B. subtilis shows growth arrest when subjected to blue light
The separation of DNA regions after their duplication during DNA replication (segregation) has been studied extensively using fluorescent repressor/operator (FROS) systems, or ParB/parS systems [10]. Repeats of specific DNA sequences are inserted at a single site on the chromosome whose segregation dynamics are to be investigated, and a specific binding protein (a transcriptional repressor or ParB protein) are expressed as fluorescent protein fusion to visualize the position of the binding cassette within the cell. We noticed adverse effects on cell growth when we imaged a B. subtilis strain (PG26), which carries a lacO array inserted in the chromosomal origin regions (by single crossover, leading to a duplication of the spo0J gene) and expresses LacI-CFP to visualize the origins [11], at 10 s intervals (100 ms exposure time) using 445 nm laser excitation (12 mW at the image plane). Cells were grown under aeration at 25 °C (doubling time of 93 ± 7 min, compared with 91 ± 7 min for cells devoid of the FROS system) and were mounted on minimal medium-containing agarose pads to continue growth under these oxygen-limiting conditions. When only subjected to bright field microscopy, they continued to grow with an average doubling time of 180 ± 10 min (movie S1). However, the use of 445 nm laser excitation for CFP imaging resulted in growth arrest. Cells stopped growing completely after just 5 min (Fig. 1a), with some dying (based on cell shrinkage seen in bright field acquisition) towards the end of the acquisition time (movies S2 and S3). In an earlier study, a strain with the same FROS system was imaged every 5 min, and no adverse effects on cell growth were noticed [11]. Interestingly, PG26 cells continued to grow under the microscope when subjected to white light illumination (movie S1), and likewise strain KS188, which carries a tetO cassette near the origin region (inserted into the yycR gene, whose deletion has no detectable phenotype) and expresses TetO-YFP [12] (the strain grew with a doubling time of 94 ± 5 min versus 91 ± 7 min for cells devoid of a FROS system), when imaged with a 514 nm laser (for YFP imaging, 100 ms exposures with 12 mW in the image plane; movie S4), or with 200 ms exposures using metal halide illumination (120 W) (movie S5). These results indicate that the growth defect was indeed caused by blue light toxicity rather than a defect in the strain itself or light microscopy per se.
Despite the toxicity of blue light in the CFP channel, 4.5% of the imaged cells (n = 300) showed separated sister origins (Fig. 1a). The panel only shows the frames of a movie from the moment the two indicated origins started separating (around 200 s) until they assumed their final positions in the cell quarters. These findings suggest that, in some cells, the cell cycle continues (in spite of a lack of cell growth), indicating that there is no immediate cell death after the first 5 min of imaging. When cells of strain KS188, carrying a tetO array at an origin-proximal position on the chromosome and expressing TetR-YFP, were imaged using the YFP channel, 20% of the cells showed segregation events over the course of the 60 min duration of the experiment, indicating that blue light strongly blocks cell cycle progression.
To further investigate the effects of blue light excitation on the growth of B. subtilis, we employed spore germination, which allows to study the cell cycle in a synchronized population. Spores contain a single chromosome and germinate by converting their coat structure into a regular cell wall, as reflected by the conversion of bright spores into dark small rods on bright field micrographs, and then commence DNA replication in a well-timed manner [13, 14]. When imaging spores of PG26, it was obvious that the first replication event occurred early in the cell cycle, because 48% of the cells (n = 400) already contained two visible origins of replication before emerging out of the spore coat, one hour after spore activation and incubation at 37 °C (Fig. 1b). By contrast, in a similar experiment using 514 nm laser or white light passed through a YFP excitation filter, 50% of KS188 cells contained 4 separated origin regions, and 40% two separated origin signals (Fig. 1c) by 60 min after spore revival. Thus, YFP imaging is permissive for live-cell imaging in this case, while CFP imaging is not.
We noticed that the spores of strain PG26 did not complete germination after illumination with multiple pulses of blue laser light, or white light passed through a CFP excitation filter (15 one-second exposures every 1 min, 45 min to 60 min after the induction of germination), because only 50% of the spores (n = 350) changed from phase-bright into dark cells, indicating conversion of the spore coat (Fig. 1d). Figure 1b shows that many spore coats broke open, but no cell elongation took place. However, after imaging with bright field or YFP excitation in an analogous manner, 91% of spores (n = 300) showed converted spore coats and 65% of cells measured more than 2 μm (Fig. 1e), while spores only measure 1.5 μm in length. Thus, illumination with blue light halts germination and growth, suggesting that the failure to separate the origin regions (Fig. 1b) is likely a consequence of growth inhibition. These findings verify that irradiation with blue and violet light quickly and permanently arrests growth and development in B. subtilis.
We wondered whether the blue light receptor YtvA or the stress-induced σB operon might be responsible for the observed cell cycle arrest. Therefore, we imaged ytvA or sigB mutant cells taking 15 images in the CFP channel (500 ms at 1 min intervals). When imaging was initiated 15 min after the induction of germination, 95% of wild-type, sigB and ytvA mutant spores germinated (as indicated by cracked spore coats), but cell growth was arrested, even after 105 min of incubation in the dark (Fig. 1f and g). By contrast, when following strain KS188, bearing the tetO/TetR-YFP FROS tag, in the YFP channel, we observed continued cell growth with unhindered origin segregation (Fig. 1h). We therefore conclude that the σB- and YtvA-dependent blue light response is not responsible for the growth and developmental arrest observed. We extended these experiments to exponentially growing cells, which were subjected to 15 exposures of 1 s CFP illumination, with 1 min intervals, analogous to the experiments with wild-type cells described above. As observed for wild-type cells (movies S2 and S3), growth of ytvA mutant cells arrested and some cells began to shrink (movie S6), showing that blue light receptor YtvA is not involved in growth arrest following blue/violet light excitation.
We wondered whether YFP imaging might induce an adaptation process that renders cells more resistant to the adverse effects of CFP imaging. We therefore analyzed if cells responded to a combination of YFP and CFP imaging. For example, cells were imaged with 10 acquisitions (1 s) in the YFP channel at 1 min intervals, during which they continued to grow, as reflected by cell elongation (Fig. 2a). After a 2 min break, they were then subjected to 10 acquisitions (1 s) in the CFP channel at 1 min intervals. Another 9 ± 2 min later (3 independent replicates) (30 min, Fig. 2a), some cells began to shrink (13% of N = 220 cells analysed), indicative of strong cell damage. We conclude that wave lengths permissive for live-cell imaging of B. subtilis do not render the cells more resistant to CFP imaging.
B. subtilis growth is highly sensitive to low violet light illumination
We next employed excitation with a 405 nm (violet) laser, wondering if the effects seen during CFP imaging also extended towards shorter wavelengths. We first let cells grow for some time (130 min; Fig. 3a) monitoring their growth by bright field imaging. Cells were then subjected to 3% laser power (50 mW laser) for 15 s (70 μW at the image plane), and further growth was monitored by bright field imaging. In all experiments performed (three biological triplicates), cell growth ceased immediately, and after 90 to 120 min cells visibly shrank (Fig. 3a) (movie S7), indicating severe physiological defects. We then moved towards a lower laser power, as usually less than 1% laser intensity is used for live-cell PALM single-molecule tracking (i.e. less than 10 μW or about 1 W/cm2) [15, 16]. We found two different scenarios: when using (a) 0.1% laser intensity (2.3 μW at the image plane) for 75 s, cell growth arrested and cell length declined, analogous to experiments using 3% laser intensity for 15 s (movie S8). However, when cells were subjected to (b) 15 s of 0.1% intensity, they continued to grow (Fig. 3b) (movie S9). These experiments suggest that a low dose of blue light can be tolerated by B. subtilis cells, but that a threshold exists (that likely depends on the imaging conditions) beyond which growth is severely affected.
Escherichia coli cells cease to grow upon blue light (CFP) illumination
We wondered whether the inhibition of cell growth might be a specific property of B. subtilis or a more general feature in bacteria. We therefore imaged E. coli cells with (a) bright field illumination, (b) 445 nm excitation or (c) 514 nm excitation 15 times using 1 s exposures at 1 min intervals. With bright field illumination, cells showed growth 1 h after the 15 min time-lapse experiment and continued growth after 2 h (Fig. 4a). YFP imaging, by contrast, resulted in a visible impairment of growth at both time points (Fig. 4b); however, most cells (85%, 3 independent replicates performed) did still show growth, revealing that these imaging conditions were not detrimental. However, CFP time-lapse microscopy suppressed growth and instead led to visible cell death for more than 80% of the cells, as judged from a drastic change in cell transparency (Fig. 4c). When the experiment was repeated using 500 ms exposures, which in our experience is the low-end used for CFP imaging (for complexes producing bright fluorescent signals), we observed growth arrest for 75% of the cells and visible changes in cell transparency for 20% of the cells (data not shown). We therefore conclude that E. coli cells are also highly sensitive to violet light but tolerate YFP time-lapse imaging.
Caulobacter crescentus cells also are sensitive to violet light illumination
In addition to E. coli and B. subtilis, violet light excitation (using a CFP filter set) also had a bacteriostatic effect on C. crescentus, one of the other common model systems for bacterial cell biology. Cells slowed down growth already during the first 5 acquisitions (1 s exposure each) in the CFP channel (Fig. 5a and b). No more cell growth was observed after 10 exposures (Fig. 5b). The bacteriostatic effect was persistent, because cells that had been subjected to 16 CFP exposures did not resume growth within a 255 min recovery period (i.e. no more CFP exposure, but 150 ms bright field exposures every 15 min in order to take phase contrast images). No cell lysis was observed during the experiment. By contrast, cells exposed to green (YFP channel) or white light continued to grow throughout the course of the experiment (Fig. 5b), although exposure to green light slowed down growth to a small extent (Fig. 5b). Collectively, these findings show that all of the three investigated bacterial model organisms show strong sensitivity to violet light illumination.