Quantitative and qualitative analysis of live cells by fluorescence microscopy is an important technology in cell biology as it broadened the knowledge on cell types, their functionality, location and interrelationship within a tissue. Importantly, the use of fluorescent dyes opened the way to image and concurrently measure a variety of properties of cells simultaneously by using multiple excitation wavelengths and fluorescent probes for particular molecules. Image cytometry enabled the quantitative analysis of complex tissues1 and sequential restaining and bleaching can yield complex phenotypic analysis2 comparable to the most complex flow cytometry assays. However, it is well known that light can induce cell damage including DNA lesions and may cause apoptosis. Photodynamic therapy has taken advantage of this effect in the treatment of various diseases including cancer and skin disorders, and photosensitizers are being used to enhance the effect3. UV light is particularly harmful as it directly induces pyrimidine dimers and 6,4-photoproducts which potentially are mutagenic. Commonly used vital DNA dyes can by themselves be genotoxic4 but additional exposure of DNA-dye labeled cells to light such as in fluorescence microscopy, imaging cytometry and possibly in flow cytometry, can further enhance photo-genotoxicity. These damaging effects can be detected by numerous assays such as the DNA comet assay5,6, analysis of cell cycle perturbation, detection of DNA damage response revealed by phosphorylation of the response mediators and its effect on cell cycle regulating proteins, with the aid of image cytometry4. After several cell cycles some of these damaged cells have the potential to survive, maintain their altered genetic information and, induce modified gene expression patterns into the progeny cells which leads, in the worst case scenario, to neoplastic transformation.
It would be expected, that in fluorescence microscopy UV but not visible light is the inducer of various DNA damages and genotoxicity because it is directly absorbed by DNA. However, recent reports show that this statement has to be revised because also blue light (480nm) modifies DNA by oxidation due to production of reactive oxygen species (ROS). This damage can be observed using the DNA comet assay when cells are treated with formamylpyrimidine-DNA glycosylase6. Somewhat surprisingly, calcein-AM, the common dye that is used in many experimental setups for long term cell visualization, for example in cell migration experiments, acts as a photo-enhancer inducing substantial DNA damage not only at the sites where fluorescence is directly excited by light but also indirectly, leading to DNA modifications in cells remote from the spot of light excitation6. This effect is presumably due to ROS formation. On the other hand, the long-term exposure of cells to the novel supravital fluorochrome DRAQ7 shows no harmful effects that can be detected by analysis of DNA damage signaling or cell cycle progression7.
With this finding in mind, the results from experiments that involve long term observation of live cells performed in fluorescence imaging systems should be taken under careful scrutiny. This includes fluorochrome labeling not only of cell nuclei but also cytoplasmic and possibly even extracellular matrix constituents that are at some distance from the cells of interest as in the case of labeling the latter with the novel highly specific label Col-F8. Of importance, prior to performing time course experiments on live cells the fluorochromes with the lowest phototoxicity need to be selected. Furthermore, low intensity excitation light with possibly diminished toxic effects but still able to excite the fluorochrome is preferable. Unfortunately, future innovative quantitative label free technologies such as UV imaging9 while reducing the need for specific labeling agents, still remain phototoxic because of cells exposure to UV. Whereas this could stand true for multiphoton microscopy, it is possible that second harmonic imaging10 may be less or not at all exerting phototoxic effects.
Taken together, careful experimental design is pivotal in future live cell analysis setups. It is even as important, to revisit previous experimental findings with regard to the dyes used for cell compartment and cell function visualization. Such comparisons may clarify potential discrepancies between the findings obtained in comparable experimental settings. This is of particular importance in the upcoming field of regenerative medicine by cell therapy, where the quality control of ex vivo manipulated cell products prior to introduction into the patient is required. Uttermost care needs to be taken to avoid any genetic modifications potentially induced by the analytical procedures.