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We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338–BHHTEGST–Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets. [ABSTRACT FROM AUTHOR]