We thank Ms. Susan Staurovsky at the UConn Health Center for Cell Analysis and Modeling Microscopy Facility for her assistance with microscopy experiments. We thank Dr. Mona Wu Orr and the Essentials of Staphylococcal Genetics and Metabolism Workshop for their protocols and advice on cloning in P. aeruginosa and S. aureus, respectively. This work was supported by funding from the National Institutes of Health (NIH; DP2GM146456-01 and 1R01AI167886-01A1 to W.W.K.M., 1F30DE032598-01A1 to P.J.H., and 1F31DK136259-01A1 to T.J.L.). The funders had no role in the design of our experiments or preparation of this manuscript.

Visualizing Antibiotic Persistence in Bacterial Pathogens

<ol>
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N., Michiels, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+persister+cells+in+chronic+infections:+clinical+relevance+and+perspectives+on+anti-persister+therapies.">Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies.</a> J Med Microbiol. 60 (pt 6), 699-709 (2011).</li><li>Van den Bergh, B., Fauvart, M., Michiels, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation,+physiology,+ecology,+evolution+and+clinical+importance+of+bacterial+persisters.">Formation, physiology, ecology, evolution and clinical importance of bacterial persisters.</a> FEMS Microbiol Rev. 41, 219-251 (2017).</li><li>Levin, B. R., Rozen, 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=Non-inherited+antibiotic+resistance.">Non-inherited antibiotic resistance.</a> Nat Rev Microbiol. 4 (7), 556-562 (2006).</li><li>Davis, K. M., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+heterogeneity+within+bacterial+populations+via+single+cell+approaches.">Defining heterogeneity within bacterial populations via single cell approaches.</a> Bioessays. 38 (8), 782-790 (2016).</li><li>Hare, P. J., LaGree, T. J., Byrd, B. A., DeMarco, A. M., Mok, W. W. 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Y., 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=agr-Dependent+interactions+of+Staphylococcus+aureus+USA300+with+human+polymorphonuclear+neutrophils.">agr-Dependent interactions of Staphylococcus aureus USA300 with human polymorphonuclear neutrophils.</a> J Innate Immun. 2 (6), 546-559 (2010).</li><li>Bose, J. L., Fey, P. D., Bayles, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+tools+to+enhance+the+study+of+gene+function+and+regulation+in+Staphylococcus+aureus.">Genetic tools to enhance the study of gene function and regulation in Staphylococcus aureus.</a> Appl Environ Microbiol. 79 (7), 2218 (2013).</li><li>Schenk, S., Laddaga, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+electroporation+of+Staphylococcus+aureus.">Improved method for electroporation of Staphylococcus aureus.</a> FEMS Microbiol Lett. 73, 133-138 (1992).</li><li>Grosser, M. R., Richardson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+Preparation+and+Electroporation+of+S.+aureus+and+S.+epidermidis.">Method for Preparation and Electroporation of S. aureus and S. epidermidis.</a> The Genetic Manipulation of Staphylococci. Methods Molecular Biology. , (2016).</li><li>Krausz, K. L., Bose, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+Transduction+in+Staphylococcus+aureus:+Broth-Based+Method.">Bacteriophage Transduction in Staphylococcus aureus: Broth-Based Method.</a> The Genetic Manipulation of Staphylococci. Methods Molecular Biology. , (2016).</li><li>Olson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+Transduction+in+Staphylococcus+aureus.">Bacteriophage Transduction in Staphylococcus aureus.</a> The Genetic Manipulation of Staphylococci. Methods Molecular Biology. , (2016).</li><li>Fey, P. 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=A+genetic+resource+for+rapid+and+comprehensive+phenotype+screening+of+nonessential+Staphylococcus+aureus+genes.">A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes.</a> mBio. 4 (1), e00537-e00612 (2013).</li><li>Coe, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-strain+Tn-Seq+reveals+common+daptomycin+resistance+determinants+in+Staphylococcus+aureus.">Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus.</a> PLoS Pathog. 15 (11), e1007862 (2019).</li><li>Figurski, D. H., Helinski, D. 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The authors have no conflicts of interest to declare.

We have found that the success of a time-lapse microscopy experiment hinges on the quality of the agarose pads and their stability throughout the course of imaging. The stainless-steel chamber-enclosed agarose pads are relatively easy to prepare, resulting in consistently planar samples that can be stably imaged over dozens of hours. This enables the imaging of tens of thousands of cells in a single experiment and increases the likelihood of detecting rare phenotypic variants, like persister cells, in a population.
This agarose pad preparation method presents an easily implementable alternative to previously published methods. Our protocol does not require the technical precision of microfluidic device fabrication or the dexterous manipulations of agarose &#34;sandwich&#34; methods, making it easier to achieve consistent preparations from run to run14,16,36. Furthermore, the system is cost-effective. The stainless-steel chamber is sterilizable and reusable (unlike single-use plastic chambers) and the setup does not require specialized equipment16,37. The chamber is easily fit to different microscopy systems using commercially available stage inserts. Additionally, because bacteria are immobilized at the agarose-cover glass interface, we have had success with tracking highly motile bacteria such as P. aeruginosa while still allowing for morphological changes (Figure&#160;4, Supplementary Video 4). Other single-cell imaging techniques, such as the &#34;mother machine&#34;, confine cells to channels that preclude observation of morphological changes other than filamentation36.
For success with this protocol, there are some critical steps and parameters to keep in mind. For pad preparation, it is important to thoroughly heat and melt the agarose, as any remaining agarose crystals will cause light diffraction and affect image quality. Similarly, one must take care to pipette the agarose into the chamber without introducing air bubbles. To ensure that the thickness of the agarose pads remains consistent and to limit sample drift, it is important to allow the pad to equilibrate&#8212;typically for 15 min&#8212;in the humidified, temperature-controlled enclosure before imaging begins. Another factor that can cause poor image quality is humidity control: low humidity will result in the agarose pad dehydrating and shrinking, whereas high humidity (or improper sample warming in the chamber) could cause the warm air to condense on the sample and distort the imaging. An example of suboptimal time-lapse imaging due to condensation can be found&#160;Supplementary Video 8.
A limitation of the current set-up is that the culture media cannot be exchanged, which prevents continuous tracking of individual bacteria before, during, and after antibiotic treatment. We anticipate that coupling the agarose pad with flow cells or microfluidic devices that permit culture media exchange could enable populations to be tracked during nutritional or environmental change. Another parameter of the current design that could be improved is sample aeration. The O-ring seal, screw-top design of the interchangeable coverslip dish enables better sample aeration compared with set-ups that require the use of wax or grease-based sealant to seal the pads16. However, aeration in the sealed chamber may still be limited and may not support the growth of obligate aerobes, though this remains to be tested.
The time-lapse imaging sample preparation protocol that we present in this article enables thousands of bacteria to be tracked as they respond to or recover from antibiotic treatment. This method is also highly generalizable and has a variety of potential applications beyond persister biology. For example, the agarose pad and divider set-up allows for the seeding of spatially separated cell samples yet permits cell-cell communication via diffusion through the agarose pad. We are currently exploring this set-up&#39;s potential for testing how the exchange of secreted products affects cell growth in multispecies communities. We anticipate that this protocol will provide a low barrier of entry to time-lapse microscopy for the new investigator and limitless variations for the seasoned microbiologist to explore.

Bacterial pathogens can evade the effects of antibiotics through two primary mechanisms: antibiotic resistance, which involves genetic changes, and phenotypic tolerance, which involves non-genetic changes. Antibiotic resistance is a genetically encoded phenomenon that confers the ability of a given bacterial cell to not only survive but also replicate in the presence of an antibiotic1. Phenotypic tolerance, which can encompass antibiotic-tolerant or antibiotic-persistent bacteria, occurs when cells withstand bactericidal antibiotic treatment without gaining the ability to replicate in the presence of an inhibitory concentration of the antibiotic1,2. What differentiates tolerance from persistence is that tolerance refers to the ability of the whole population to survive treatment, whereas persistence refers to a subset of an isogenic but phenotypically heterogeneous population that survives antibiotic treatment. When a clonal culture is treated with bactericidal antibiotics and survivors that remain in the culture are plotted against time on a log-linear scale, a biphasic curve is usually detected when persisters are present. On these curves, the first phase shows that the majority of the population is killed relatively quickly, and the second phase indicates that an antibiotic persistent fraction is killed at a slower rate or not at all1,2.
Antibiotic persistence presents a major burden on global healthcare systems. For instance, Staphylococcus aureus and Pseudomonas aeruginosa persisters, which are the focus of this article, are thought to cause antibiotic-recalcitrant infections, including recurrent airway infections in patients with cystic fibrosis and chronic wound infections3,4. Therefore, further elucidating persister cell biology and phenotypic programs is critical. While progress has been made in understanding how persisters form and resuscitate, critical knowledge gaps pertaining to the coordination of metabolic reprogramming and molecular events in individual cells that underlie persistence remain5,6,7,8.
Effectively studying persistence has proven to be a technical challenge. Since persistence is only observable in a small subset of a bacterial population, techniques that sample bulk bacterial populations often fail to capture relevant biological information1,2,8,9,10. Furthermore, since phenotypic changes that underlie persistence are transient and not heritable, tracking the fate of persister cells can be complex1,8,9,10,11. Once bacterial persisters resume growth, they can divide and give rise to both persisters and non-persisters, which makes it impossible to enrich for pure persister populations by culturing. These challenges highlight the need for techniques that can fulfill the following criteria: 1) the ability to capture biological information of live, single cells and 2) the ability to be used in tandem with fluorescent dyes, probes, sensors, and reporters that allow phenotypes of individual cells in heterogeneous populations to be interrogated over time.
Recent advancements in single-cell technologies have provided an avenue to effectively investigate bacterial heterogeneity and overcome these hurdles in studying persistence12,13. Some of these techniques include fluorescence microscopy, flow cytometry/fluorescence-activated cell sorting, microfluidics, and single-cell RNA sequencing12,13. Here, we describe protocols for elucidating single-cell persister physiology using epifluorescence time-lapse microscopy of transcriptional or translational reporter strains. Fluorescence microscopy is a powerful technique that fulfills the criteria for studying persister phenotypes, namely, the ability to identify which individual cells in a large population propagate after antibiotic removal and can thus be defined as persisters. With the introduction of automated camera technologies and incubated chambers, capturing live bacterial cells is widely accessible across the field of microbiology. Crucially, time-lapse microscopy offers the ability to visualize single cells in real-time, over the course of hours and even days, which makes it possible to track bacteria before, during, and after antibiotic treatment14,15,16. Insights from these investigations harnessing time-lapse microscopy have immense potential to generate insight into the complex mechanisms of persister biology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BaSiC</td>
			<td>GitHub</td>
			<td>https://github.com/marrlab/BaSiC</td>
			<td></td>
		</tr>
		<tr>
			<td>Certified Molecular Biology Agarose</td>
			<td>Biorad</td>
			<td>1613101</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji-ImageJ</td>
			<td>NIH</td>
			<td>https://imagej.net/software/fiji/downloads</td>
			<td></td>
		</tr>
		<tr>
			<td>Interchangeable Coverglass Dish</td>
			<td>Bioptechs</td>
			<td>190310-35</td>
			<td>35 mm ICD for preparing agarose pads; comes with 30 mm (#1.5) coverslips</td>
		</tr>
		<tr>
			<td>Lumencor Spectra 7 LED light engine</td>
			<td>Lumencor</td>
			<td>https://lumencor.com/products/spectra-light-engine</td>
			<td>Spectra 7 LED light engine</td>
		</tr>
		<tr>
			<td>MetaMorph</td>
			<td>Molecular Devices</td>
			<td>Premier version 7.10.5</td>
			<td></td>
		</tr>
		<tr>
			<td>pco.edge 4.2 bi sCMOS camera</td>
			<td>Excelitas</td>
			<td>https://www.excelitas.com/product/pcoedge-42-bi-usb-scmos-camera</td>
			<td>sCMOS camera</td>
		</tr>
		<tr>
			<td>PeCon live cell incubation chamber</td>
			<td>peCon</td>
			<td>https://www.pecon.biz/</td>
			<td></td>
		</tr>
		<tr>
			<td>Thomas Scientific Round cover glass, #1.5 thickness, 25 mm, 100 pack</td>
			<td>Fisher Scientific</td>
			<td>NC1272770</td>
			<td>25 mm (#1.5) coverslips</td>
		</tr>
		<tr>
			<td>Zeiss Axiovert 200M microscope</td>
			<td>Zeiss</td>
			<td>https://www.zeiss.com/microscopy/en/products/light-microscopes/widefield-microscopes/axiovert-for-materials.html</td>
			<td>Inverted microscope with Plan-Apochromat 63x/1.40 Oil Ph3 M27 objective, Lumencor Spectra 7 LED light engine, and pco.edge 4.2 bi sCMOS camera (6.5 mm pixel size)</td>
		</tr>
	</tbody>
</table>

time-lapse epifluorescence microscopy imaging of pseudomonas aeruginosa and staphylococcus aureus heterogeneous phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

Successful introduction of reporter plasmids into P. aeruginosa and S. aureus is indicated by growth on the correct selective antibiotics and can be confirmed by colony PCR and/or sequencing. The modified strains should be verified as phenotypic reporters by subjecting them to conditions in which the gene of interest is known to be induced, and the resultant fluorescence can be measured by flow cytometry, spectrophotometry, or epifluorescence microscopy (Figure 1).
To facilitate the selection of an antibiotic dose(s) that will be used for subsequent experiments, perform concentration-dependent antibiotic persister assays for the P. aeruginosa or S. aureus strains of interest. Concentration-dependent assays typically result in a biphasic curve with a steep initial slope at lower antibiotic concentrations and a plateau or less steep slope at higher concentrations. However, for some antibiotic-species pairs, a distinct biphasic curve may not result. For example, the curve for S. aureus delafloxacin curve is clearly biphasic (Figure 3A), but the P. aeruginosa levofloxacin curve is not (Figure 3B)15. In this scenario, we would choose a concentration that is at least 10x the MIC (e.g.,&#160;5&#160;&#181;g/mL, which is about 15x the MIC for P. aeruginosa)15. However, because 15x levofloxacin MIC results in only ~0.001% P. aeruginosa survivors, we use 1 &#181;g/mL levofloxacin treatment if we want to see persisters when imaging cells as they recover on antibiotic-free agarose pads (Supplementary Video 6); otherwise, the number of fields of view needed to image multiple persisters becomes prohibitive.
At the start of imaging, the ideal sample and agarose pad preparation should appear planar throughout the field of view, free of large debris, wrinkles, or air bubbles, and with evenly distributed single cells. Obtaining well-distributed single cells may require optimization of the sample dilution or resuspension. For S. aureus, cells tend to form small clusters and need to be vortexed thoroughly before seeding to the agarose pad (Figure 4 and Figure 5). For P. aeruginosa, cells may form aggregates encased in a sticky extracellular matrix in suspension; it is necessary to pipette these samples thoroughly and disrupt the aggregates for imaging single cells. 
After an imaging experiment&#39;s conclusion, a successful time-lapse of images will appear in focus, stably illuminated, and with minimal drift in the x-y plane throughout the experiment. Supplementary Video 7 represents an optimal image acquisition: t is the phase channel of Supplementary Video 4 before shade or drift correction. Loss of focus can occur if condensation (from over-humidification or insufficient sample warming) causes water droplets to form on the top 25 mm coverslip, distorting the light and pushing the focal plane outside of the autofocus algorithm&#39;s maximal search range (Supplementary Video 8). Variation in illumination usually indicates insufficient immersion oil at the time of imaging. If the stage moves too quickly, the oil on the objective may drag behind and still be catching up when the images are acquired. This can be mitigated by adjusting the acquisition controls to slow the speed of movement or adding a pause between the movement to the next position and the image acquisition. Major sample drift will look like many cells streaking across the field of view while some stay in place (Supplementary Video 9). This typically occurs later in experiments because the agarose pad has dehydrated due to insufficient humidity control. The agarose pad preparation presented in this paper was designed to facilitate sample stability, but properly warming/humidifying the sample and its surrounding environment is necessary for optimal image acquisition.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67617/67617fig1.jpg" /> 
Figure 1: Fluorescent reporter strains illuminate the expression of a gene of interest. (A) S. aureus was transduced with a GFP transcriptional reporter for a gene of interest per Protocol 1. The reporter strain was treated for 24 h with antibiotic, washed with PBS, and then seeded onto an agarose pad made from CA-MHB plus propidium iodide (1.6 &#181;M) and chloramphenicol (10 &#181;g/mL for reporter plasmid maintenance) for imaging during recovery (Supplementary Video 1). (B) P. aeruginosa was transformed with a plasmid bearing a mScarlet-linked translational reporter for a protein of interest26. The reporter strain was treated for 5 h with antibiotic, washed with PBS, and then seeded onto an agarose pad made from BSM plus Tet (75 &#181;g/mL; for reporter plasmid maintenance) for imaging during recovery (Supplementary Video 2). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/67617fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67617/67617fig2.jpg" /> 
Figure 2: Propagating and harvesting bacteriophage. (A) The six plates show six different amounts of diluted phage stock on the lawns of S. aureus RN4220. The red outlines indicate the three plates that would be harvested, from the plate with the most clearing (bold red outline; 1 x109 PFU/mL) to the next two dilutions (1 x108 and 1 x 107 PFU/mL). The black arrows point to individual plaques. (B) To harvest phage from the plates, scrape the soft agar layer (left), transfer the slurry to the next dilution plate (center), and, after pooling the soft agar from all three plates together, combine into a conical tube for centrifugation (right). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/67617fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67617/67617fig3.jpg" /> 
Figure 3: Representative concentration-dependent persister assays. Concentration-dependent fluoroquinolone persistence was assessed in stationary-phase (A)&#160;S. aureus (against delafloxacin) and (B)&#160;P. aeruginosa (against levofloxacin). Subsequent experiments utilize 5 &#181;g/mL delafloxacin (red circle) because S. aureus killing had plateaued at this concentration. A dosage of at least 1 &#181;g/mL levofloxacin (red circle) would be utilized for subsequent experiments with P. aeruginosa. Note that the bacterial killing does not plateau for P. aeruginosa, but there is still a less steep &#34;second phase&#34; of the biphasic curve that indicates a persistent subpopulation. Panel 3B has been adapted with permission from Hare et al.15. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/67617fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67617/67617fig4.jpg" /> 
Figure 4: Imaging bacterial phenotypes during antibiotic treatment. Stationary-phase&#160;(A)&#160;S. aureus and&#160;(B)&#160;P. aeruginosa cells were seeded onto agarose pads containing fluoroquinolone antibiotics and monitored during treatment: 5 &#181;g/mL delafloxacin for S. aureus (Supplementary Video 3) and 5 &#181;g/mL levofloxacin for P. aeruginosa (Supplementary Video 4)15. Propidium iodide (PI; 16 &#181;M for P. aeruginosa, 1.6 &#181;M for S. aureus) was added to the pads to mark dead or dying cells. S. aureus cells remain largely intact and alive in the presence of the FQ, whereas most P. aeruginosa cells undergo drastic morphological changes, including forming round spheroplasts, before they lyse and die. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/67617fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67617/67617fig5.jpg" /> 
Figure 5: Tracking persisters during recovery. (A)&#160;S. aureus and&#160;(B)&#160;P. aeruginosa populations were seeded onto agarose pads containing fresh media after they had been treated with fluoroquinolones (5 &#181;g/mL delafloxacin for S. aureus and 1 &#181;g/mL levofloxacin for P. aeruginosa) and monitored during their post-treatment recovery (Supplementary Video 5 and Supplementary Video 6). The perisisters seen are indicated with green arrows in the first two frames in each panel, and they remained intact and viable during antibiotic treatment. After an initial lag period, the persisters started to divide and gave rise to new progeny (indicated with green circles). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/67617fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67617/67617fig6.jpg" /> 
Figure 6: Microscope sample preparation. (A)&#160;Schematic of the sample preparation workflow using an interchangeable coverslip dish (&#34;chamber&#34;). (B) Picture of the disassembled chamber and its individual components. (C) Picture of the fully assembled chamber. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/67617fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: S. aureus persister. Video file containing the images in Figure 1A. In brief, S. aureus bearing a GFP transcriptional reporter for a gene of interest was treated for 24 h with antibiotic, washed with PBS, then seeded onto an agarose pad made with CA-MHB plus propidium iodide (1.6 &#181;M) and chloramphenicol (10 &#181;g/mL) for imaging during recovery. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS1_S-aureus_persister_video.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 2: P. aeruginosa persister. Video file containing images in Figure 1B.&#160;In brief, P. aeruginosa bearing an mScarlet-linked translational reporter for a protein of interest was treated for 5 h with antibiotic, washed with PBS, then seeded onto an agarose pad made with BSM plus Tet (75 &#181;g/mL) for imaging during recovery26. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS2_Paeru-fluorescent-persister.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 3: S. aureus during antibiotic treatment. A stationary-phase culture of S. aureus grown in rich chemically defined media was seeded to agarose pads made from the culture&#39;s cell-free conditioned media with propidium iodide (1.6 &#956;M) and delafloxacin (5 &#956;g/mL). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS3_S-aureus_treatment.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 4: P. aeruginosa during antibiotic treatment. A stationary-phase culture of P. aeruginosa grown in BSM was seeded to agarose pads made from cell-free conditioned media from a culture of P. aeruginosa grown in BSM in parallel; the agarose pad also contained propidium iodide (16 &#181;M) and levofloxacin (5 &#181;g/mL). This video has been adapted with permission from Hare et al.15. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS4_P-aeruginosa_treatment.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 5: S. aureus during post-antibiotic recovery. S. aureus was&#160;grown to&#160;stationary phase in rich chemically defined media. The stationary-phase cultures were treated with 5 &#181;g/mL delafloxacin in test tubes for 24 h, washed with PBS, and then seeded to antibiotic-free CA-MHB agarose pads containing propidium iodide (1.6 &#181;M) for imaging. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS5_S-aureus_recovery.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 6: P. aeruginosa during post-antibiotic recovery. A stationary-phase culture&#160;of P. aeruginosa grown in BSM was treated with 1 &#181;g/mL levofloxacin in test tubes for 7 h, washed with PBS, then seeded to antibiotic-free BSM agarose pads containing propidium iodide (16 &#181;M) for imaging. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS6_P-aeruginosa_recovery.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 7: Example of optimal image acquisition. This video is the phase channel of Supplementary Video 4 before image processing as an example of an optimal time-lapse acquisition. Note the minimal drift, stable illumination, and maintenance of focus throughout the experiment. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS7_uncorrected-Paeru.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 8: Example of suboptimal image acquisition due to condensation. This video shows part of an experiment when the image acquisition was affected by poor focus, likely due to condensation on the chamber due to improper heating of the sample and/or over-humidification of the imaging environment. The sample being imaged was levofloxacin-treated P. aeruginosa during post-antibiotic recovery on a BSM agarose pad. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS8_Suboptimal_Imaging_Condensation.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 9: Example of suboptimal image acquisition due to drift. This video shows part of an experiment when the image acquisition was affected by sample drift, likely due to dehydration and the shrinking/lifting of the agarose pad from the coverslip. The sample being imaged was P. aeruginosa on an agarose pad containing levofloxacin and propidium iodide. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/VideoS9_Suboptimal_Drift.avi" target="_blank">Please click here to download this video.</a>
Supplementary File 1: 25mm-3D-divider-for-35mmBioptechs.stl <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/67617/25mm-3D-divider-for-35mmBioptechs.stl" target="_blank">Please click here to download this file.</a>

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

time-lapse-epifluorescence-microscopy-imaging-pseudomonas-aeruginosa

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

671 Views.  UConn Health. In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize phenotypic variants, such as persisters, by time-lapse epifluorescence microscopy.

Video: Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

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.

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

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 ...

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

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 ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...