Abstract
Fluorescence imaging in halophilic archaea is technically challenging due to high-salt conditions, presence of S-layer and small cellular dimensions, which all limit signal detection and structural
interpretation. To evaluate feasible labeling strategies, we systematically screened a wide panel
of commercial dyes under live, fixed, and permeabilized conditions. A clear
permeability-dependent trend emerged: methanol permeabilization consistently produced strong
intracellular signals, fixation often yielded moderate labeling, whereas most dyes produced little
to no signal in live cells. Disruption of the S-layer markedly enhanced dye entry, confirming the
S-layer as a central barrier controlling labeling efficiency. Additionally, cross-species
comparisons revealed substantial variability in staining outcomes, underscoring the diversity of
membrane and S-layer architectures among halophilic archaea.
We further applied STED super-resolution microscopy to image HaloTag-PCNA foci
representing replication sites in Haloferax volcanii. STED successfully resolved replication sites
once merged under confocal imaging, enabling more accurate quantification of active replication
sites number, size, and spatial arrangement. Together, these results outline practical strategies for
fluorescent labeling in halophilic archaea and demonstrate STED microscopy as a powerful tool
for resolving nanoscale archaeal structures.