Quantum Dye® Papers

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Quantum Dye® preprints to appear in the Journal of Biomedical Optics

D. Jin, J.A. Piper, R.C. Leif, S. Yang, B.C. Ferrari, J. Yuan, G. Wang, L.M. Vallarino, and J.W. Williams, Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples to appear in the Journal of Biomedical Optics (2009).

Abstract

A fundamental problem for rare-event cell analysis is autofluorescence from non-target particles and cells. Time-gated flow cytometry is based upon the temporal-domain discrimination of long-lifetime (> 1 μs) luminescence-stained cells, and can render invisible all non-target cells and particles. This paper aims to further evaluate the technique, focusing on detection of ultra-rare-event 5-μm calibration beads in environmental water dirt samples. Europium-labeled 5-μm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria™ flow cytometer was used to sort accurately a very-low number of beads (< 100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%±30% and 91%±3% with 10 and 100 target beads respectively that were mixed with over one million non-target autofluorescent background particles. Under the same conditions, a conventional flow cytometer was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex bio-labels in flow cytometry. This approach may augment the current method that uses multi-fluorescence-channel flow cytometry gating.


R.C. Leif, S. Yang, D. Jin, J.A. Piper, L.M. Vallarino, J.W. Williams, and R. M. Zucker, Calibration Beads Containing Luminescent Lanthanide Ion Complexes, to appear in the Journal of Biomedical Optics (2009).

Abstract

The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, will be enhanced by the availability of narrow-band emitting, UV excited lanthanide calibration beads. 0.5, 3, and 5 micron (μm) beads containing a luminescent europium-complex were manufactured; and the luminescence distribution of the 5 μm beads was measured with a timedelayed- light-scatter-gated luminescence flow cytometer to have a 7.0% coefficient of variation, CV. The spacial distribution of the europium-complex in individual beads was determined to be homogeneous by confocal microscopy. Emission peaks were found at 592, 616 (width 9.9 nm), and 685 nm with a PARISS® spectrophotometer. The kinetics of the luminescence bleaching caused by UV irradiation of the 0.5 and 5 μm beads measured under LED excitation with a fluorescence microscope indicated that bleaching did not interfere with their imaging. The luminescence lifetimes in water and in air were 340 and 460 microseconds (μs), respectively. Thus, these 5 μm beads can be used for spectral calibration of microscopes equipped with a spectrograph, as test particles for time-delayed luminescence flow cytometers, and possibly as labels for macromolecules and cells.

Quantum Dye® papers that can be obtained from Wiley-Liss

R. C. Leif, L. M. Vallarino, M. C. Becker, and S. Yang, Increasing the Luminescence of Lanthanide Complexes, Cytometry Vol. 69A, pp 767-778 (2006).

Abstract

This review compares the chemical and physical properties of lanthanide ion complexes and of other narrow-emitting species that can be used as labels for cytometry. A series of luminescent lanthanide ion macrocyclic complexes, Quantum Dyes®, which do not release or exchange their central lanthanide ion, do accept energy transfer from ligands, and are capable of covalent binding to macromolecules, including proteins and nucleic acids, is described and their properties are discussed.
Two methods are described for increasing the luminescence intensity of lanthanide ion complexes, which intrinsically is not as high as that of standard fluorophores or quantum dots. One method consists of adding a complex of a second lanthanide ion in a micellar solution (columinescence); the other method produces dry preparations by evaporation of a homogeneous solution containing an added complex of a second lanthanide ion and/or an excess of an unbound antenna ligand. Both methods involve the Resonance Energy Transfer Enhanced Luminescence, RETEL, effect as the mechanism for the luminescence enhancement.


R. C. Leif, L. M. Vallarino, M. C. Becker, and S. Yang, Increasing Lanthanide Luminescence by use of the RETEL Effect, Cytometry Vol. 69A, 940-946, (2006).

Abstract

Background: Luminescent lanthanide complexes produce emissions with the narrowest-known width at half maximum; however, their significant use in cytometry required an increase in luminescence intensity. The companion Review (1) described a new technique for the enhancement of lanthanide luminescence, the Resonance Energy Transfer Enhanced Luminescence (RETEL) effect, which increases luminescence and is compatible with standard slide microscopy.

Methods: The luminescence of the europium ion macrocyclic complex, EuMac, was increased by employing the RETEL effect. After adding the non-luminescent gadolinium ion complex of the thenoyltrifluoroacetonate (TTFA) ligand and/or the sodium salt of TTFA in ethanol solution, the EuMac-labeled sample was allowed to dry. Both a conventional arc lamp and a time-gated UV LED served as light sources for microscopic imaging. The emission intensity was measured with either a UV box or a CCD camera. Multiple time-gated images were summed with special software to permit analysis and effective presentation of the final image.

Results: With the RETEL effect, the luminescence of the EuMac-streptavidin conjugate increased at least six-fold upon drying. Nuclei of apoptotic cells were stained with DAPI and tailed with 5BrdUrd to which a EuMac-Anti-5BrdU conjugate was subsequently attached. Time-gated images showed the long-lived EuMac luminescence but did not show the short-lived DAPI fluorescence. Imaging of DNA-synthesizing cells with an arc lamp showed that both S phase and apoptotic cells were labeled, and that their labeling patterns were different. The images of the luminescent EuMac and fluorescent DAPI were combined to produce a color image on a white background. This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes®, as molecular diagnostics for cytological and histopathological microscopic imaging.

Quantum Dye® papers that can be obtained from the SPIE or final drafts from here

R. C. Leif, M. C. Becker, A. Bromm Jr., N. Chen, A. E. Cowan, L. M. Vallarino, S. Yang, and R. M. Zucker, Lanthanide Enhanced Luminescence (LEL) with one and two photon excitation of Quantum Dyes® Lanthanide(III)-Macrocycles,, in Manipulation and Analysis of Biomolecules, Cells, and Tissues, D. V. Nicolau, J. Enderlein, R. C. Leif, and D. Farkas, Editors, SPIE Proceedings Vol. 5322 pp. 187-199(2004).

Abstract

Improvements in the lanthanide enhanced luminescence (LEL) protocol have facilitated the use of the recently synthesized Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye®, as a label. It was discovered that a homogeneous solution in ethanol or other solvent could be used to produce the lanthanide enhanced luminescence (LEL) effect, provided that the solution was permitted to evaporate. This protocol has been applied to the direct staining of cells in S phase, and was optimized for solid phase assays with Quantum Dye labeled streptavidin. Preliminary studies indicate that cells stained with the europium Quantum Dye can be observed both by conventional UV laser excitation and by infrared two-photon confocal microscopy. An enhancer has been found that enables the observation of simultaneous emissions from both the europium and terbium Quantum Dyes.


R. C. Leif, M. C. Becker, L. M. Vallarino J. W. Williams, and S. Yang, Progress in the Use of Quantum Dye® Eu(III)-Macrocycles,in Manipulation and Analysis of Biomolecules, Cells and Tissues, D. V. Nicolau, J. Enderlein, and R. C. Leif, Editors, SPIE Proceedings Vol. 4962 pp. 341-353 (2003).

Abstract

A Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye®, has been synthesized that has minimal contamination with the Eu(III)-macrocycle-di-isothiocyanate, which cross-links proteins. The mono-isothiocyanate has been conjugated to streptavidin (EuMac-Strept). An indirect assay with EuMac-Strept and biotinylated anti5BrdU has been used to observe apoptotic cells. This system and cells directly labeled with the Eu(III)-macrocycle-di-isothiocyanate have been employed in fading studies and reagent stability tests. The fading of cells mounted in a plastic medium was much slower than that observed when the cells were in the aqueous, micellar Lanthanide Enhanced Luminescence (LEL) solution. The fading was not the result of the photo-destruction of the Eu(III)-macrocycle, since the luminescence returned after a second addition of the LEL solution. A time-gated, peltier cooled, monochrome CCD camera has been combined with a flashlamp to eliminate imaging of the emission of fluorescein while maintaining the images of EuMac staining. This was demonstrated with both separate preparations of fluorescein and EuMac stained cells and mixtures thereof. Time-gating was employed to produce an EuMac image of cells that were stained with both the EuMac and DAPI.


R. C. Leif, M. C. Becker, A. J. Bromm Jr., L. M. Vallarino, J. W. Williams, S. A. Williams, and S. Yang, Optimizing the Luminescence of Lanthanide(III) Macrocyclic Complexes for the Detection of Anti5BrdU, Optical Diagnostics of Living Cells V, D. L. Farkas and R. C. Leif, Editors, SPIE Proceedings Vol. 4622 pp. 250-261 (2002).

Abstract

A Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye®, has been coupled to a monoclonal antibody against 5BrdU. Since Quantum Dyes do not undergo concentration quenching, the coupling conditions were optimized to achieve the maximum number of Eu(III) macrocyles bound to the antiBrdU, without decrease in solubility or loss of antigen-binding ability. In order to optimize the coupling conditions, a colorimetric method for the quantitation of the Eu(III)-macrocycle-mono-isothiocyanate has been developed.

A simple mixture composed of an ethanolic solution and a Gd(III)-containing aqueous solution is now used to provide lanthanide enhanced luminescence, LEL. Under LEL conditions, the specific binding of Eu(III) macrocycles to apoptotic cells has been observed in both aqueous and mounted slide preparations. A comparison between measurements of the same LEL model system, obtained in both time-gated luminescence and standard fluorescence modes, has demonstrated that time-gating significantly improves the signal to noise ratio.


R. C. Leif, M. C. Becker, A. J. Bromm Jr., L. M. Vallarino, S. A. Williams, and S. Yang,Increasing the Luminescence of Lanthanide(III) Macrocyclic Complexes by the Use of Polymers and Lanthanide Enhanced Luminescence, Optical Diagnostics of Living Cells IV, D. L. Farkas and R. C. Leif, Editors, SPIE BIOS Proceeding Volume 4260 pp. 184-197 (2001).

Abstract

A Eu(III)-macrocycle-isothiocyanate, Quantum Dye®, has been reacted with lysine homo- and hetero-peptides to give polymers with multiple luminescent side chains. Contrary to the concentration quenching that occurs with conventional organic fluorophores, the attachment of multiple Quantum Dyes to a polymer results in a concomitant increase in luminescence. The emission intensity of the peptide-bound Quantum Dye units is approximately linearly related to their number. The attachment of peptides containing multiple lanthanide(III)-macrocycles to analyte-binding species is facilitated by employing solid-phase technology. Bead-bound peptides are first labeled with multiple Quantum Dye units, then conjugated to an antibody, and finally released from the bead by specific cleavage with Proteinase K under physiological conditions. Since the luminescence of lanthanide(III) macrocycles is enhanced by the presence of Gd(III) or Y(III) ions in a micellar system, a significant increases in signal can be achieved by attaching a polymer labeled with multiple Quantum Dye units to an analyte-binding species, such as a monoclonal antibody, or by taking advantage of the luminescence enhancing effects of Gd(III) or Y(III), or by both approaches concomitantly. A comparison between the integrated intensity and lifetime measurements of the Eu(III)-macrocycle under a variety of conditions show that the signal increase caused by Gd(III) can not be explained solely by the increase in lifetime, and must result in significant part from an energy transfer process involving donors not directly bound to the Eu(III).


J. R. Quagliano, R. C. Leif, L. M. Vallarino, and Steven A. Williams, Methods to Increase the Luminescence of Lanthanide(III) Macrocyclic Complexes, Optical Diagnostics of Living Cells III, D. L. Farkas and R. C. Leif, Editors, Proceedings of SPIE Vol. 3921. pp. 124-133 (2000).

Abstract

Simultaneous detection of both a Eu(III) and a Sm(III) Quantum Dye® is now possible because the enhanced luminescence (cofluorescence) of the Eu(III) and Sm(III) macrocycles occurs in the same solution and with excitation at the same wavelengths between 350 to 370 nm. Since DAPI is also excited between 350 to 370 nm, it is possible to use common excitation optics and a single dichroic mirror for measuring two molecular species and DNA. The narrow emissions of these macrocycles can be detected with negligible overlap between themselves or with DAPI-stained DNA. This will permit precise pixel by pixel ratio measurements of the Eu(III) macrocycle to Sm(III) macrocycle, and of each macrocycle to DNA. This technology should be applicable to antibodies, FISH, comparative genomic hybridization, and chromosome painting. Cofluorescence of the Tb(III)-macrocycle has also been obtained under different conditions. The luminescence of these lanthanide macrocycles can be observed with conventional fluorescence instrumentation at previously unattainable low levels. Thus, it will be possible to employ narrow bandwidth lanthanide luminescent tags to identify three molecular species with a conventional microscope.


A. J. Bromm Jr., R. C. Leif, J. R. Quagliano, and L. M. Vallarino, The Addition of a Second Lanthanide Ion to Increase the Luminescence of Europium(III) Macrocyclic Complexes, Proceedings of Optical Diagnostics of Living Cells II, D. L. Farkas, R. C. Leif, B. J. Tromberg, Editors, SPIE Progress in Biomedical Optics,. A. Katzir series Editor, Vol. 3604, ISBN 0-8194-3074-9, pp. 263-272, 1999.

Abstract

At present, the microscopic visualization of luminescent labels containing lanthanide(III) ions, primarily europium(III), as light-emitting centers is best performed with time-gated instrumentation, which by virtually eliminating the background fluorescence results in an improved signal to noise ratio. However, the use of the europium(III) macrocycle, Quantum Dye®, in conjunction with the strong luminescence enhancing effect (cofluorescence) of yttrium(III) or gadolinium(III), can eliminate the need for such specialized instrumentation. In the presence of Gd(III), the luminescence of the Eu-macrocycles can be conveniently observed with conventional fluorescence instrumentation at previously unattainable low levels. The Eu(III) 5D0−>7F2 emission of the Eu-macrocycles was observed as an extremely sharp band with a maximum at 619 nm and a clearly resolved characteristic pattern. At very low Eu-macrocycle concentrations, another sharp emission was detected at 614 nm, arising from traces of Eu(III) present in even the purest commercially available gadolinium products. Discrimination of the resolved emissions of the Eu-macrocycle and Eu(III) contaminant should provide a means to further lower the limit of detection of the Eu-macrocycle.


A. M. Adeyiga, P. M. Harlow, L. M. Vallarino, and R. C. Leif, Advances in the Development of Lanthanide Macrocyclic Complexes as Luminescent Bio-MarkersAdvanced Techniques in Analytical Cytology, Optical Diagnosis of Living Cells and Biofluids, Ed. T. Askura, D. L. Farkas, R. C. Leif, A. V. Priezzhev, and B. J. Tromberg. A. Katzir Series Editor, Progress Biomedical Optics Series Editor SPIE Proceedings Series, Vol. 2678, pp 212-220 (1996).

Abstract

The development of peripherally substituted europium(III)-macrocycles suitable as luminescent bio-markers was continued in three related areas. (1) Protocols were established for the coupling of NCS-substituted Eu-macrocycles to proteins and for the mounting on microscope slides of particles labeled with luminescent Eumacrocycles. The emission/excitation spectra of the dried, slide-mounted particles were investigated. (2) A procedure was developed for the synthesis of lanthanide-macrocycles having a single peripheral functionality. The structure and properties of the mono-functionalized macrocyclic complexes were established. (3) A study was undertaken to explore whether the emission intensity of the Eu-macrocycles can be increased by energy transfer from yttrium(III) complexes. Preliminary results have shown that a considerable luminescence enhancement can be achieved by this method.

The results obtained in these three areas are evaluated in the light of the research reported by other investigators.


R. C. Leif, P. M. Harlow, and L. M. Vallarino; Production, Fixation, and Staining of Cells on Slides for Maximum Photometric Sensitivity. Proceedings of Biochemical Diagnostic Instrumentation, Progress in Biomedical Optics. Ed. R. F. Bonner, G. E. Cohn, T. M. Laue, and A. V. Priezzhev. SPIE Proceedings Series 2136, pp. 255-262 (1994).

Abstract

The need to detect increasingly low levels of antigens or polynucleotides in cells requires improvements in both the preparation and the staining of samples. The combination of centrifugal cytology with the use of glyoxal as cross-linking fixative produces monolayers of cells having minimum background fluorescence. Detection can be further improved by the use of a recently developed type of luminescent tags containing a lanthanide(III) ion as the light-emitting center. These novel tags are macrocyclic complexes functionalized with an isothiocyanate group to allow covalent coupling to a biosubstrate. The Eu(III) complex possesses a set of properties - water solubility, inertness to metal release over a wide pH range, ligand-sensitized narrow-band luminescence, large Stoke's shift, and long excited-state lifetime - that provide ease of staining as well as maximum signal with minimum interference from background autofluorescence. Luminescence efficiency studies indicate significant solvent effects.


L. M. Vallarino, P. M. Harlow and R. C. Leif; Lanthanide Macrocyclic Complexes, "Quantum Dyes® Optical Properties and Significance". Proceedings of Advances in Fluorescence Sensing Technology, J. R. Lakowicz and R. B. Thompson Editors, A. Katzir Progress in Biomedical Optics Series Editor, SPIE Proceedings Series 1885 376-385 (1993).

Abstract

Macrocyclic complexes of the lanthanide(III) ions were functionalized to permit their attachment to antibodies, nucleic acid probes, and any other species capable of specific binding. The Eu(III) complex was found to possess a combination of properties (water solubility, inertness to metal release, ligand sensitized luminescence, reactive peripheral functionalities) that make it suitable as a luminescent marker for bio substrates. Its coupling to avidin was achieved, and the properties of the resulting conjugate were investigated.


R. C. Leif and L. M. Vallarino, Rare-Earth Chelates as Fluorescent Markers in Cell Separation and Analysis. ACS Symposium Series 464, Cell Separation Science and Technology, D. S. Kompala and P. W. Todd Editors, American Chemical Society, Washington, DC, pp 41- 58 (1991).

Abstract

The synthesis of a novel series of functionalized macrocyclic complexes of the lanthanide(III) ions is reported. The Eu(III) complexes possess a set of properties (water solubility, inertness to metal release, ligand-sensitized luminescence, reactive peripheral functionalities) that make them suitable as luminescent markers for bio-substrates. Currently employed organic fluorophores give efficient signal production, but this is accompanied by interference from background fluorescence and, if multiple fluorophores are used, from spectral overlap. The long lifetimes and narrow-band emissions of the luminescent lanthanide complexes will minimize background interference; however, long lifetimes will also result in a significantly reduced signal for flow cytometry or cell sorting.


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