STED nanoscopy of KK114‐stained pathogenic bacteria

Abstract

Super‐resolution microscopy techniques can provide answers to still pending questions on prokaryotic organisms but are yet to be used at their full potential for this purpose. To address this, we evaluate the ability of the rhodamine‐like KK114 dye to label various types of bacteria, to enable imaging of fine structural details with stimulated emission depletion microscopy (STED). We assessed fluorescent labeling with KK114 for eleven Gram‐positive and Gram‐negative bacterial species and observed that this contrast agent binds to their cell membranes. Significant differences in the labeling outputs were noticed across the tested bacterial species, but importantly, KK114‐staining allowed the observation of subtle nanometric cell details in some cases. For example, a helix pattern resembling a cytoskeleton arrangement was detected in Bacillus subtilis. Furthermore, we found that KK114 easily penetrates the membrane of bacterial microorganism that lost their viability, which can be useful to discriminate between living and dead cells.

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

“…To expand the palette of FR-emitting dyes available for bacterial labeling, an N -hydroxysuccinimidyl (NHS)-containing oxazine (namely, KK 1905-NHS) and two rhodamines (namely, KK 1116 and KK 1518) have been generated, and their performances were compared to a previously synthesized set of dyes never applied for bacterial imaging before (i.e., KK 114S, KK 1115, KK 1517, KK 1517-NHS, KK 1558-NHS, KK 1905, and STAR RED in its deactivated form) or previously employed for STED bacterial imaging (i.e., KK 114-NHS and/or STAR RED-NHS) ( 18 ). Figure 1 displays the chemical structure of the fluorophores used in this work.…”

“…The resolving power of conventional fluorescence microscopy depends on the wavelength of the fluorescence light and constraints on both the lateral ( xy ~200–250 nm) and axial ( z ~500–600 nm) resolutions ( 4 , 22 ). Different approaches have been proposed to overcome the diffraction limit, and various nanoscopic techniques have been successfully used to generate accurate images of bacterial cells, which themselves only marginally exceed the diffraction limit ( 4 , 18 , 23 ). Currently, super-resolution methods are not often used in microbiological laboratories because of the shortage of suitable dyes ( 1 , 18 , 23 – 25 ).…”

“…Different approaches have been proposed to overcome the diffraction limit, and various nanoscopic techniques have been successfully used to generate accurate images of bacterial cells, which themselves only marginally exceed the diffraction limit ( 4 , 18 , 23 ). Currently, super-resolution methods are not often used in microbiological laboratories because of the shortage of suitable dyes ( 1 , 18 , 23 – 25 ). Stimulated-emission depletion (STED) microscopy is a super-resolution technique able to increase the optical resolution of confocal laser scanning microscopy (CLSM) up to one order of magnitude ( 26 ).…”

“…However, this resolution improvement comes at the cost of using high laser intensities, and it requires photostable dyes with high fluorescence quantum yields capable of resisting photobleaching ( 26 ). This limitation restricted the use of STED microscopy in bacteriology to visualizing bacterial division machinery, membrane microdomains, and cytoskeletons, primarily in Escherichia coli and Bacillus subtilis ( 18 , 23 ).…”

Advanced Biological Imaging for Intracellular Micromanipulation: Methods and Applications

“…Super-resolution microscope technology is advanced in observing small-scale intracellular components (such as protein [117,118], lysosomal [119,120], bacteria [121,122]). However, there are few attempts to use SIM and STEDM for micromanipulation because the current robot-aided manipulation tools can only handle objects at as low as sub-micrometers.…”