Luminescence Bioimaging Research Articles

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Many microorganisms have a very low threshold (<10 cells) to trigger infectious diseases, and, in these cases, it is important to determine the absolute cell count in a low-cost and speedy fashion. Fluorescent microscopy is a routine method; however, one fundamental problem has been associated with the existence in the sample of large numbers of nontarget particles, which are naturally autofluorescent, thereby obscuring the visibility of target organisms. This severely affects both direct visual inspection and the automated microscopy based on computer pattern recognition. We report a novel strategy of time-gated luminescent scanning for accurate counting of rare-event cells, which exploits the large difference in luminescence lifetimes between the lanthanide biolabels, >100 μs, and the autofluorescence backgrounds, <0.1 μs, to render background autofluorescence invisible to the detector. Rather than having to resort to sophisticated imaging analysis, the background-free feature allows a single-element photomultiplier to locate rare-event cells, so that requirements for data storage and analysis are minimized to the level of image confirmation only at the final step. We have evaluated this concept in a prototype instrument using a 2D scanning stage and applied it to rare-event Giardia detection labeled by a europium complex. For a slide area of 225 mm(2) , the time-gated scanning method easily reduced the original 40,000 adjacent elements (0.075 mm × 0.075 mm) down to a few "elements of interest" containing the Giardia cysts. We achieved an averaged signal-to-background ratio of 41.2 (minimum ratio of 12.1). Such high contrasts ensured the accurate mapping of all the potential Giardia cysts free of false positives or negatives. This was confirmed by the automatic retrieving and time-gated luminescence bioimaging of these Giardia cysts. Such automated microscopy based on time-gated scanning can provide novel solutions for quantitative diagnostics in advanced biological, environmental, and medical sciences.

Fluorine-18 labeled rare-earth nanoparticles for positron emission tomography (PET) imaging of sentinel lymph node

Rare-earth-based nanoparticles have attracted increasing attention for their unique optical and magnetic properties. However, their application in bioimaging has been limited to photoluminescence bioimaging and magnetic resonance imaging. To facilitate their use in other bioimaging techniques, we developed a simple, rapid, efficient and general synthesis strategy for 18F-labeled rare-earth nanoparticles through a facile inorganic reaction between rare-earth cations and fluoride ions. The 18F-labeling process based on rare-earth elements was achieved efficiently in water at room temperature with an 18F-labeling yield of >90% and completed within 5 min, with only simple purification by aqueous washing and centrifugation, and without the use of organic agents. The effectiveness of 18F-labeled rare-earth nanoparticles was further evaluated by positron emission tomography (PET) imaging of their in vivo distribution and application in lymph monitoring. In addition, this strategy is proposed for the creation of a dual-model bioimaging technique, combining upconversion luminescence bioimaging and PET imaging.

Artificial luminescent protein as a bioprobe for time-gated luminescence bioimaging

An artificial luminescent protein, apoferritin-encapsulated luminescent europium complex, has been designed/fabricated and displays good biocompatibility and long-lived luminescence, which means it can be used as a bioprobe to image living cells with a time-gated mode.

Facile Synthesis of Amine-Functionalized Eu(3+)-Doped La(OH)3 Nanophosphors for Bioimaging.

Here, we report a straightforward synthesis process to produce colloidal Eu3+-activated nanophosphors (NPs) for use as bioimaging probes. In this procedure, poly(ethylene glycol) serves as a high-boiling point solvent allowing for nanoscale particle formation as well as a convenient medium for solvent exchange and subsequent surface modification. The La(OH)3:Eu3+ NPs produced by this process were ~3.5 nm in diameter as determined by transmission electron microscopy. The NP surface was coated with aminopropyltriethoxysilane to provide chemical functionality for attachment of biological ligands, improve chemical stability and prevent surface quenching of luminescent centers. Photoluminescence spectroscopy of the NPs displayed emission peaks at 597 and 615 nm (λex = 280 nm). The red emission, due to 5D0 → 7F1 and 5D0 → 7F2 transitions, was linear with concentration as observed by imaging with a conventional bioimaging system. To demonstrate the feasibility of these NPs to serve as optical probes in biological applications, an in vitro experiment was performed with HeLa cells. NP emission was observed in the cells by fluorescence microscopy. In addition, the NPs displayed no cytotoxicity over the course of a 48-h MTT cell viability assay. These results suggest that La(OH)3:Eu3+ NPs possess the potential to serve as a luminescent bioimaging probe.

Development of a Visible-Light-Sensitized Europium Complex for Time-Resolved Fluorometric Application

The time-resolved luminescence bioassay technique using luminescent lanthanide complexes as labels is a highly sensitive and widely used bioassay method for clinical diagnostics and biotechnology. A major drawback of the current technique is that the luminescent lanthanide labels require UV excitation (typically less than 360 nm), which can damage living biological systems and is holding back further development of time-resolved luminescence instruments. Herein we describe two approaches for preparing a visible-light-sensitized Eu(3+) complex in aqueous media for time-resolved fluorometric applications: a dissociation enhancement aqueous solution that can be excited by visible light for ethylenediaminetetraacetate (EDTA)-Eu(3+) detection and a visible-light-sensitized water-soluble Eu(3+) complex conjugated bovine serum albumin (BSA) for biolabeling and time-resolved luminescence bioimaging. In the first approach, a weakly acidic aqueous solution consisting of 4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedion-6''-yl)-o-terphenyl (BHHT), 2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine (DPBT), and Triton X-100 was prepared. This solution shows a strong luminescence enhancement effect for EDTA-Eu(3+) with a wide excitation wavelength range from UV to visible light (a maximum at 387 nm) and a long luminescence lifetime (520 micros), to provide a novel dissociation enhancement solution for time-resolved luminescence detection of EDTA-Eu(3+). In the second approach, a ternary Eu(3+) complex, 4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedion-6''-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu(3+)-DPBT, was covalently bound to BSA to form a water-soluble BSA-BHHCT-Eu(3+)-DPBT conjugate. This biocompatible conjugate is of the visible-light excitable feature in aqueous media with a wide excitation wavelength range from UV to visible light (a maximum at 387 nm), a long luminescence lifetime (460 micros), and a higher quantum yield (27%). The conjugate was successfully used for streptavidin (SA) labeling and time-resolved luminescence imaging detection of three environmental pathogens, Giardia lamblia , Cryptosporidium muris , and Cryptosporidium parvum , in water samples. Our strategy gives a general idea for designing a visible-light-sensitized Eu(3+) complex for time-resolved luminescence bioassay applications.

Preparation and time-gated luminescence bioimaging application of ruthenium complex covalently bound silica nanoparticles

Luminescent ruthenium(II) complex covalently bound silica nanoparticles have been prepared and used as a probe for time-gated luminescence bioimaging. The new nanoparticles were prepared by copolymerization of a luminescent Ru(II) complex tris(5-amino-1,10-phenanthroline)ruthenium(II) conjugated with 3-aminopropyl(triethoxy)silane (APS-Ru conjugate), free (3-aminopropyl)triethoxysilane (APS) and tetraethyl orthosilicate (TEOS) in a water-in-oil reverse microemulsion consisting of Triton X-100, n-octanol, cyclohexane and water in the presence of aqueous ammonia. Characterization by transmission electron microscopy indicates that the nanoparticles are monodisperse, spherical and uniform in size, 64 ± 4 nm in diameter. Compared with the dye-doping nanoparticles, dye leakage of the new nanoparticles was remarkably decreased. In addition, it was found that the Ru(II) complex luminescence could be effectively enhanced with a longer luminescence lifetime (∼2.3 μs) after forming the nanoparticles, which enables the nanoparticles to be suitable as a bioprobe for time-gated luminescence bioimaging applications. The nanoparticle-labeled streptavidin was prepared and successfully used for time-gated luminescence imaging detection of an environmental pathogen, Giardia lamblia, with high specificity and sensitivity.

Visible-light-sensitized highly luminescent europium nanoparticles: preparation and application for time-gated luminescence bioimaging

Time-gated luminescence bioimaging based on microsecond-lifetime luminescent biolabels can provide complete background-free conditions for detecting target cells in an autofluorescence biosample matrix. However, a major drawback of the current lanthanide biolabels is the requirement for UV excitation (<370 nm), which leads to damage to many biological systems and greatly affects the improvement of time-gated luminescence instruments. Herein we describe luminescent europium nanoparticles that have an excitation peak around 406 nm with high quantum yield (∼66%) and fine monodispersity in aqueous solutions. The nanoparticles were prepared by copolymerization of a visible-light-sensitized Eu3+ complex 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)chlorosulfo-o-terphenyl-Eu3+-2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine conjugated with 3-aminopropyl(triethoxy)silane, free 3-aminopropyl(triethoxy)silane and tetraethyl orthosilicate in a water-in-oil reverse microemulsion. Characterization by transmission electron microscopy and luminescence spectroscopy indicates that the nanoparticles are monodisperse, spherical and uniform in size, <50 nm in diameter, and show strong visible-light-sensitized luminescence with a large quantum yield and a long luminescence lifetime. The new nanoparticles were successfully applied to distinguish an environmental pathogen, Giardia lamblia, within a concentrate of environmental water sample using a time-gated luminescence microscope with pulsed visible light excitation. The method resulted in highly specific and sensitive imaging for Giardia lamblia. These results suggest a broad range of potential bioimaging applications where both long time microscopy observation and high signal-to-background ratio are required for samples containing high concentrations of autofluorescence background.

Luminescent europium nanoparticles with a wide excitation range from UV to visible light for biolabeling and time-gated luminescence bioimaging

Silica-encapsulated highly luminescent europium nanoparticles with a wide excitation range from UV to visible light (200-450 nm) have been prepared and used for streptavidin labeling and time-gated luminescence imaging of an environmental pathogen, Giardia lamblia.