This study was supported by the National Science Centre grant Opus 2018/29/B/NZ6/00539 to J.Z.C.

1. Preparation of B. bacteriovorus lysate for microscopic analysis
<ol>
	<li>In a 250 mL flask, set up the coculture by combining 1 mL of fresh 24 h B. bacteriovorus culture (e.g., HD100 DnaN-mNeonGreen/PilZ-mCherry) and 3 mL of overnight prey culture (e.g., E. coli S17-1 pZMR100) with 50 mL of Ca-HEPES buffer (25 mM HEPES, 2 mM CaCl2 [pH = 7.6]) supplemented with antibiotics when needed (here, 50 &#181;g/mL kanamycin).</li>
	<li>Incubate the coculture for 24 h at 30 &#176;C with shaking at 200 rpm.</li>
	<li>Check that the prey cells are fully lysed by liquid mounted phase-contrast microscopy. At this point, numerous motile attack phase B. bacteriovorus cells should be present, and no E. coli cell or bdelloplast should be visible.</li>
	<li>Filter the B. bacteriovorus culture through a 0.45 &#181;m pore size filter to remove any remaining host cells. Predatory cells (0.3&#8211;0.5 &#181;m x 0.5&#8211;1.4 &#181;m) freely penetrates 0.45 &#181;m pores, whereas host cells are retained on the filter surface.</li>
	<li>Spin down the filtrate for 20 min at 2469 x g and 30&#176;C in a 50 mL conical tube to collect predatory cells. Resuspend the B. bacteriovorus pellet in 3 mL of Ca-HEPES buffer (to a final OD600 of approximately 0.2) and incubate at 30 &#176;C and 200 rpm for 30 min.</li>
</ol>
2. Preparation of host cells for B. bacteriovorus invasion
<ol>
	<li>Use a single colony of the host strain (E. coli S17-1 pZMR100 is recommended due to various cell sizes resulting in the formation of unequally sized bdelloplasts) to inoculate 10 mL of YT medium (0.8% Bacto tryptone, 0.5% yeast extract, 0.5% NaCl [pH = 7.5]) that has been supplemented with antibiotics, if needed (here, 50 &#181;g/mL kanamycin). Culture cells overnight at 37 &#176;C and 180 rpm.</li>
	<li>Transfer 2 mL of overnight E. coli culture (OD600 of ~3.0) to a 2 mL test tube and centrifuge at room temperature (RT) and 2469 x g for 5 min. Resuspend the pellet in 200 &#181;L of Ca-HEPES buffer.</li>
</ol>
3. Set-up of the microscope (see Table of Materials)
<ol>
	<li>Pre-warm a microscopic chamber with a 100x oil immersion objective to 30 &#176;C for at least 1 h before use. This step is required to prevent errors in maintaining focus during the experiment.</li>
	<li>Turn on both the microscope and microscopy automation. Initialize the control software and select the appropriate filters to acquire brightfield/DIC/phase-contrast and fluorescence images, as needed (here: BF images, GFP/mCherry polychromic filter, and emission/excitation for mCherry and GFP).</li>
</ol>
4. Assembly of layouts for time-lapse fluorescence microscopy
<ol>
	<li>Mix 200 mg of low fluorescent molecular grade agarose with 20 mL of Ca-HEPES buffer (1% final agarose concentration) and dissolve the agarose in a microwave. The batch can be reused several times after it solidifies, so simply repeat the heating step.</li>
	<li>Pour 3 mL of melted agarose into a 35 mm glass-bottom dish (Table of Materials). Allow the agarose to solidify.</li>
	<li>Using a laboratory micro-spatula, carefully remove the agarose pad from the 35 mm dish without disturbing the center pole of the agarose pad. Flip the pad bottom side facing up and place it in a Petri dish cover (Figure 2).</li>
	<li>Put 5 &#181;L of concentrated E. coli suspension on top of the flipped pad and spread cells in the center pole using an inoculation loop. Add 5 &#181;L of concentrated B. bacteriovorus suspension (OD600 of ~0.2) onto the E. coli-coated surface. Do not spread the cells.</li>
	<li>Quickly return the agarose gel to the 35 mm glass-bottom dish (top side facing down), then cover with a lid. If necessary, remove any air bubbles by gently pressing down the agar against the plate.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61105/61105fig02.jpg" /> 
Figure 2: Schematic depiction of the experimental workflow.&#160;The agarose pad is removed from the 35 mm dish and flipped so that the bottom side faces upwards. Fresh suspensions of predatory cells and overnight culture of prey cells are placed on the flipped pad to coat the &#8220;bottom&#8221; side. The agarose pad is then flipped to its original orientation and placed back in the 35 mm dish, which is mounted onto the inverted microscope stage in the microscope chamber. <a href="https://www.jove.com/files/ftp_upload/61105/61105fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Conduction of time-lapse fluorescence microscopy
<ol>
	<li>Place the 35 mm dish in the Petri dish holder such that it will not move during the course of the experiment. Place one drop of immersion oil (1.518 refractive index) onto the objective as well as the bottom of the 35 mm dish.</li>
	<li>Mount the holder onto the stage of the inverted microscope in the microscope chamber.</li>
	<li>Set up the options in the microscope control software to collect multiple images at multiple stage positions over time.
	<ol>
		<li>Set the magnification (100x) and polychromic lens (GFP/mCherry). Using an eye piece and brightfield (BF), find the focal plane and open the point list manager.</li>
		<li>Select at least 10 positions of interest by moving the stage and storing the coordinates for each position in the microscope software by clicking the Mark point button. Avoid positions located close to each other to prevent photobleaching and phototoxicity. Turn the camera valve from the eyepiece to the monitor and calibrate each point by setting the focal plane and clicking the Replace point button in the point list manager.</li>
		<li>Open the experiment designer and set all experimental parameters in each tab as follows:
		<ol>
			<li>Unmark the Z-stacking box.</li>
			<li>Choose fluorescent channels and set the optimal illumination settings. Here, the following settings were used: for the mCherry channel, mCherry filter sets (EX575/25; EM625/45), 50% intensity, and 200 ms exposure time; for the GFP channel, GFP filter set (EX475/28; EM525/48), 50% intensity, and 80 ms; and for the BF images, POL channel, 5% intensity, and 50 ms.</li>
			<li>Select intervals between acquiring images and the total time of the experiment. Here, 5 min intervals were performed over the 10 h period of the experiment.</li>
			<li>Enable focus maintenance for the chosen positions (for the system used here, by marking the Maintain focus with UltimateFocus check box).</li>
			<li>Select the data folder in which the image files should be automatically saved.</li>
			<li>Recheck all settings in the microscope software control, then start the time-lapse experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After the first hour of the experiment, check that all stage positions are still in focus. Adjust the focus during time intervals between image acquisition, if needed.</li>
	<li>When the time-lapse experiment is finished, remove the Petri dish and utilize the 35 mm dish with an agarose pad according to the biosafety protocol. 
	NOTE: All substeps after step 5.2 are specific for DeltaVision Elite users. Researchers using other systems need to adjust the settings according to individual systems.</li>
</ol>
6. Processing of time-lapse images and generation of movies using Fiji software
<ol>
	<li>Transfer experimental data to a computer loaded with the Fiji software. Launch the Fiji program and open an image using File | Open or by simply dragging and dropping the image file into Fiji.</li>
	<li>In Bio-Formats Import Options, choose View stack with Hyperstack in the stack viewing options, Click on Composite color mode in the color options, and mark Autoscale.</li>
	<li>By scrolling through the image stacks, assess whether the cells and bdelloplasts remained in focus over the course of the experiment. Check whether green fluorescent spots from DnaN-mNeonGreen are present within B. bacteriovorus cells inside bdelloplasts throughout the growth phase, and ensure that all stages of the predatory life cycle can be visualized.</li>
	<li>To analyze a particular B. bacteriovorus cell/bdelloplast, mark it using the selection tools in the Fiji menu (Rectangle, Oval, Polygon, or Freehand). Duplicate the chosen region using Image | Duplicate or by using Ctrl + Shift + D.</li>
	<li>Adjust the brightness and contrast for each fluorescence channel as follows:
	<ol>
		<li>To choose a single channel, open Channel tools by clicking Image | Color | Channels tools or Ctrl + Shift + Z, and select the channel of interest (Channel 1: mCherry, Channel 2: mNeonGreen, Channel 3: brightfield).</li>
		<li>Adjust the brightness and contrast of images in the fluorescence channel by opening the B&#38;C menu in Image | Adjust | Brightness/Contrast or by clicking Ctrl + Shift + C.</li>
	</ol>
	</li>
	<li>Add scale bar using Analyze | Tools | Scale bar. Add the time stamper to the images by clicking Images | Stacks | Time Stamper.</li>
	<li>To save modified images, go to File | Save As and choose TIFF to save as an image sequence, AVI to produce a movie, or PNG to save a single image of the current frame.</li>
</ol>

<ol>
	<li>Shatzkes, K., 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=Predatory+Bacteria+Attenuate+Klebsiella+pneumoniae+Burden+in+Rat+Lungs.">Predatory Bacteria Attenuate Klebsiella pneumoniae Burden in Rat Lungs.</a> mBio. 7 (6), (2016).</li><li>Iebba, V., 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=Bdellovibrio+bacteriovorus+directly+attacks+Pseudomonas+aeruginosa+and+Staphylococcus+aureus+Cystic+fibrosis+isolates.">Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates.</a> Frontiers in Microbiology. 5, (2014).</li><li>Willis, A. R., 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=Injections+of+Predatory+Bacteria+Work+Alongside+Host+Immune+Cells+to+Treat+Shigella+Infection+in+Zebrafish+Larvae.">Injections of Predatory Bacteria Work Alongside Host Immune Cells to Treat Shigella Infection in Zebrafish Larvae.</a> Current biology: CB. 26 (24), 3343-3351 (2016).</li><li>Lambert, 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=Characterizing+the+flagellar+filament+and+the+role+of+motility+in+bacterial+prey-penetration+by+Bdellovibrio+bacteriovorus.">Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus.</a> Molecular Microbiology. 60 (2), 274-286 (2006).</li><li>Lambert, 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=A+Predatory+Patchwork:+Membrane+and+Surface+Structures+of+Bdellovibrio+bacteriovorus.">A Predatory Patchwork: Membrane and Surface Structures of Bdellovibrio bacteriovorus.</a> Advances in Microbial Physiology. 54, 313-361 (2008).</li><li>Makowski, &. #. 3. 2. 1. ;., 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=Initiation+of+Chromosomal+Replication+in+Predatory+Bacterium+Bdellovibrio+bacteriovorus.">Initiation of Chromosomal Replication in Predatory Bacterium Bdellovibrio bacteriovorus.</a> Frontiers in Microbiology. 7, 1898 (2016).</li><li>Makowski, &. #. 3. 2. 1. ;., 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=Dynamics+of+Chromosome+Replication+and+Its+Relationship+to+Predatory+Attack+Lifestyles+in+Bdellovibrio+bacteriovorus.">Dynamics of Chromosome Replication and Its Relationship to Predatory Attack Lifestyles in Bdellovibrio bacteriovorus.</a> Applied and Environmental Microbiology. 85 (14), (2019).</li><li>Fenton, A. K., Kanna, M., Woods, R. D., Aizawa, S. I., Sockett, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shadowing+the+actions+of+a+predator:+backlit+fluorescent+microscopy+reveals+synchronous+nonbinary+septation+of+predatory+Bdellovibrio+inside+prey+and+exit+through+discrete+bdelloplast+pores.">Shadowing the actions of a predator: backlit fluorescent microscopy reveals synchronous nonbinary septation of predatory Bdellovibrio inside prey and exit through discrete bdelloplast pores.</a> Journal of Bacteriology. 192 (24), 6329-6335 (2010).</li><li>Kuru, E., 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=Fluorescent+D-amino-acids+reveal+bi-cellular+cell+wall+modifications+important+for+Bdellovibrio+bacteriovorus+predation.">Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation.</a> Nature Microbiology. 2 (12), 1648-1657 (2017).</li><li>Dashiff, A., Junka, R. A., Libera, M., Kadouri, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predation+of+human+pathogens+by+the+predatory+bacteria+Micavibrio+aeruginosavorus+and+Bdellovibrio+bacteriovorus.">Predation of human pathogens by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus.</a> Journal of Applied Microbiology. 110 (2), 431-444 (2011).</li></ol>

The authors have nothing to disclose.

Due to the increased interest in using B. bacteriovorus as a living antibiotic, new tools for observing the predatory life cycle, particularly predator-pathogen interactions, are needed. The presented protocol is used to track the entire B. bacteriovorus life cycle, especially during its growth inside the host, in real-time. Moreover, the application of a strain producing fluorescently tagged beta clamp of DNA polymerase III holoenzyme enabled monitoring of chromosome replication progression throughout ...

Bdellovibrio bacteriovorus is a small (0.3&#8211;0.5 &#181;m by 0.5&#8211;1.4 &#181;m) gram-negative bacterium that preys on other gram-negative bacteria, including harmful pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Shigella flexneri1,2,3. Since B. bacteriovorus kills pathogens, it is considered a potential living antibiotic that can be applied to combat bacterial infections, particularly those caused by multidrug-resistant strains.
B. bacteriovorus exhibits a peculiar life cycle consisting of two phases: a free-living non-replicative attack phase and an intracellular reproductive phase (Figure 1). In the free-living phase, this highly motile bacterium, which moves at speeds of up to 160 &#181;m/s, searches for its prey. After attaching to the prey&#8217;s outer membrane, it enters the periplasm4,5. During the interperiplasmic reproductive phase, B. bacteriovorus uses a plethora of hydrolytic enzymes to degrade the host&#8217;s macromolecules and reuse them for its own growth. Soon after invading the periplasm, the host cell dies and bloats into a spherical structure called a bdelloplast, inside which the predatory cell elongates and replicates its chromosomes. The replication process starts at the replication origin (oriC)6 and proceeds until several copies of the chromosome have been completely synthesized7. Interestingly, replication of each chromosome is not followed by cell division. Instead, the predator elongates to form a long, multinucleoid and filamentous cell. Upon nutrient depletion, the filament undergoes synchronous septation and progeny cells are released from the bdelloplast8.
Before B. bacteriovorus can be used as a living antibiotic against bacterial infections, it is crucial to understand the major stages of its life cycle, particularly those related to its proliferation inside the prey. Live-cell imaging of B. bacteriovorus has been challenging, due to the various morphological forms of the predator and its prey during the complex life cycle. So far, the interactions between B. bacteriovorus and its host cell have been mainly studied by electron microscopy and snap-shot analysis2,9,10, both of which have limitations, especially when they are used to monitor successive stages of the predatory life cycle. These methods provide high-resolution images of B. bacteriovorus cells and enable observation of a small predator during the attack or growth phase. However, they do not allow tracking of single B. bacteriovorus cells throughout both life cycle phases.
Presented here is a comprehensive protocol for using time-lapse fluorescence microscopy (TLFM) to monitor the complete life cycle of B. bacteriovorus. A system consisting of an agarose pad is used in combination with a cell-imaging dish, in which the predatory cells can move freely beneath the agarose pad while the immobilized prey cells are able to form bdelloplasts (Figure 2). This set-up is prepared based on specific strains of both E. coli and B. bacteriovorus, but the protocol may be easily altered to fit a user&#8217;s individual strains (e.g., carrying different selection markers, proteins fused with different fluorophores, etc.).
In this case, to visualize B. bacteriovorus during the attack phase, a specific strain (HD100 DnaN-mNeonGreen/PilZ-mCherry) was constructed that expresses a fluorescently tagged version of the cytoplasmic protein, PilZ (available in our laboratory upon request)7. This strain additionally produces DnaN (the &#946;-sliding clamp), a subunit of DNA polymerase III holoenzyme, fused with a fluorescent protein. This enables ongoing DNA replication to be monitored inside the predatory cells as they grow within bdelloplasts.
Although the described protocol and software used for image acquisition refer to an inverted microscope provided by a specific manufacturer (see Table of Materials), this technique may be adjusted for any inverted microscope equipped with an environmental chamber or other external heating holder and capable of time-lapse imaging. For data analysis, users may choose any available software compatible with the individual output formats.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61105/61105fig01.jpg" /> 
Figure 1: B. bacteriovorus life cycle in E. coli as a host cell.&#160;During the attack phase, a free-swimming B. bacteriovorus cell searches for and attaches to a host E. coli cell. After the invasion, the predatory cell becomes localized in the prey&#8217;s periplasm, changing the host cell&#8217;s shape and forming a bdelloplast. The reproductive phase starts with bdelloplast formation. The predatory cell digests the prey cell and reuses simple compounds to build its own structures. B. bacteriovorus grows as a long single filament inside the host&#8217;s periplasm. When the prey cell&#8217;s resources are exhausted, the B. bacteriovorus filament synchronously septates and forms progeny cells. After the progeny cells develop their flagella, they lyse the bdelloplast. <a href="https://www.jove.com/files/ftp_upload/61105/61105fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Centrifuge</td>
			<td>MPW MED. INSTRUMENTS</td>
			<td>MPW-260R</td>
			<td>Rotor ref. 12183</td>
		</tr>
		<tr>
			<td>CertifiedMolecular Biology Agarose</td>
			<td>BIO-RAD</td>
			<td>161-3100</td>
			<td>low fluorescence agarose for agarose pad</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>ImageJ</td>
			<td>https://imagej.net/Fiji</td>
			<td>Open source image processing package</td>
		</tr>
		<tr>
			<td>Glass Bottom Dish 35 mm</td>
			<td>ibidi</td>
			<td>81218-200</td>
			<td>uncoated glass</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>GE</td>
			<td>DeltaVision Elite</td>
			<td>Microtiter Stage, ultimate focus laser module, DV Elite CoolSnap HQ2 Camera, SSI assembly FP DV, kit obj. Oly 100x oil 1.4 NA, prism Nomarski 100x LWD DIC, ENV ctrl IX71 uTiter opaQ 240 V</td>
		</tr>
		<tr>
			<td>Minisart Filter 0.45 &#181;m</td>
			<td>Sartorius</td>
			<td>16555----------K</td>
			<td>Cellulose Acetate, Sterile, Luer Lock Outlet</td>
		</tr>
		<tr>
			<td>Start SoftWoRx</td>
			<td>GE</td>
			<td></td>
			<td>Manufacturer-supplied imaging software</td>
		</tr>
	</tbody>
</table>

live-cell imaging of the life cycle of bacterial predator bdellovibrio bacteriovorus using time-lapse fluorescence microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. Therefore, it is considered a living antibiotic. To apply B. bacteriovorus as a living antibiotic, it is first necessary to understand the major stages of its complex life cycle, particularly its proliferation inside prey. So far, it has been challenging to monitor successive stages of the predatory life cycle in real-time. Presented here is a comprehensive protocol for real-time imaging of the complete life cycle of B. bacteriovorus, especially during its growth inside the host. For this purpose, a system consisting of an agarose pad is used in combination with cell-imaging dishes, in which the predatory cells can move freely beneath the agarose pad while immobilized prey cells are able to form bdelloplasts. The application of a strain producing a fluorescently tagged &#946;-subunit of DNA polymerase III further allows chromosome replication to be monitored during the reproduction phase of the B. bacteriovorus life cycle.

The described TLFM-based system allows individual cells of B. bacteriovorus to be tracked in time (Figure 3, Movie 1) and provides valuable information about each stage of the complex predatory life cycle. The PilZ-mCherry fusion enables the entire predatory cell to be labeled in the attack phase as well as early stage of the growth phase (Figure 3). The transition from the attack to replicative phase was visualized not only by the host...

Watch this Scientific Journal Video about Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy at JoVE.com

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Presented here is a protocol that describes monitoring of the complete life cycle of predatory bacterium Bdellovibrio bacteriovorus using time-lapse fluorescence microscopy in combination with an agarose pad and cell-imaging dishes.

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...

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JoVE Journal

Biology

8.0K Views.  University of Wroclaw. Presented here is a protocol that describes monitoring of the complete life cycle of predatory bacterium Bdellovibrio bacteriovorus using time-lapse fluorescence microscopy in combination with an agarose pad and cell-imaging dishes.

Video: Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

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 Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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 ...

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, ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

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

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

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

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 ...