Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging
Massimiliano Lucidi, Giulia Capecchi, Daniela Visaggio, Tecla Gasperi, Miranda Parisi, Gabriella Cincotti, Giordano Rampioni, Paolo Visca, Kirill Kolmakov
fluorophores, far-red, multicolor imaging, FR-emitting dyes
Our understanding of bacteria is increasingly dependent on our ability to visualize cellular processes at the single-cell level with high spatial and temporal resolution. Advances in fluorescence microscopy are accompanied by an increasing demand for novel fluorophores that enable tagging specific bacterial components. In this context, new fluorescent probes emitting in the far-red (FR) are of particular interest as they reduce possible interference caused by sample autofluorescence and increase flexibility in multicolor imaging experiments. In this study, an extended set of previously reported and newly synthesized FR-emitting dyes has been characterized for their applicability in live single-cell imaging of the Gram-negative and Gram-positive prototype bacteria Escherichia coli and Bacillus subtilis. Toxicity tests demonstrated that these dyes do not interfere with the growth kinetics of both species, opening up the possibility of using them in live-cell imaging. Moreover, confocal laser-scanning microscopy imaging revealed that all the tested dyes can distinguish viable from dead bacterial cells. Among the newly synthesized fluorophores, the oxazine derivative KK 1905-NHS was particularly efficient in membrane staining and was effectively employed to monitor membrane biogenesis using a two-step labeling protocol on living cells. In addition, KK 1905-NHS was successfully used in super-resolution stimulated emission depletion microscopy. Overall, the new fluorophores presented in this study expand the microscopy toolbox, which is an asset for the investigation of fundamental bacterial processes.