Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen
de Jong, I. G., Beilharz, K., Kuipers, O. P., Veening, J. W. Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy. J. Vis. Exp. (53), e3145, doi:10.3791/3145 (2011).
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.
1. Preparation of B. subtilis cultures
2. Preparation of the microscope sample (also see Figure 2)
One hour before cells reach mid-exponential growth, prepare the microscope slide as follows:
3. Time-lapse fluorescence microscopy (also see Figure 3 and Movie 1)
The following equipment (provided by DeltaVision, UK) was used for the time-lapse microscopy experiments published in de Jong et al. 2010 5: IX71 Microscope (Olympus), CoolSNAP HQ2 camera (Princeton Instruments), 300W Xenon Light Source, 60x bright field objective (1.25 NA), GFP filterset (Chroma, excitation at 470/40 nm, emission 525/50 nm), mCherry filterset (Chroma, excitation at 572/35 nm, emission 632/60 nm). Autofocus was performed using diascopic light and using the autofocus routine present in Deltavision's Softworx software. It should be noted that there are now a number of other autofocus systems that are also suitable such as the Zeiss Definite Focus, the Nikon Perfect Focus System and the Leica Adaptive Focus Control.
The following settings were used for the time-lapse microscopy experiments published in de Jong et al. 2010 5: snapshots for movies were taken at intervals of 8 or 12 minutes using 10 % APLLC White LED light and 0.05 s exposure for bright field pictures, 10% Xenon light and 0.5 s exposure for GFP detection, and 32% Xenon light and 0.8 s exposure for mCherry detection, respectively. Raw data were stored using softWoRx 3.6.0 (Applied Presicion). The autofocus was programmed for 0.06 μm steps and a total range of 1.2 μm.
4. Data analysis of promoter activity dynamics using ImageJ
We note that other good software packages are available which are specialized in analyzing time-lapse microscopy images such as BHV software 9, 4, Schnitzcell 10, PSICIC 11, and Microbe-Tracker 12, but here we focus on the freely available ImageJ package.
5. Producing movies for publication with ImageJ
Alternative Protocol Adaptations for Streptococcus pneumoniae (Figure 4 and Movie 2):
6. Preparation of S. pneumoniae cultures
7. Preparation of the microscope sample
8. Time-lapse phase-contrast microscopy
Adjust the microscope settings for S. pneumoniae: use phase-contrast microscopy since S. pneumoniae is difficult to identify using bright-field microscopy. Continue the protocol as described for B. subtilis (follow steps 2.9 - 3.7). S. pneumoniae cells can be grown at either 30°C or 37°C (they grow faster at 37°C).
9. Representative results:
The time-lapse fluorescence experiment has been carried out successfully, if the bacteria grew into a microcolony monolayer, which is completely located within the field of view at the end of the experiment (see Figure 5A-C). If cells grew on top of each other, it is not only impossible to trace back their history accurately, but also the fluorescence levels of overlapping cells can not be measured correctly. Cells tend to grow on top of each other, if the spotted cells were not dried sufficiently (step 2.9) or if the medium composition needs to be adjusted to obtain slower growth. If a microcolony grew out of view, then the distribution of fluorescence signals within one colony can not be determined. Causes for "microcolony movement" can be insufficient drying of spotted cells (step 2.9), or if the software was not programmed to track the microcolony during development. Furthermore, it is important that local patches of increased fluorescence are not detectable in the medium as this obscures the fluorescence signals coming from the cells (see Figure 5D-F). Background related problems can arise from media compounds, airbubbles or undissolved agarose clumps. To visualize this, we show in Fig. 5F background signals of this specific slide when the image was taken using excitation/emission filters for red fluorescent dyes. As seen, bright autofluorescent spots are present which could hamper imaging. To prevent such spots, make sure the agarose is completely dissolved and there are no airbubbles when placing the coverslip on the microscope slide.
Figure 1: Experimental overview
Figure 2: Preparation of the microscope sample
Figure 3: Time-lapse fluorescence microscopy of B. subtilis cells harboring a PkinB-gfp fusion. Snapshots are taken from Movie 1. Top panels: bright field, bottom panels: GFP channel.
Figure 4: Time-lapse phase-contrast microscopy of S. pneumoniae wild type strain R6. Snapshots are taken from Movie 2.
Figure 5: Illustration of possible (time-lapse) microscopy outcomes. A-C shows factors that need to be considered for data obtained with diascopic light settings. (A) Brightfield micrograph of a microcolony monolayer (positive outcome) of sporulating B. subtilis cells (B) Brightfield image of a B. subtilis microcolony in which some cells grew on top of each other (negative outcome) (C) Brightfield image of a sporulating B. subtilis microcolony that grew out of the field of focus (negative outcome). D-F show factors that need to be considered for data obtained with episcopic light settings (D) Phase contrast picture of B. subtilis cells in exponential phase depicted to visualize where the fluorescence signals in E and F originate from (E) GFP signals of the cells shown in D. Note that the background signals are similar in each pixel (positive outcome). Also note that the exposure time might be too much since one cell shows a saturated signal (negative outcome) (F) Signals obtained through the red channel of the cells shown in D. Note that the background contains areas with increased red fluorescence levels (negative outcome).
Movie 1. Time-lapse fluorescence microscopy of B. subtilis cells harboring a PkinB-gfp fusion. Snapshots were taken in 8 min intervals. Left: bright field, Right: GFP channel. Click here to watch the movie.
Movie 2. Time-lapse phase-contrast microscopy of S. pneumoniae wild type strain R6. Snapshots were taken in 10 min intervals. Click here to watch the movie.
In contrast to many other single cell techniques, the time-lapse fluorescence microscopy method described here can be used to follow the history of a specific cell with regard to its ancestors, its behaviour, and division events. In combination with fluorescently labelled target promoters or proteins, specific developmental pathway activation can be followed in time and protein localization as well as protein dynamics can be monitored during bacterial development.
As indicated above, studies concentrating on different bacterial species can be performed by adapting the growth conditions according to the requirements for a specific bacterium. The only limitations we encountered are related to the growth conditions and sample size. Due to a sealed environment, medium conditions cannot be changed during the experiment. Furthermore, a maximum of four strains per experiment can be monitored efficiently.
Considering a few critical steps, the single cell analysis method described here can easily be applied using any automated microscope. In the following, an overview of these critical steps will be given. Detailed information can be found in the main text. General preparation: It is wise to check the autofocus settings required for a specific bacterium prior to the experiment. Likewise, approximate optimal settings for the visualization of fluorescence should be determined in advance, if possible. Furthermore, following a prepared time-line helps to have all material ready to use in time (pre-warming the microscope chamber, programming the microscope settings, preparing the slide one hour before the cells are in the desired growth phase, see Figure 1). Growth of B. subtilis in TLM and CDM: TLM and CDM are chemically defined starvation media in which B. subtilis only grows slowly. The time-period in which the cells are grown in the media might have to be prolonged depending on the specific strain. The slow growth prevents cells from piling up on each other. Preparation of the microscope sample: Air bubbles between the gene frame, the glass slide and the cover slip have to be prevented to prevent extensive drying of the agarose-based medium. The same holds for the medium/cover slip interface. It is crucial to let the cells dry enough, to prevent swimming and/or multiple layer growth. Time-lapse fluorescence microscopy: Pre-warming of the slide as well as the environmental chamber is crucial to prevent major autofocus problems. Cells should be selected in the middle of an agar pad, since these have the highest chance to stay in field and focus during the experiment (provided the sample was dried well enough). A maximum of 10 locations per experiments still works properly. After having selected the first cell of interest only use the software to adjust the focus (see text for details). Check whether the cells are still in focus during the first hours of the experiment in 30 min intervals. Analysis: It is important to check prior to extended analysis procedures whether the background of the medium has similar values in the fluorescence channels. Small dust particles, medium components, dirty lenses or tiny agarose clumps can contribute to locally increased fluorescence, making the movie difficult or impossible to analyse. Trouble shooting: If the cells grow on top of each other, this might either indicate that the coverslip was attached too soon or that the medium is not suited for growth of microcolony monolayers. If cells of interest continuously die prematurely, whereas other cells on the slide divide happily, you might want to check whether you put the UV-filter in position. It might also help to decrease the exposure time or light intensity during long experiments.
No conflicts of interest declared.
Work in the group of JWV is supported by an EU Marie-Curie Reintegration Fellowship, a Sysmo2 Grant (NWO-ALW/ERASysBio), a Horizon grant (ZonMW) and by a VENI fellowship (NWO-ALW). The group of OPK is supported by several STW grants (NWO), a SYSMO1 (IGdeJ) and SYSMO2 grant, an ESF Eurocores SynBio grant (SynMod) and by the Kluyver Center for Genomics of Industrial Fermentation and the Top Institute for Food and Nutrition.
|Gene Frame||ABgene Limited||AB-0578||1.7 x 2.8 cm|
|high-resolution low melting agarose||Sigma-Aldrich||A4718|
|big cover slip||Multiple Suppliers||24 x 50 mm|
|if desired, membrane dye, e.g. FM 5-95||Invitrogen||T23360||other membrane dyes are also available: http://probes.invitrogen.com/media/pis/mp34653.pdf|
|Time-lapse microscope with environmental chamber||Multiple Suppliers||see details for our device in the corresponding sections|