Abstract
BackgroundFluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. Intensive irradiation of bacteria with UV, blue and violet light has been shown to be able to kill cells, but very little information is available on the effect of blue or violet light during live-cell imaging.ResultsWe show here that in the model bacterium Bacillus subtilis chromosome segregation and cell growth are rapidly halted by standard violet (405 nm) and blue light (CFP) (445–457 nm) excitation, whereas they are largely unaffected by green light (YFP). The stress sigma factor σB and the blue-light receptor YtvA are not involved in growth arrest. Using synchronized B. subtilis cells, we show that the use of blue light for fluorescence microscopy likely induces non-specific toxic effects, rather than a specific cell cycle arrest. Escherichia coli and Caulobacter crescentus cells also stop to grow after 15 one-second exposures to blue light (CFP), but continue growth when imaged under similar conditions in the YFP channel. In the case of E. coli, YFP excitation slows growth relative to white light excitation, whereas CFP excitation leads to cell death in a majority of cells. Thus, even mild violet/blue light excitation interferes with bacterial growth. Analyzing the dose-dependent effects of violet light in B. subtilis, we show that short exposures to low-intensity violet light allow for continued cell growth, while longer exposures do not.ConclusionsOur experiments show that care must be taken in the design of live-cell imaging experiments in that violet or blue excitation effects must be closely controlled during and after imaging. Violet excitation during sptPALM or other imaging studies involving photoactivation has a threshold, below which little effects can be seen, but above which a sharp transition into cell death occurs. YFP imaging proves to be better suited for time-lapse studies, especially when cell cycle or cell growth parameters are to be examined.
Highlights
Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes
We investigated in more detail if blue light could affect cell cycle progression and investigated excitation with violet (405 nm) light as used for photoactivated localization microscopy (PALM)-based single-molecule tracking
B. subtilis shows growth arrest when subjected to blue light The separation of DNA regions after their duplication during DNA replication has been studied extensively using fluorescent repressor/operator (FROS) systems, or ParB/parS systems [10]
Summary
Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. It has been shown that light-induced chemical changes in pyrrole compounds, which are present in vital cellular compounds such as vitamin B12, heme, cytochromes and other tetrapyrroles, can be a cause of cell death, but it is possible that flavin compounds could absorb photons and give rise to singlet oxygen species [3, 4]. Because of these toxic effects, many microorganisms have evolved specific responses to light, especially a stress response to blue light, to protect themselves from light-induced cellular damage. Photosynthetic organisms need to respond to light in order to maximize or restrict light reactions, and heterotrophic species need to protect themselves from light-induced cellular damage
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