Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging

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. IMPORTANCE By harnessing the versatility of fluorescence microscopy and super-resolution imaging, bacteriologists explore critical aspects of bacterial physiology and resolve bacterial structures sized beyond the light diffraction limit. These techniques are based on fluorophores with profitable photochemical and tagging properties. The paucity of available far-red (FR)-emitting dyes for bacterial imaging strongly limits the multicolor choice of bacteriologists, hindering the possibility of labeling multiple structures in a single experiment. The set of FR fluorophores characterized in this study expands the palette of dyes useful for microbiologists, as they can be used for bacterial LIVE/DEAD staining and for tagging the membranes of viable Escherichia coli and Bacillus subtilis cells. The absence of toxicity makes these dyes suitable for live-cell imaging and allows monitoring of bacterial membrane biogenesis. Moreover, a newly synthesized FR-fluorophore can be employed for imaging bacterial membranes with stimulated emission depletion microscopy, a super-resolution technique capable of increasing the resolving power of conventional microscopes.