Name: vertical immobilization method for time-lapse microscopy analysis in filamentous cyanobacteria
Uploaded: 2023-09-25T14:30:00.000+00:00
Duration: 7 min 26 s
Description: Watch this Scientific Journal Video about Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria at JoVE.com

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

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

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

There is no conflict of interest.

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

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

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

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

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

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

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

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

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

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

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

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

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

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

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 ...

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source 1.
In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In ...

Developmental Biology

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

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

Summary

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Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography