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Increasing lanthanide luminescence by use of the RETEL effect
Abstract
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, Leif et al., Cytometry 2006;69A:767-778, 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.
The luminescence of the europium ion macrocyclic complex, EuMac, was increased by employing the RETEL effect. After adding the nonluminescent gadolinium ion complex of the thenoyltrifluoroacetonate (TTFA) ligand 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 a CCD camera. Multiple time-gated images were summed with special software to permit analysis and effective presentation of the final image.
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.
... Lu et al., 2012; instruments and provides improved sensitivity and specificity. The recent development of highly luminescent lanthanide complexes (Petoud et al., 2003;Weibel et al., 2004;Deiters et al., 2009), methods to enhance lanthanide luminescence (Leif et al., 2006b), responsive lanthanide-based luminescent probes (Song et al., 2006;Thibon and Pierre, 2009a,b), functionalized lanthanide-ion nanocomposites (Makhluf et al., 2008), and nano-encapsulation of lanthanide-containing biolabels (Harma et al., 2001;Wu et al., 2008Wu et al., , 2009Song et al., 2009) further enhance the potential of lanthanide-based cellular imaging. Efficient lanthanide complexes and protein-conjugated complexes are also commercially available from vendors such as Sigma-Aldrich and the Invitrogen division of Life Technologies (Jin, 2011b). ...
... This digital summation technique has a protracted acquisition time. The time required to obtain 2,500 images at a rate of approximately 10 frames per sec was 250 sec (Leif et al., 2006b). At this rate, the actual acquisition time for 250 frames was 250/10 (25) sec, or 50 times the sum of acquisition (1 msec) and signal local storage time (1 msec), which is 2 msec or a total of 0.5 sec. ...
The sensitivity of filter-based fluorescence microscopy techniques is limited by autofluorescence background. Time-gated detection is a practical way to suppress autofluorescence, enabling higher contrast and improved sensitivity. In the past few years, three groups of authors have demonstrated independent approaches to build robust versions of time-gated luminescence microscopes. Three detailed, step-by-step protocols are provided here for modifying standard fluorescent microscopes to permit imaging time-gated luminescence. Curr. Protoc. Cytom. 67:2.22.1-2.22.36. © 2014 by John Wiley & Sons, Inc.
... The variation of luminescence intensity amongst individual beads was recorded by imaging the luminescence beads under UV LED excitation with a luminescence microscope 15 . Images were obtained with essentially continuous excitation from a Nichia UV LED, Model NCCU033 16 . ...
... Other available europium complexes exist that have excitation maxima that match the emission of the UV LED. One of these is the europium Quantum Dye® with its TTFA enhancer 15,19 (TTFA is the mono-negative anion of thenoyltrifluoroacetone). The presence of excess complexes of Gd(TTFA) 3 in a micellar solution also can be used to increase the luminescence of the europium Quantum Dye 16,20 . ...
Flow cytometric detection of specific rare-event targets within high-background samples such as water or food are frequently defeated by the extremely large population of non-target background particles. Time-gated detection of long lifetime fluorescence (>10mus) labeled microbial targets has been proven highly efficient in suppressing this non-target autofluorescent (10 million non-target particles). The recovery rate for counting these very-rare-event particles using the TGL flow cytometer was then found to be 100%+/-20% between bead concentrations evaluated.
... The increased popularity of FRET is due to several factors including the availability of brighter, more photostable dyes, quantum dots, and color variants of fluorescent proteins, and the development of new features, add-ons and dedicated hardware and software that are available for commercial microscopes (11) and flow cytometers (12), and facilitate the smooth application of FRET techniques. In addition to the fact that FRET measurements can be applied to detect molecular associations (2-9,13), the FRET phenomenon can be used to improve spectral characteristics of luminescent probes to be utilized in cytometry (14,15). In this commentary we will address two concerns frequently encountered in FRET imaging: the need for being able to dissect molecular populations displaying different FRET efficiencies (16), and the surmounting of artifacts that may arise due to molecular diffusion during the FRET experiment (17,18). ...
... The increased popularity of FRET is due to several factors including the availability of brighter, more photostable dyes, quantum dots, and color variants of fluorescent proteins, and the development of new features, add-ons and dedicated hardware and software that are available for commercial microscopes (11) and flow cytometers (12), and facilitate the smooth application of FRET techniques. In addition to the fact that FRET measurements can be applied to detect molecular associations (2)(3)(4)(5)(6)(7)(8)(9)13), the FRET phenomenon can be used to improve spectral characteristics of luminescent probes to be utilized in cytometry (14,15). In this commentary we will address two concerns frequently encountered in FRET imaging: the need for being able to dissect molecular populations displaying different FRET efficiencies (16), and the surmounting of artifacts that may arise due to molecular diffusion during the FRET experiment (17,18). ...
... . Phase shift methods have application when fluorescence lifetimes are typically less than a couple of microseconds (4,5). Time-domain methods, which utilize micro/millisecond luminescence lifetimes, are well suited to suppress short-lived autofluorescence (6,(7)(8)(9) and to eliminate the effects of light scatter (1). ...
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%.
... The emission from Europium beads yields peaks (595, 619, and 685 nm) which can also be used as reference markers to validate a spectroscopic imaging machine. It is beneficial to have a bead that has the characteristics of the Europium Quantum Dye with its chemically defined emission peak and very small FWHM (4,21,34). The combination of the position of the peaks and the width of the histograms are parameters that can be used to determine if the spectroscopic system is working correctly. ...
There is a need for a standardized, impartial calibration, and validation protocol on confocal spectral imaging (CSI) microscope systems. To achieve this goal, it is necessary to have testing tools to provide a reproducible way to evaluate instrument performance.
We evaluated fluorescent spectral beads (FocalCheck) from Molecular Probes/Invitrogen that consist of four pairs with emissions between 500 and 725 nm and a europium macrocycle quantum dye bead. These bead tools compliment our previously published protocol for testing spectral imaging systems that used an inexpensive multi-ion discharge lamp (MIDL) that contains Hg(+), Ar(+), and inorganic fluorophores that emits distinct, stable spectral features.
We acquired the spectra of the FocalCheck beads on a Zeiss 510 Meta, a Leica TCS-SP1, a Leica SP2 AOBS, an Olympus FV 1000, and a Nikon C1Si confocal systems and a PARISS microscopic spectral system and of the europium beads on the Leica TCS-SP1 and PARISS spectral imaging systems. A lack of performance with some equipment between 650 and 750 nm was identified using the far red pair of the FocalCheck beads. The position of the slider in front of PMT 2 that reflects light into PMT 1 and PMT 3 affected the measurement of all bead intensities. Unmixing algorithms were used to separate beads with different fluorochromes and separate two fluorochromes on the same bead.
The FocalCheck multi-spectral beads yielded similar profiles on four CSI systems and a PARISS spectral system. The utilization of the spectral FocalCheck beads is helpful to evaluate proper spectral performance, especially in the far red region. Europium beads provide a very narrow spectrum that can help to identify machines that have spectral problems. The dyes located on individual beads or mixed together in ring-core configuration can be used as test particles to demonstrate spectral unmixing with various algorithms.
... Phase shift methods have application when fluorescence lifetimes are typically less than a couple of microseconds (4,5). Time-domain methods, which utilize micro/millisecond luminescence lifetimes, are well suited to suppress short-lived autofluorescence (6,(7)(8)(9) and to eliminate the effects of light scatter (1). ...
The method of time‐gated detection of long‐lifetime (1–2,000 μs) luminescence‐labeled microorganisms following rapid excitation pulses has proved highly efficient in suppressing nontarget autofluorescence (<0.1 μs), 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‐μm 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. © 2007 International Society for Analytical Cytology
... . Phase shift methods have application when fluorescence lifetimes are typically less than a couple of microseconds (4,5). Time-domain methods, which utilize micro/millisecond luminescence lifetimes, are well suited to suppress short-lived autofluorescence (6,(7)(8)(9) and to eliminate the effects of light scatter (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 μs LED pulsed excitation and ∼60 μs delay-gated detection. The performance of the TGL flow cytometer was evaluated by enumerating 5.7 μm Eu3+ 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. © 2007 International Society for Analytical Cytology
A series of Lanthanide complexes Nd(btc).4H2O,
Eu(btc).3H2O, Tb(btc).3H2O, Er(btc).H2O, Yb(btc).
3H2O and heterometallic 3d-4f complexes, Nd(btc )
(Zn)(C2H5O)2.5H2O, Eu(btc)(Zn)(C2H5O)2.5H2O, Tb
(btc)(Zn)(C2H5O)2.H2O, Er(btc)(Zn)(C2H5O)2.H2O, Yb
(btc)(Zn)(C2H5O)2.H2O have been prepared by one
step reaction of LnIII and LnIII
-ZnII with 1,3,5-
benzenetricarboxylic acid (H3btc) by reflux method. All
the synthesized complexes were incorporated into
poly(vinyl pyrrolidone) (PVP) matrix.
All these complexes (1-10) have been characterized by
elemental analyses (C,H,N), FT-IR-spectra,
Transmission electron microscopy (TEM),
fluorescence spectra and powder X-ray diffraction
(PXRD) analyses to arrive at conclusion regarding
their geometrical structure. Transmission electron
microscopy (TEM) showed the shape and size of the
complexes, which were homogeneously dispersed in
the PVP matrix. TEM results showed the complexes
were of rod shaped with a mean diameter about 20 nm.
The powder X-ray diffraction (PXRD) patterns of 3d-4f
complexes showed well defined crystalline peaks.
Keywords:fluorescence lifetime;flow cytometry;time-correlated single photon counting (TCSPC);burst integrated fluorescence lifetime (BIFL);time-tagged time-resolved (TTTR);fluorescence resonance energy transfer (FRET)
Metalloproteomics is the large-scale study of metal-binding proteins. These proteins, which represent about one quarter of all the proteins in the Protein Data Bank, play important roles in all biological systems and all biological processes. Metalloproteomics provides the latest information on all major families of metal-binding proteins, including their structural, physico-chemical, and functional properties, enabling readers to better understand these proteins. Moreover, the book demonstrates how understanding the structures, properties, and functions of intracellular and extracellular metal-binding proteins may unlock the key to drug development for the treatment of a myriad of diseases.
Problem: Lanthanide complexes have significantly lower extinction coefficients than organic fluorophores and require time-gated luminescence measurements to produce background-free images. Two solutions to the low extinction problem have been previously reported. 1) Resonance energy transfer enhanced luminescence (RETEL), particularly, in the solid state, allowed multiple antennae molecules to absorb light and then transfer the energy to a complexed lanthanide ion. 2) It has been possible to take advantage of the lack of concentration quenching of polymers of lanthanide complexes by the attachment of peptides containing multiple Lanthanide(III)-macrocycles to analyte-binding species A third solution is the use of recently developed lanthanide labeled nanoparticles. Previous images of time-gated luminescence have been obtained with a cooled CCD camera by digitally summing a series of sequential images. The data acquisition rate of approximately 10 one millisecond exposure images per second was rate limiting and too slow for standard research and clinical use. These summed images contained an annoying undulating background, which could not be totally removed by subtraction of an unexposed, control image. Two inexpensive solutions to the analog summation of time-gated images have been developed. For both, a pulsed (ca. 500 Hz) inexpensive Nichia UV LED is used. The use of either an interline transfer, electronically shuttered camera or an inexpensive mechanical chopper permitted the analog summation of multiple images that were acquired during the dark period after each illumination. Results and Conclusions: The interline transfer camera produced useful time-gated luminescent images of five micron and 0.5 micron uniform europium complex stained microspheres. The mechanical shutter system produced useful time-gated luminescent images of environmental pathogen microorganisms. The development of new chemistries together with improved instrumentation permit and encourage the use of lanthanide labels in cytometry. Either a software change to present commercial interline cameras can be made or a mechanical shutter can be employed to produce background-free, narrow spectral band width images of biological objects of interest. The use of lanthanide, background free labels is now both practical and desirable.
The features of co-luminescence (sensitized luminescence) of lanthanide ions and dye molecules inserted in nanoparticles of complexes formed by trivalent metals with β-diketones are discussed. The mechanisms of energy transfer in these nanoparticles that lead to sensitization of Ln3+ ion luminescence and dye molecule fluorescence are considered. It is shown that the process of nanoparticle formation and their morphology in aqueous solutions can be studied by analyzing the mechanism of their co-luminescence and co-fluorescence. Examples of chemical, biological and medicinal applications of nanoparticles containing lanthanide ions and dye molecules with enhanced luminescence brightness are given. The bibliography includes 131 references.
Problem: Previous images of time-gated luminescence have been obtained with a cooled CCD camera by digitally summing a series of sequential images. The data acquisition rate of approximately 10 one millisecond exposure images per second was rate limiting and too slow for standard research and clinical use. An annoying undulating background was present, which could not be totally removed by subtraction of an unexposed, control image. Solution: An analog approach to this problem is to use an interline transfer, electronically shuttered camera. After each exposure, the storage line is not readout; instead, the electrons from the acquisition pixels are transferred to the storage pixels and thus are added to those previously stored. The length of the exposure is limited by the capacity of the storage pixels and the rate of generation of background (noise) electrons. This electronic concept was tested with a Point Grey Dragonfly2 640 by 480 pixel monochrome camera equipped with a Sony 1/3" progressive interline scan, electronically shuttered CCD, which since it did not have any cooling, was operated at room temperature. Pulsed excitation was from a Nichia UV LED. Results: Five and 0.5 micron uniform europium complex stained microspheres could at room temperature be imaged with time-gated excitation and acquisition times of 1 millisecond each and analog summation of 50 images. Conclusion: The analog integration solution apparently works; however, a cooled scientific grade camera with the same capacity for multiple transfers into storage pixels would be better suited for use with dimmer luminescent objects.
Time-gated luminescence images were obtained by analog summation of a series of sequential images that were obtained with a cooled modified interline CCD camera, and a fluorescence microscope modified to use a UV LED for illumination. The interline CCD camera obtains an analog sum of a multi-frame image by not reading out the storage line after each frame is acquired; instead, the charges from the acquisition pixels are transferred to the storage pixels, which adds them to those previously stored; subsequently, the sum of the images is readout from the storage pixels and digitized. The length of the exposure is limited by the capacity of the storage pixels and the rate of generation of background (noise). Previously, the quality of the images obtained with the room temperature camera was degraded by the buildup of thermal noise. The interline transfer, electronically shuttered, cooled astronomy CCD camera, which was modified for analog summation rapidly produced low noise images; yet permitted long exposures. The past problems with lanthanide dyes of low extinction coefficients and equipment cost have now been solved.
Lanthanide bioprobes offer a number of novel advantages for advanced cytometry, including the microsecond luminescence lifetime, sharp spectral emission, and large stokes shift. However, to date, only the europium-based bioprobes have been broadly studied for time-gated luminescence cell imaging, though a wide range of efficient terbium bioprobes have been synthesized and some of them are commercially available. We analyze that the bottleneck problem was due to the lack of an efficient microscope with pulsed excitation at wavelengths of 300-330 nm. We investigate a recently available 315 nm ultraviolet (UV) light emitting diode to excite an epifluorescence microscope. Substituting a commercial UV objective (40×), the 315 nm light efficiently delivered the excitation light onto the uncovered specimen. A novel pinhole-assisted optical chopper unit was attached behind the eyepiece for direct lifetime-gating to permit visual inspection of background-free images. We demonstrate the use of a commercial terbium complex for high-contrast imaging of an environmental pathogenic microorganism, Cryptosporidium parvum. As a result of effective autofluorescence suppression by a factor of 61.85 in the time domain, we achieved an enhanced signal-to-background ratio of 14.43. This type of time-gating optics is easily adaptable to the use of routine epifluorescence microscopes, which provides an opportunity for high-contrast imaging using multiplexed lanthanide bioprobes.
Time-resolved luminescence (TRL) microscopy can image signals from lanthanide coordination complexes or other probes with long emission lifetimes, thereby eliminating short-lifetime (<100 ns) autofluorescence background from biological specimens. However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, making it difficult to rapidly acquire high quality images at probe concentrations that are relevant to live cell experiments. This article describes the development and characterization of a TRL microscope that employs a light-emitting diode (LED, λ(em) = 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection. Europium chelate-impregnated microspheres were used to evaluate instrument performance in terms of short-lifetime fluorescence background rejection, photon collection efficiency, image contrast, and signal-to-noise ratio (SNR). About 200 nm microspheres were imaged within the time resolution limit of the ICCD (66.7 ms) with complete autofluorescence suppression. About 40 nm microspheres containing ~400 chelate molecules were detected within ~1-s acquisition times. A luminescent terbium complex, Lumi4-Tb®, was introduced into the cytoplasm of cultured cells at an estimated concentration of 300 nM by the method of osmotic lysis of pinocytic vesicles. Time-resolved images of the living, terbium complex-loaded cells were acquired within acquisition times as short as 333 ms, and the effects of increased exposure time and frame summing on image contrast and SNR were evaluated. The performance analyses show that TRL microscopy is sufficiently sensitive and precise to allow high-resolution, quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions.
A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (>1 micros) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-microm calibration beads in environmental water dirt samples. Europium-labeled 5-microm 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 nontarget autofluorescent background particles. Under the same conditions, a conventional FCM 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 biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.
The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, would be enhanced by the availability of narrowband emitting, UV excited lanthanide calibration beads. 0.5-, 3-, and 5-microm beads containing a luminescent europium-complex are manufactured. The luminescence distribution of the 5-microm beads is measured with a time-delayed 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 is determined to be homogeneous by confocal microscopy. Emission peaks are 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-microm beads measured under LED excitation with a fluorescence microscope indicate that bleaching does not interfere with their imaging. The luminescence lifetimes in water and air were 340 and 460 micros, respectively. Thus, these 5-microm 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.
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 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. © 2006 International Society for Analytical Cytology
A Eu (III)-macrocycle-isothiocyanate, Quantum DyeTM, 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 unedr 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 increase 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 invloving donors not directly bound to the Eu(III).
Improvements in the lanthanide enhanced luminescence (LEL) protocol have facilitated the use of the recently synthesized Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye(Registered), 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.
Methods for increasing the luminescence intensity of lanthanide macrocycles, Quantum Dyes®, by the Fluorescence Res-onance Energy Transfer Enhanced Luminescence (FRETEL) effect in the solid state have been developed. A homoge-neous solution containing the europium or terbium Quantum Dye and an excess of selected energy transfer species is evaporated to dryness, resulting in a thin film that surrounds and embeds the Quantum Dye or its conjugates. Under these conditions, in the presence of the gadolinium-thenoyltrifluoroacetonate complex as the energy transfer species, the lumi-nescence of the europium Quantum Dye increased approximately 6-fold upon drying. However, the presence of a non-emitting lanthanide such as gadolinium is not always required for this effect. In studies employing the 2,6-pyridinedicar-boxylate ion as the energy transfer species, where both the terbium and the europium Quantum Dyes could be simulta-neously excited at 280 nm, the presence of gadolinium actually decreased the luminescence compared to that obtained with the 2,6-pyridinedicarboxylate alone. The simplest explanation for the FRETEL effect is that fluorescence resonance energy transfer occurs between the photo-trapping energy transfer species, either unbound or complexed with the non-luminescent gadolinium ion. The energy being finally transferred to the luminescent lanthanide ion complexes with con-sequent increase in emission intensity. This new method for the enhancement of luminescence intensity in the solid state has the significant advantage of eliminating the need for the previously required aqueous emulsion, which was difficult to make and transport.
Photo-Optical Instrumentation Engineers. This paper will be published in SPIE BIOS Proceeding Volume 4622 and is made available as an electronic preprint with permission of SPIE. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited. 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 maxi-mum 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-isothio-cyanate has been developed. A simple mixture composed of an ethanolic solution and a Gd(III)-containing aqueous solution is now used to provide lan-thanide 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.
CytometryML is a proposed new Analytical Cytology (Cytomics) data standard, which is based on a common set of XML schemas for encoding flow cytometry and digital microscopy text based data types (metadata). CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and PCS keywords. Flow Cytometry Standard (PCS) list-mode has been mapped to the DICOM Waveform Information Object. The separation of the large binary data objects (list mode and image data) from the XML description of the metadata permits the metadata to be directly displayed, analyzed, and reported with standard commercial software packages; the direct use of XML languages; and direct interfacing with clinical information systems. The separation of the binary data into its own files simplifies parsing because all extraneous header data has been eliminated. The storage of images as two-dimensional arrays without any extraneous data, such as in the Adobe® Photoshop® RAW format, facilitates the development by scientists of their own analysis and visualization software. Adobe Photoshop provided the display infrastructure and the translation facility to interconvert between the image data from commercial formats and RAW format. Similarly, the storage and parsing of list mode binary data type with a group of parameters that are specified at compilation time is straight forward. However when the user is permitted at run-time to select a subset of the parameters and/or specify results of mathematical manipulations, the development of special software was required. The use of CytometryML will permit investigators to be able to create their own interoperable data analysis software and to employ commercially available software to disseminate their data.
Photo-Optical Instrumentation Engineers. This paper was published in SPIE BIOS Proceeding Volume 3604 and is made available as an electronic preprint with permission of SPIE. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited. 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 fluo-rescence results in an improved signal to noise ratio. However, the use of the europium(III) macrocycle, Quantum Dye TM , 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 con-veniently observed with conventional fluorescence instrumentation at previously unattainable low levels. The Eu(III) 5 D 0 → 7 F 2 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 detec-tion of the Eu-macrocycle.
Photo-Optical Instrumentation Engineers. This paper will be published in SPIE BIOS Proceeding Volume 4962 and is made available as an electronic preprint with permission of SPIE. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited. 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 streptavi-din (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 emis-sion 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.
Simultaneous detection of both a Eu(III) and a Sm(III) Quantum Dye TM 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 wave-lengths 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.
Solvent polarity and the local environment have profound effects on the emission spectral properties of fluorophores. The effects of solvent polarity are one origin of the Stokes shift, which is one of the earliest observations in fluorescence. Emission spectra are easily measured, resulting in numerous publications on emission spectra of fluorophores in different solvents, and when bound to proteins, membranes, and nucleic acids. One common use of solvent effects is to determine the polarity of the probe binding site on the macromolecule. This is accomplished by comparison of the emission spectra and/or quantum yields when the flu-orophore is bound to the macromolecule or dissolved in solvents of different polarity. However, there are many additional instances where solvent effects are used. Suppose a fluorescent ligand binds to a protein or membrane. Binding is usually accompanied by spectral shift or change in quantum yield due to the different environment for the bound ligand. Alternatively, the ligand may induce a spectral shift in the intrinsic or extrinsic protein fluorescence. In either case the spectral changes can be used to measure the extent of binding. The effects of solvent and environment on fluorescence spectra are complex, and are due to several factors in addition to solvent polarity. The factors that affect fluorescence emission spectra and quantum yields include:
Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of
pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent.
These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis.
The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between
the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive timeresolved fluorescence
measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium,
and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.
We have developed and interfaced to a computer an automated instrument (the AMAC III) which is designed to observe simultaneously several physical parameters of cells. Typical parameters include electronic cell volume (Coulter effect), RF amplitude (opacity), multiwavelength fluorescence of cytological stains, and cell light-scattering. The use of a new ultraviolet laser combined with a holographic grating spectrograph promises to increase the number of fluorescing species that can be detected simultaneously. This number can be further increased by use of special rare-earth-based fluorochromes, that emit well-defined, spectrally distinct peaks.
A new method is described which combines the identification of DNA replicating and apoptotic cells in a single measurement by flow cytometry. The detection of DNA replicating cells is based on incorporation of 5-bromo-2'-deoxyuridine or 5-iodo-2'-deoxyuridine, followed by selective photolysis at the site of incorporation of the halogenated DNA precursors. Single-strand breaks in DNA resulting from the photolysis are subsequently labeled with digoxygenin or biotin-conjugated dUTP in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The double-stranded DNA breaks in apoptotic cells resulting from activation of the endonuclease can be labeled in this reaction as well. However, in contrast to the photolysed DNA, the low molecular weight fraction of DNA of apoptotic cells is extractable from the cells, and the degree of DNA elution can be modulated by cross-linking with formaldehyde. Thus, apoptotic cells can be distinguished and quantified by virtue of their fractional DNA content. Replication of less than 1% of a genome of a cell in the presence of 5-bromo-2'-deoxyuridine (equivalent of a 5-min 10 microM 5-bromo-2'-deoxyuridine pulse) can be detected by the selective photolysis method. The method was applied to study apoptosis and proliferation of human leukemic HL-60 cells and normal, mitogen-stimulated lymphocytes. Whereas apoptosis of HL-60 cells induced by the DNA topoisomerase I inhibitor camptothecin was selective to DNA replicating cells, apoptosis induced by hyperthermia showed no such selectivity. Lymphocytes that preferentially underwent apoptosis in cultures stimulated by phytohemagglutinin did not initiate DNA replication. By offering the possibility for identification of both DNA replicating and apoptotic cells in a single measurement, the method may find an application in studies of the prognostic value of both cell proliferation and death in human tumors and the apoptotic response of DNA replicating vs. nonreplicating cells to different treatments.
Comparative genomic hybridization was applied to 5 breast cancer cell lines and 33 primary tumors to discover and map regions of the genome with increased DNA-sequence copy-number. Two-thirds of primary tumors and almost all cell lines showed increased DNA-sequence copy-number affecting a total of 26 chromosomal subregions. Most of these loci were distinct from those of currently known amplified genes in breast cancer, with sequences originating from 17q22-q24 and 20q13 showing the highest frequency of amplification. The results indicate that these chromosomal regions may contain previously unknown genes whose increased expression contributes to breast cancer progression. Chromosomal regions with increased copy-number often spanned tens of Mb, suggesting involvement of more than one gene in each region.
Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. Compared with
conventional fluorophores, the nanocrystals have a narrow, tunable, symmetric emission spectrum and are photochemically stable.
The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling
experiment on mouse fibroblasts. These nanocrystal probes are thus complementary and in some cases may be superior to existing
fluorophores.
An optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described in this paper. It relies on the use of two unique families of fluorophores, namely energy-transfer fluorescent beads (TransFluoSpheres) and semiconductor nanocrystal quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A multicolor sample-scanning confocal microscope was constructed that allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps with a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread function of the microscope. We present results of two-dimensional colocalization of TransFluoSpheres (40 nm in diameter) and of nanocrystals (3-10 nm in diameter) and demonstrate distance-measurement accuracy of better than 10 nm using conventional far-field optics. This ruler bridges the gap between fluorescence resonance energy transfer, near- and far-field imaging, spanning a range of a few nanometers to tens of micrometers.
The major reason for sorting or separating Cells is to make a subsequent measurement on a purified population(s). Since, the quantity of cells separated by sorting and other techniques is often quite small, the conventional technique of making a smear on a glass slide produces a very sparse, essentially unusable, dispersion. In fact, the Centrifugal Cytology technique for preparing cells on a microscope slide, described below, was invented to permit conventional morphological analysis of separated cells. Initially, this was for density gradient separation of cells. Subsequently, the fluorescence activated cell sorter was invented, and Centrifugal Cytology was employed to determine the cellular composition of sorted fractions.
Two novel six-nitrogen-donor macrocyclic complex cations of yttrium(III) were obtained from the metal-templated Schiff-base condensation of 1,2-diaminoethane with either 2,6-diacetylpyridine or 2,6-diformylpyridine. The complex cations were isolated as salts of coordinating as well as non-coordinating anions. Single crystal X-ray analysis was carried out for the mixed acetate perchlorate salt of composition Y(C22H26N6) (CH3COO)2(H2O)0.5ClO4, which crystallized in the triclinic system, space group P with Z = 2, in a unit cell having a = 13.052(2), b = 11.362(2), c = 11.426(2) Å, α = 108.87(4), β = 104.73(4) and γ = 100.26(4)°. The structure was characterized by the presence of two slightly different coordination entities, statistically distributed in the crystal lattice in a 1 : 1 ratio. Both entities contained a yttrium(III) ion linked to the six-nitrogen-donor atoms of the macrocycle, which was slightly folded into a butterfly configuration. A bidentate chelating acetate on the convex side of the macrocycle and either a monodentate acetate or a molecular of water on the opposite side, completed the coordination sphere, resulting in coordination number nine. Ionic acetate and perchlorate balanced the residual charges of the complex cations. The yttrium(III) macrocyclic cations remained undissociated in solution, as shown by the chemical behaviour and NMR spectra.
The present invention provides luminescent lanthanide metal chelates comprising a metal ion of the lanthanide series and a complexing agent comprising at least one phthalamidyl moiety. Also provided are probes incorporating the phthalamidyl ligands of the invention and methods utilizing the ligands of the invention and probes comprising the ligands of the invention.
The relaxation of excited molecular states is often followed by the emission of light. Molecular excited states are related to the orbital, vibrational, and rotational levels of the orbital electrons. In addition, the electron spin determines the singlet and triplet states. In the singlet state the spin of an electron is paired, whereas in the triplet state the spin is not paired. When a molecule is excited to an upper singlet energy level, such as S2 (Figure 1), it can rapidly (10 sec) go to the lowest excited state S1 without emission of light. From S1 the molecule may go to any of the rotational and vibrational levels of the ground electronic state S0. This happens either by fluorescence emission or by nonradiative internal processes, referred to as intersystem crossing. From T1, the molecule can return to S0 by nonradiative processes or by a radiative process called phosphorescence. Under suitable conditions S1 and T1 can also transfer their excitation energy to other molecules.
A sensitive co-fluorescence enhancement system was developed for the fluorimetric detection of europium, terbium, samarium and dysporium ions. The fluorescence intensities of the ternary chelates of Eu3+ (and Tb3+, Sm3+ or Dy3+) with pivaloyltrifluoroacetone and 1,10-phenanthroline in a solution containing Triton X-100 and ethanol were greatly enhanced by the addition of excess of Y3+, Lu3+, Gd3+ or La3+. Y3+ enhanced the fluorescence intensities of Eu3+, Tb3+, Sm3+ and Dy3+ chelates 130-, 1078-, 61- and 102-fold, respectively. The limits of detection of Eu3+, Tb3+, Sm3+ and Dy3+ in the developed co-fluorescence enhancement solution were 0.035, 0.34, 7.9 and 46 pM, respectively, when measured with a sensitive time-resolved fluorimeter. The present enhancement system is well suited for the fluorimetric determination of lanthanide ions used, e.g., as labels in time-resolved fluorimetric immunoassays. As compared with a direct enhancement system, the co-fluorescence enhancement solution benefits from higher fluorescence intensities and prolonged decay times. Therefore, it is particularly well suited for a multi-label immunoassay, where several analytes are to be measured simultaneously from a single sample by employing three or four chelate labels.
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-link- ing 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 li ght-emit- ting center. These novel tags are macrocyclic complexes functionalized with an isothiocyanate group to allow covalent coupling to a biosubstrate. The Eu(I II) 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 inter ference from background autof- luorescence. Luminescence efficiency studies indicate significant solvent effects.
Lanthanide complexation chemistry in solution is an active research theme because of the importance of gadolinium complexes as contrast agents in magnetic resonance imaging, and of luminescent europium and terbium complexes as probes in biochemistry. The physicochemical principles and theory underpinning this work are being refined to account for recent developments such as the reappraisal of complex hydration state.
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 fluo-rescence and, if multiple fluorophores are used, from spectral overlap. The long lifetimes and narrow-band emissions of the luminescent lan-thanide complexes will minimize background interference; however, long lifetimes will also result in a significantly reduced signal for flow cytometry or cell sorting.
Functionalized water soluble macrocyclic complexes of lanthanide, actinide and yttrium ions were obtained by metal templated, Schiff-base, cyclic condensation of: (1) a functionalized 1,2-diaminoethane and a dicarbonyl compound selected from the group consisting of 2,6-dicarbonylpyridine, 2,6-diformylpyridine, 2,5-dicarbonylfuran, 2,5-diformylfuran, 2,5-dicarbonylthiophene and 2,5-diformylthiophene; or (2) 1,2-diaminoethane and a ring-substituted heterocyclic dicarbonyl compound selected from a group consisting of substituted 2,6-dicarbonylpyridine, substituted 2,6-diformylpyridine, substituted 2,5-dicarbonylfuran, substituted 2,5-diformylfuran; substituted 2,5-dicarbonyl thiophene, and substituted 2,5-diformylthiophene. Coordination complexes thus formed are kinetically stable in dilute aqueous solution. They are further reacted, or coupled, through a substituent on the 1,2-diaminoethane or on the pyridine, furan, or thiophene moieties, to one of the following: proteinaceous materials, polysaccharides, other biologically compatible macromolecules or bridging molecules which, can be further reacted or coupled to the above mentioned substrates. These macrocyclic complexes are suitable in the preparation of reporter molecules and for magnetic resonance, radiation imaging and radiation therapy.
The present invention provides luminescent lanthanide metal chelates comprising a metal ion of the lanthanide series and a complexing agent comprising at least one salicylamidyl moiety. Also provided are probes incorporating the salicylamidyl ligands of the invention and methods utilizing the ligands of the invention and probes comprising the ligands of the invention.
The characteristic line fluorescence of trivalent europium is excited in certain organoeuropium compounds by irradiation with light absorbed only by the organic part of the compound. The efficiency of excitation varies greatly with the nature of the compound, temperature, and solvent. Under optimum conditions, i.e., solution of a covalent compound at liquid‐air temperature, almost unit efficiency has been obtained. The decay time of the fluorescence is independent of quenching; quenching prevents excitation of the europium ion. There seems to be a steady gradation of efficiency of transfer from covalent to ionic compounds.
The photoacoustic (PAS) spectra and luminescence spectra of Eu(Hba)3 Phen and coprecipitates Eu0.6Ln0.4(Hba)3 Phen complexes (Ln:Y, Nd; Hba: benzoic acid; Phen: phenanthroline) have been measured, and the co‐luminescence effect of the coprecipitates are reported. The PAS intensities of the central lanthanide ions are interpreted in terms of the probability of nonradiative transitions. It is found that the PAS intensity of the ligand bears a relationship with the energy transfer processes. The PAS intensity in the region of ligand absorption increases in the order of Eu0.6Nd0.4(Hba)3 Phen > Eu(Hba)3 Phen > Eu0.6Y0.4(Hba)3 Phen, which indicates that addition of the second lanthanide ions in each case changes the relaxation processes of the complexes. The energy transfer and relaxation processes are discussed in combination with the luminescence spectra.
Western or immunoblotting analysis of protein expression in cells and tissues has been the major analytical tool for assessing molecular biological functions in basic cell biological research, pathology and drug discovery and development. With the rapid growth of our understanding of gene and protein expression pathways and limited supplies of tissue, there is an ever-increasing need for more sensitive, quantitative methods capable of multiplex detection of proteins or protein states from a single western blot. Quantum Dot Corporation's (QDC) new western blot detection kits harness the high brightness, extreme stability and multiple colors of the Qdot Secondary Antibody Conjugates to quantitatively detect several proteins in a single gel. When used in combination with existing user-defined primary antibodies, the kit combines ease of use with sensitivity to picogram levels of protein using several existing gel imaging systems.
Referee: C. Morgan, School of Environmental and Life Sciences. University of Salford, U.K.
A new approach to the chemical synthesis of polypeptides was investigated. It involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by a covalent bond to a solid resin particle. This provided a procedure whereby reagents and by-products were removed by filtration, and the recrystallization of intermediates was eliminated. The advantages of the new method were speed and simplicity of operation. The feasibility of the idea was demonstrated by the synthesis of the model tetrapeptide L-leucyl-L-alanylglycyl-L-valine. The peptide was identical with a sample prepared by the standard p-nitrophenyl ester procedure.
Although conventional fluorometry has found use as the detection technology in both cDNA and protein microarrays, time-resolved fluorometry has not as yet been widely applied to these analyses. Here we describe sensitive, laser-mediated, solid phase detection of a novel time-resolved fluorescent compound, prepared by multiply labeling a polyvinylamine-streptavidin complex with a europium chelate of BCPDA. We demonstrate its use as a universal detection reagent in an antibody based assay, which can be further miniaturized for use in microarray grids, designed to quantify multiple protein species present in complex biological mixtures (blood, tissue extracts). Sensitivity of the immunoassay, as tested with 8 different biotinylated protein-monoclonal antibody pairs, extended down to 1 pg/mL. Extracts of 28 different human tissues were examined. Potential miniaturization of the assay was demonstrated by the ability to detect 10 pg of adsorbed biotinylated mouse immunoglobulin applied in 10 nL microspots and the ability to resolve the time resolved fluorescent signal among 10 nL microspots applied 25 µm apart. The assay is sufficiently sensitive to be applied to microarray formats to detect multiple proteins at physiological concentration in complex biological mixtures. Microarrays have already found important applications in DNA sequencing and gene expression studies. 1-8 Commercial products and instrumentation are now available. On these microdevices, it is possible to immobilize either short oligonucleotides or longer DNA sequences. After target-probe hybridization, the signal is detected by using either radioactivity or fluorescence. It is usually possible to immobilize many thousands of DNA molecules in relatively small areas, and, with newer techniques, the density can reach the level of whole genomes. 6-12 Despite the rapid improvements and progress on DNA microarrays, protein microarrays are much less widely used. 13 Multi-analyte immunoassays, a type of form of protein microarray technology are based on either specific detection of multiple emission signals from fluors or fluorescence emitted from spatially distributed microspots. 14-18 The procedure of screening expression cDNA libraries with antibodies is also a form of a protein microarray technology. Other possible protein microarray formats include solid-phase immobilization of large number of monospecific antibodies, which could capture specific proteins from complex mixtures in an addressable format. With the development of efficient methods for detection of such microspots with high sensitivity and specificity, protein microarrays could find important new applications in future studies. The clinical utility of time-resolved fluorescence to detect biomarkers is well established. 27-31 Time-resolved fluorometry is 100-1,000 fold more sensitive than conventional fluorometry due to background signal rejection from the application of pulsed excitation and time-gated detection. One of the attractive features of lanthanide fluorescent chelates is that quenching effects from multiple labeling are not usually observed. This has led many investigators to explore multiple labeling to improve the signal and sensitivity of immunochemical and other assays. 16,17,19-23 More recently, polyvinylamine complexes of streptavidin that can detect spatially separated fluorescent microspots have been developed. 18,22-24,26 In this paper we describe the application of such a multiply labeled time-resolved fluorescent compound to protein detection antibody based assays carried out in conventional 96 well titer plates and in a miniaturized format involving 10 nL microspots. The preliminary data suggest that the general principles used in this study would be applicable to high-density measurements in protein microarray formats.
A novel method to identify DNA replicating cells is described. In this method DNA strand breaks at sites of incorporation of 5-bromo-2-deoxyuridine (BrdUrd) are induced by photolysis and labeled with digoxygenin- or biotin conjugated dUTP. The reaction is catalyzed by exogenous terminal deoxynucleotidyl transferase. This approach, in conjunction with DNA content analysis by flow cytometry, was applied to studies of the proliferation kinetics of human leukemic HL-60 or MOLT-4 cells during pulse and pulse-chase incubation with BrdUrd. A 30-60 min incubation with BrdUrd led to selective labeling of S phase cells and the progression of a cohort of labeled cells through late S, G2 and G1 was revealed by pulse (30 min) - chase (8 h) labeling with the precursor. The presence of apoptotic cells did not interfere with identification of DNA replicating cells, as the subpopulation of apoptotic cells could be distinguished by low DNA content resulting from extraction of DNA during the procedure. The technique could be applied as well to human breast cancer tissue incubated in vitro with BrdUrd. There is no need for DNA denaturation as is required for the conventional immunocytochemical detection of BrdUrd, which can often impair analysis of the cell phenotype. The SBIP methodology may be uniquely advantageous when there is a need for characterization of the phenotype of proliferating cells, or preservation of some features that would otherwise be destroyed during the step of DNA denaturation.
Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased.
We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed.
A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix.
The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehydefixed, wax-embedded specimens.
A new luminescent marker for the immunochemical detection of proteins and nucleic acids on filters is reported. The label consists of inorganic crystals, generally called phosphors, with a particle size of 0.1-0.3 microns, stabilized in suspension with polycarboxylic acids and subsequently conjugated to immunoreactive macromolecules. Immunophosphor conjugates exhibit slowly decaying fluorescence that is strong and practically nonfading and not sensitive to quenching by water molecules. They are therefore suited for conventional fluorescence detection as well as for time-resolved detection. The lifetime of the phosphors was in the micro/milliseconds range upon excitation with ultraviolet light. Proteins or nucleic acids immobilized on nitrocellulose filters were detected immunochemically or by hybridization, using haptenized nucleic acid probes followed by immunochemical detection, respectively. The ultimate detection limit of proteins, using phosphor-labeled macromolecules including an immunochemical amplification step, was found to be 10 fg. The detection limit of nucleic acids was 300 fg for demonstration of hapten-labeled probes and 10 pg in hybridization formats with hapten-labeled probes. The sensitivity of methods using phosphor-labeled macromolecules was in all cases as good as or better than that of methods using alkaline phosphatase developed to NBT/BCIP. The use of immunophosphors for detection of proteins and nucleic acids on Western and Southern blots is demonstrated. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.
The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 micron and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads.
Oligonucleotide probes containing multiple non-radioactive labels have been prepared by utilising and extending the methods
used to prepare polyamide-oligonucleotide conjugates. The probes were prepared by incorporating suitable amino acid residues,
such as lysines, in the polyamide, which were then used as sites for the attachment of the non-radioactive labels. The procedures
developed give control over the distance of the label from the oligonucleotide, and also the inter-label distance. The labels
can be conveniently introduced while the substrate is still on the solid support. Even though fluorescent oligonucleotide
probes prepared in this way carrying multiple carboxyfluorescein labels gave low levels of fluorescence due to quenching,
the probes containing ten biotin labels gave a detection sensitivity of approximately 5 attomole (3 million molecules).
A new strongly luminescent marker consisting of inorganic crystals is described for time-resolved microscopy. These crystals, known as phosphors, show delayed luminescence, unlike prompt fluorescent labels such as FITC, TRITC and phycobiliproteins, and are therefore potentially suitable for time-resolved microscopy. The luminescence of these phosphors is strong and non-fading in comparison to FITC/TRITC, and not significantly influenced by pH or temperature. The phosphor yttriumoxisulfide activated with europium emits maximally at 620 nm with a typical half lifetime of approximately 700 μsec, upon excitation with near ultraviolet light (360 nm).
Phosphors for immunocytochemical staining were made by ball milling and were stabilized in suspension with polycarboxylic acids. Proteins such as avidin, protein A or immunoglobulins were allowed to adsorb to the surface of the phosphors. The immunocytochemical properties of the conjugates were evaluated in a model system of latex beads with defined surface antigens and in a cellular system containing fixed human lymphocytes or erythrocytes. Specific cytochemical staining was observed in suspension as well as on glass slides. A specially constructed time-resolved microscope was used to suppress the fast decaying fluorescence, thereby permitting visualization of the specific, slowly decaying luminescence of the phosphor label without the necessity of integration. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.
Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.
The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin- or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Cell fluorescence was measured by flow cytometry as well as by a microscope-based laser scanning multiparameter cytometer. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin's lymphoma cells treated with prednisolone were easily identified utilizing BODIPY-conjugated dUTP (B-dUTP). Apoptotic cells were also recognized using fluorescein-conjugated dUTP or dATP, although the discrimination was more pronounced with B-dUTP. The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique.(ABSTRACT TRUNCATED AT 250 WORDS)
Partial alkylation of polylysine with 4-(iodoacetamido)-2,6-dimethylpyridine dicarboxylate (IADP), followed by exhaustive reaction with succinic anhydride, yielded polymers (PLDS, polymer of lysine, dipicolinate, and succinate) containing large numbers (50-100) of 4-substituted dipicolinic acid moieties per molecule, with the remaining lysyl side chains succinylated. Competition experiments showed that PLDS binds Tb(III) ions with much higher affinity than does EDTA and strongly enhances the visible luminescence they emit when excited with ultraviolet light. Carbodiimide-mediated coupling to proteins, including bovine serum albumin, ovalbumin, and protein A, yielded PLDS-protein conjugates whose Tb(III) chelates displayed intense green luminescence and millisecond excited state lifetimes. These conjugates retained sufficient immunoreactivity to allow their use in sensitive luminescence-based immunodetection schemes for proteins immobilized on nitrocellulose. The presence of 10 ng of ovalbumin could be easily visualized by eye when probed with rabbit anti-ovalbumin and PLDS-protein A-Tb(III). The ease of preparation of PLDS-protein-Tb(III) conjugates, and their favorable luminescence properties, make them promising reagents for use in time-resolved luminescence immunoassays and other ultrasensitive detection schemes for macromolecules.
A series of new fluorescent labeling reagents based on sulfoindocyanine dyes has been developed. We describe the synthesis and properties of these reagents. They contain succinimidyl ester reactive groups and can be readily conjugated to antibodies, avidin, DNA, lipids, polymers, and other amino-group-containing materials. The labeling reagents are water soluble, pH insensitive, and show much reduced dye aggregation under labeling conditions. One of the reagents, Cy3, can be excited with the 488-, 514- and 532-nm laser lines and is optimally excited with the 546-nm mercury arc line. Another, Cy5, can be excited with the 633-nm HeNe and 647-nm Kr laser lines available with many flow cytometers and confocal laser-scanning microscopes. New laser diodes emitting near 650 nm should also be excellent excitation sources for Cy5.
Two different kinds of europium(III) chelates, luminescent and nonluminescent, were prepared. The chelates were coupled to bioanalytical reagents, such as antibodies, after activations of the amino group on the chelates with thiophosgene,2,4,6-trichloro-1,3,5-triazine, or iodoacetic anhydride. The reactivities of the activated luminescent chelates in the labeling of antibodies as well as the effects of both the coupling ratio and the linkage group to the luminescence quantum yield of the antibody-bound chelate were studied in aqueous buffer solution.
A time‐resolved flow cytometer capable of measuring a luminescence with a decay time in the range of 10 μs to 2 ms, typical for some lanthanide chelates, is presented. The instrument permits acquisition of conventional light scatter and prompt fluorescence signals as well as detection of slowly decaying luminescence by a photon counting unit for a selectable time period of 1 μs to 1 ms. During photon counting, the laser beam is turned off by an acoustooptic deflector. The design of a flow chamber with an average geometrical light collection efficiency of 35% over a distance of 1.7 mm is presented and analyzed by ray tracing. A pulse processing system featuring digital integration of the conventional signals and a transputer system for the acquisition and the transfer of the measured parameter values to a host computer is described.
Instrument function is verified with lyophilized human lymphocytes stained for the CD8 antigen with dye‐loaded liposomes. Quantitation of cell‐associated europium chelate fluorescence, displaying a decay time of 1.6 ms, is demonstrated. Elimination of fast decaying background emission generated by DNA‐associated ethidium bromide is shown. The background generated by instrument components in the time‐gated measurement channel is characterized, and measures for its complete elimination are discussed. © 1994 Wiley‐Liss, Inc.
Highly luminescent semiconductor quantum dots (zinc sulfide–capped cadmium selenide) have been covalently coupled to biomolecules
for use in ultrasensitive biological detection. In comparison with organic dyes such as rhodamine, this class of luminescent
labels is 20 times as bright, 100 times as stable against photobleaching, and one-third as wide in spectral linewidth. These
nanometer-sized conjugates are water-soluble and biocompatible. Quantum dots that were labeled with the protein transferrin
underwent receptor-mediated endocytosis in cultured HeLa cells, and those dots that were labeled with immunomolecules recognized
specific antibodies or antigens.
We have incorporated peptides selected by combinatorial library [Peterson, J. J., and Meares, C. F. (1998) Bioconjugate Chem. 9, 618-626) into peptide-linked radiolabeled immunoconjugates of the form DOTA-peptide-antibody. Decapeptide linkers -GFQGVQFAGF- and -GFGSVQFAGF-, selected for cleavage by human liver cathepsin B, were rapidly digested in vitro when compared to the simple model tetrapeptide motif of the prototype -GGGF- [Li, M., and Meares, C. F. (1993) Bioconjugate Chem. 4, 275-283]. Cleavage properties of these library-selected substrates for cathepsin B compared favorably with decapeptide linkers -GLVGGAGAGF- and -GGFLGLGAGF-, which incorporate two of the most labile extended cathepsin B substrates from the literature. The decapeptide linker -GFGSTFFAGF-, selected from the library for cleavage by human liver cathepsin D, was rapidly digested by cathepsin D while the others were not.
Substituted 3-aroylcoumarins incorporated in a polyethylenoxy cryptand (2.2.2) by the 3-aroyl group were synthesized with the purpose of developing new markers to be used in time-resolved fluorimetric bioaffinity assays based on the unique luminescence properties of Eu(III) and Tb(III) ions. Some spectral properties and luminescence intensities of the complexes were measured in acetonitrile and MeOH. The mechanism of metal sensitization depended on lanthanide and will be discussed in detail. Copyright
In general, sensitization of lanthanide(III) ions by organic sensitizers is regarded to take place via the triplet state of the sensitizers. Herein, we show that in dansyl- and lissamine-functionalized Nd3+ complexes energy transfer occurs from the singlet state of the sensitizers to the Nd3+ center. No sensitized emission was observed in the corresponding complexes with Er3+, Yb3+, and Gd3+ ions. Furthermore, the fluorescence of the sensitizers was quenched only in the Nd3+ complex and not in the complexes with the other ions. Only Nd3+ centers can accept energy from the singlet state of the dyes, because the excited states of Nd3+ have a high spectral overlap with the fluorescence of the dansyl and lissamine sensitizers, and because the selection rules allow a fast energy transfer, which apparently is competitive with the fluorescence.
The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.