td>&#160;The thin razor blades is used to cut the agarose matrix, allowing us to obtain the disks with the sample while maintaining the integrity of the matrix.</td>
		</tr>
	</tbody>
</table>

vertical immobilization method for time-lapse microscopy analysis in filamentous cyanobacteria

A main event in bacterial cell division is the septation process, where the protein FtsZ is the key element. FtsZ polymerizes forming a ring-like structure (Z-ring) in the middle of the cell that serves as a scaffold for other division proteins. Super-resolution microscopy in bacterial models Escherichia coli and Bacillus subtilis showed that the Z-ring is discontinuous, while live cell imaging studies demonstrated that FtsZ moves along the ring by a mechanism known as treadmilling. To study the dynamics of FtsZ in vivo, a special cell placement in a vertical position is necessary for imaging the complete structure of the ring in the XY plane. In the case of FtsZ imaging in multicellular cyanobacteria, such as Anabaena sp. PCC7120, maintaining the filaments in a vertical position is challenging because of the size of the cells and the filaments' length. In this article, we describe a method that allows the vertical immobilization of Anabaena sp. PCC 7120 filaments using low melting point agarose and syringes, to record the Z-ring in a mutant that expresses a FtsZ-sfGFP fusion protein. This method is a rapid and inexpensive way to register protein dynamics at the division site using confocal microscopy.

Visualization of the Z-ring in Anabaena sp. using the vertical immobilization method 
To study the dynamics of Z-ring components in bacteria, it is necessary to acquire images in vertically oriented cells. In this position, it is possible to visualize the Z-ring and the main proteins of the divisome to monitor protein dynamics by time-lapse microscopy. The classic sample preparation for bacteria does not work for filamentous cyanobacteria. In the case of Anabaena sp., the filaments...

Watch this Scientific Journal Video about Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria at JoVE.com

Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria

We present a simple and accessible method for filamentous cyanobacterial visualization in the XY plane. A low-melting point agarose matrix was used, allowing the acquisition of images of proteins involved in the division, in a vertical orientation. Therefore, this methodology can be applied to any filamentous organism and different kinds of proteins.

A main event in bacterial cell division is the septation process, where the protein FtsZ is the key element. FtsZ polymerizes forming a ring-like ...

vertical-immobilization-method-for-time-lapse-microscopy-analysis

Research

JoVE Journal

Biology

830 Views.  Pontificia Universidad Católica de Chile. We present a simple and accessible method for filamentous cyanobacterial visualization in the XY plane. A low-melting point agarose matrix was used, allowing the acquisition of images of proteins involved in the division, in a vertical orientation. Therefore, this methodology can be applied to any filamentous organism and different kinds of proteins. 

Video: Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...