Quantum Dye® Papers
Quantum Dye® Papers |
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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.
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.
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.