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.

Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.

We describe the application of a synthetically developed tetradentate β-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells.

We describe the synthesis of a novel hydrophilic derivative of a tetradentate β-diketone europium ligand that was used to prepare an immunoconjugate probe against Giardia lamblia cysts. We used a Gated Autosynchronous Luminescence Detector (GALD) to obtain high quality delayed luminescence images of cells 30-fold faster than ever previously reported.

BACKGROUNDAs a proof-of-concept, the feasibility of detecting Staphylococcus aureus faster than previous whole-cell fluorescent in situ hybridization (FISH) methods was tested.METHODSIsolates of Staphylococcus were treated with three rapid slide-based FISH protocols and DNA probes. Protocols were shortened by optimizing, combining or omitting steps.RESULTSAll FISH protocols detected S. aureus and not the phenotypically similar Staphylococcus epidermidis. The express FISH assay was completed in 24 minutes. The one-step FISH assay with NaCl and the one-step with phosphate buffered saline (PBS) assay took 19 minutes, but yielded a weaker signal.CONCLUSIONSThe exploratory study identified S. aureus two to three times faster than previous methods. Additional testing in a clinical laboratory scenario (for example with positive blood-culture bottles) is warranted.

The sensitive detection of delayed luminescence (τ > 100 μs) can be achieved with an optomechanical instrument, the gated autosynchronous luminescence detector (GALD). The device effectively combines an excitation chopper, a detection chopper, and a dichroic mirror into a single element. With dimensions of 12 mm (H), 32 mm (W), and 90 mm (L), it is designed for insertion into the differential interference contrast (DIC) prism slot of a BX51 Olympus microscope. The GALD described here employed a compact high-power UV LED as the excitation source to capture images of Giardia lamblia cysts indirectly labeled with a europium chelate/streptavidin conjugate. Labeled cells were clearly visible in the complete absence of autofluorescence and signal intensity was sufficient to capture high-resolution color images within several seconds. Shorter exposure intervals of 100 ms on a monochrome Andor iXON camera delivered time-gated luminescence images with a signal-to-noise ratio better than 114:1.

Background: Aspects of the fluorescence in situ hybridization (FISH) method for the detection of clinically important bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, were investigated for optimization. Methods: Various approaches to optimizing the FISH procedure were taken and different methods were compared. To save time, hybridization and washing buffers were prepared beforehand and stored at -20 degrees C and mixed to their final formamide and NaCl concentrations just before use. The use of 50-ml tubes for hybridization incubation reduced drying out, reagent wastage, and reaction times. Results: A two-step permeabilization FISH assay was developed that used phosphate-buffered saline as a buffer for lysostaphin. It could detect bacteria with DNA probes conjugated to fluorophores with a higher signal intensity and the less expensive biotinylated DNA probes with minimal cell lysis in 1 hr. Conclusions: The two-step assay might be used when the FISH signal is weak, bacterial numbers are low or if there is a need to use other reporter molecules. J. Clin. Lab. Anal. 25: 359-365, 2011. (C) 2011 Wiley-Liss, Inc.

Background: Staphylococcus aureus is a clinically important pathogen. A small number of whole-cell fluorescence in situ hybridization (FISH) probes have been reported to detect S. aureus. New online computational tools for in silico design and testing make it possible to assess candidate FISH probes for S. aureus.Materials and Methods: Six online tools, NCBI-Nucleotide, Ribosomal Database Project, NCBI-Blast, Reverse-Complement, Probecheck, and mathFISH, were employed in a workflow to evaluate FISH probes for S. aureus. A previously reported probe, Staaur-16S69, was compared to a new probe, KT18-16S68, predicted by mathFISH to have the same performance,Results: A number of new probes for S. aureus were predicted to perform as well or better in silico as those previously reported. When tested in a FISH assay, Staaur and a new probe, KT18, were found to have the same performance.Conclusion: Existing and new FISH probes for S. aureus were found to be accurately identified and characterized with online computational tools. In silico evaluation of probes has the potential to reduce the time spent evaluating probes in the laboratory.

To detect with whole-cell fluorescence in situ hybridization (FISH), Staphylococcus aureus is typically permeabilized with lyozyme and lysostaphin. We tested whether it was feasible to detect S. aureus and differentiate it from Staphylococcus epidermidis with lysozyme-only permeabilization. We compared lysozyme permeabilization to S. aureus permeabilized with lysozyme in combination with lysostaphin. It was determined that S. aureus treated with agarose, methanol, and lysozyme could be detected with FISH. The 1 hr protocol is a useful alternative to conventional FISH. J. Clin. Lab. Anal. 25:142-147, 2011. (C) 2011 Wiley-Liss, Inc.

Fluorescence microscopy is a powerful tool for the rapid identification of target organisms. However, natural autofluorescence often interferes with identification. Time-gated luminescence microscopy (TGLM) uses luminescent labels with long persistence in conjunction with digital imaging to regain discriminative power. Following the excitation pulse, short-lived autofluorescence decays rapidly whereas the long-lived emission from lanthanide doped polymer beads persists for hundreds of microseconds. After a short resolving period, a gated high gain camera captures the persistent emission in the absence of short-lived fluorescence. We report on the development of a TGLM software system for automated scanning of microscope slides, and show its use to resolve luminescent microspheres within a matrix of autofluorescent algae.

Many naturally occurring materials are autofluorescent, a property that can reduce the discriminative ability of fluorescence methods, sometimes to the point where they cannot be usefully applied. Shifting from the spectral to the temporal domain, it is possible to discriminate fluorophores on the basis of their fluorescence decay lifetime. Luminophores with sufficiently long lifetimes can be discriminated from short-lived autofluorescence using time-gated luminescence (TGL). This technique relies upon the application of a brief excitation pulse followed by a resolving period to permit short-lived autofluorescence to decay, after which detection is enabled to capture persistent emission. In our studies, a high-power UV LED was mounted in the filter capsule of an Olympus BX51 microscope to serve as the excitation source. The microscope was fitted with an Andor DV885 electron-multiplying CCD (EM-CCD) camera with the trigger input synchronized to UV LED operation. Giardia lamblia cysts labeled with the europium chelate BHHST were analyzed against an autofluorescent background with the TGL microscope. The EM-CCD camera captured useful TGL images in real time with a single exposure cycle. With 4x frame averaging, images acquired in TGL mode showed a 30-fold improvement in SNR compared with conventional fluorescence microscopy.

Autofluorescent algal samples were spiked with europium beads for analysis on a novel all‐solid‐state, time‐gated luminescence (TGL) microscope. Pulsed UV excitation (365 nm) was provided by a high‐power UV‐LED source fitted to an Olympus BX51 microscope. An “Impactron” electron multiplying charge‐coupled‐device (CCD) camera acquired images in delayed luminescence mode. Second, we evaluated sensitivity of the instrument by acquiring images of immunofluorescently labeled Giardia cysts with a single‐exposure period of 3 ms. The camera was triggered 3 μs after the LED had extinguished to yield a 14‐fold increase in signal‐to‐noise ratio within a single 33 ms capture cycle. This novel instrument could be switched instantly from prompt epifluorescence mode to TGL mode for suppression of short‐lived fluorescence.

The method of time-gated detection of long-lifetime (1-2,000 micros) luminescence-labeled microorganisms following rapid excitation pulses has proved highly efficient in suppressing nontarget autofluorescence (<0.1 micros), scatterings, and other prompt stray light (Hemmila and Mukkala, Crit Rev Clin Lab Sci 2001;38:441-519). The application of such techniques to flow cytometry is highly attractive but there are significant challenges in implementing pulsed operation mode to rapid continuous flowing sample to achieve high cell analysis rates (Leif R, Vallarino L, Rare-earth chelates as fluorescent markers in cell separation and analysis, In: Cell Separation Science and Technology, ACS Symposium Series 464, American Chemical Society, 1991, pp 41-58; Condrau et al., Cytometry 1994;16:187-194; Condrau et al., Cytometry 1994;16:195-205; Shapiro HM, Improving signals from labels: Amplification and other techniques, In: Practical Flow Cytometry, 4th ed., Wiley, New York, 2002, p 345). We present here practical approaches for achieving high cell analysis rates at 100% detection efficiency, using time-gated luminescence (TGL) flow cytometry. In particular, we report that new-generation UV LEDs are practical sources in TGL flow cytometry. Spatial effects of long-lived luminescence from the target fluorophore in a fast-flowing sample stream have been investigated; excitation and detection requirements in TGL flow cytometry were theoretically analyzed; two practical approaches, a triggered model and a continuous flow-section model, were considered as a function of flow speed, sizes and relative positions of the excitation/detection spots, label lifetime, excitation pulse duration/intensity, and detection duration. A particular configuration using LED excitation to detect europium dye-labeled targets in such a system has been modeled in detail. In the triggered model, TGL mode is confined to a low repetition rate (<1 kHz) and engaged only while a target particle is present in the excitation zone. In the flow-section model, TGL mode is engaged continuously at high repetition rates to permit much higher cell arrival rates. The detection of 5.7-microm europium calibration beads in a UV LED-excited TGL flow cytometer has been shown to be feasible with a calculated signal-to-background ratio up to 11:1.

In the previous article [Part 1 (8)], we have modelled alternative approaches to design of practical time-gated luminescence (TGL) flow cytometry and examined the feasibility of employing a UV LED as the excitation source for the gated detection of europium dye labelled target in rapid flow stream. The continuous flow-section approach is well suited for rare-event cell counting in applications with a large number of nontarget autofluorescent particles. This article presents details of construction, operation and evaluation of a TGL flow cytometer using a UV LED excitation and a gated high-gain channel photomultiplier tube (CPMT) for detection. The compact prototype TGL flow cytometer was constructed and optimised to operate at a TGL cycle rate of 6 kHz, with each cycle consisting of 100 micros LED pulsed excitation and approximately 60 micros delay-gated detection. The performance of the TGL flow cytometer was evaluated by enumerating 5.7 microm Eu(3+) luminescence beads (having comparable intensity to europium-chelate-labeled Giardia cysts) in both autofluorescence-rich environmental water concentrates and Sulforhodamine 101 (S101) solutions (broadband red fluorescence covering the spectral band of target signals), respectively. The prototype TGL flow cytometer was able to distinguish the target beads, and a maximum signal to background ratio of 38:1 was observed. Neither the environmental water concentrates nor S101 solution contributed to the background in the TGL detection phase. The counting efficiency of the TGL flow cytometer was typically >93% of values determined using conventional counting methods.

This paper presents experimental and theoretical studies of time-gated discrimination of long-lived luminescence (lifetime: 12000 Vs) labelled target-organisms against non-target autofluorescence background (lifetime: < 100 ns) in flow cytometry. A theoretical model of such a TGL flow cytometer is developed which takes account of flow speed, illumination and detection apertures, fluorescence label lifetime, and pulsed illumination and gated detection timing sequences. Ultraviolet LED and channel photomultiplier were found to be practical as pulsed excitation sources and gated detector for TGL flow cytometry. The prototype cytometer was constructed and optimized to operate at 6 k Hz repetition rate of TGL cycles consisting of 100 Vs LED excitation and similar to 60 mu s gated detection. The spatial counting efficiency was evaluated by enumerating 5.5 mu m diameter europium microspheres resulting in a counting accuracy approaching 100%.