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 &amp;micro;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. ...

live-cell-imaging-life-cycle-bacterial-predator-bdellovibrio

Research

JoVE Journal

Biology

8.1K 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

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Bioengineering

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Immunology and Infection

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Bacterial cells are spatially highly organized. To follow this organization over time in slow growing Myxococcus xanthus cells, a set-up for fluorescence live-cell imaging with high spatiotemporal resolution over several generations was developed. Using this method, spatiotemporal dynamics of important proteins for chromosome segregation and cell division could be determined.

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

Biochemistry

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

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

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

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

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

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

Summary

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Chapters in this video

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Following Cell-fate in E. coli After Infection by Phage Lambda

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Super-resolution Imaging of the Bacterial Division Machinery

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions