Liposome biodistribution by time resolved fluorimetry of lipophilic europium complexes

The use of conventional fluorophores suffers from some limitations in biological fluids due to low signal/background ratio. Today, this sensitivity issue might be reasonably improved thanks to lanthanide chelates, by selective detection of long decay fluorescence. Use of pulsed light source time-resolved fluorimetry takes into account the fluorescence decay time of the lanthanide chelates to gain sensitivity in biological media. Lipid-DTPA: Eu compounds have been prepared and incorporated into liposomes to evaluate europium based detection of liposomes in biological media. Fluorescence emission was not modified by this incorporation. Europium labelled liposomes were used for biodistribution studies and showed their use in this context.     

Europium(III) cryptate: a fluorescent label for the detection of DNA hybrids on solid support.

We report here a new detection method for DNA hybrids on dot blots. The process utilizes DNA or oligonucleotide probes labeled with biotin, followed by recognition with a conjugate of streptavidin and europium cryptate, a time-resolved fluorescent label. Unlike the other lanthanide chelates, this label is an organic molecule embedding a europium ion into an intramolecular cavity. This structure has a better stability in diluted assay media, a good sensitivity even on solid support, and an elevated fluorescence lifetime which allows elimination of most of the background generated by other species present in the assay medium. This procedure is quantitative and detects down to 2 amol of a model DNA, which is similar to other nonisotopic (especially colorimetric) methods. The main advantages of this method are easy automation, quantitation, and rapidity of measurements.     

Time-resolved Fluorescence in Immunocytochemical Detection of Prostate-specific Antigen in Prostatic Tissue Sections

Chelates with fluorescent lanthanides such as europium and terbium are widely used in immunofluorometric assays, e.g. for the measurement of different molecular forms of prostate-specific antigen (PSA) in serum for detection and monitoring of prostate cancer. These chelates have also been introduced as non-radioactive labels in immunocytochemistry and in situ hybridization. In the present study, sections of non-malignant prostate were investigated using monoclonal IgGs against PSA. Detection of specific immunostaining employing time-resolved fluorescence with europium-labeled streptavidin was compared with conventional detection by streptavidin conjugated to horse-radish peroxidase. The high PSA concentration in the tissue produced high intensity, specific time-resolved fluorescence signals in the epithelial cells of the prostate gland without disturbance from non-specific tissue autofluorescense. This allowed short exposure times to be used which resulted in insignificant photobleaching. Two of the three europium-chelates evaluated yielded high signal intensities. Counterstaining was found to be optimal with Gill No. 1-Haematoxylin solution and Merckoglas was the best mounting medium for the europium chelates tested. In conclusion, time-resolved fluorescence imaging is an attractive alternative to conventional detection of streptavidin conjugated to horse-radish peroxidase, as it provides linear, high intensity, specific signals subsequent to the decay of non-specific tissue autofluorescence.     

Time-resolved fluoroimmunoassay of steroid hormones

The use of europium chelates as labels in immunoassays and their sensitive quantitation based on time-resolved fluorescence is reviewed. The technique is applied on competitive solid-phase immunoassays for direct determination of progesterone and estradiol in serum samples. Both antigen- and antibody-labelled competitive assays are described. The nonisotopic label technology, which provides a very high specific activity, as well as the antibody-labelled competitive assays, present several advantages in the assay of haptens as e.g. steroids. As the optimal sensitivity of competitive methods is not limited by the specific activity of the label the steroid assays which employ europium chelates as labels do not show any marked increase in sensitivity as compared to that achieved by using 125I. The potential sensitivity provided by the high specific activity of the label is optimally utilized in noncompetitive immunometric assays.     

Attachment of fluorescent metal chelates to macromolecules using “bifunctional” chelating agents

The use of “bifunctional” chelating agents to covalently attach stable chelates of terbium and europium to human serum albumin results in products whose lanthanide fluorescence may be studied easily at micromolar concentrations with standard instrumentation. The lanthanide ions may be added specifically and quantitatively to the protein-bound chelating groups in 0.1 M citrate, pH 6.5. The use of these reagents should greatly reduce ambiguities in the determination of distances between sites on macromolecules by energy transfer measurements.     

Detection of biotinylated DNA probes by using Eu-labeled streptavidin and time-resolved fluorometry

Europium has been used as a label in immunoassays as it can be measured with high sensitivity by means of time-resolved fluorometry. Here we have used streptavidin labeled with europium chelates in the detection of adenovirus type 2 DNA bound to microtiter wells after hybridization with a biotinylated probe. The method gave quantitative results and a sensitivity of about 10 pg of the specific DNA.     

Labeling of proteins and oligopeptides with luminescent lanthanide(III) chelates.

Synthesis of a building block that allows introduction of photoluminescent europium(III) and samarium(III) chelates to synthetic oligopeptides on solid phase using standard Fmoc chemistry is described. Upon completion of the oligopeptide synthesis, these conjugates were converted to the corresponding lanthanide(III) chelates by treatment with appropriate lanthanide(III) salt. Also synthesis of a new terpyridine-based europium(III) chelate designed for solution phase protein labeling is demonstrated.     

Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM).

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.     

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.     

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays

Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates. The ability to selectively impart terbium or europium luminescence to fusion proteins in complex physiological mixtures bypasses the need for specific antibodies and simplifies sample preparation.     

Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6

Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.     

Time-gated in vivo autofluorescence imaging of dental caries.

Laser-induced time-resolved autofluorescence from carious lesions of human teeth was studied by means of ultrashort pulsed laser systems, time-correlated single photon counting and time-gated imaging. Carious regions exhibited a slower fluorescence decay with a main 17 ns fluorescence lifetime than healthy hard dental tissue. The long-lived fluorophore present in carious lesions only emits in the red spectral region. Fluorescence decay time and spectral characteristics are typical of fluorescent metal-free porphyrin monomers. The spatial distribution of the long-lived endogenous porphyrin fluorophore within the tooth material was detected by time-gated nanosecond autofluorescence imaging. In particular, high contrast video images were obtained with an appropriate time delay of 15 ns to 25 ns between excitation and detection due to the suppression of short-lived autofluorescence of healthy tissue. First in vivo applications are reported indicating the potential of time-resolved fluorescence diagnostics for early caries- and dental plaque detection.     

An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues

Interference by autofluorescence is one of the major shortcomes of immunofluorescence analysis by confocal laser scanning microscopy (CLSM). CLSM requires minimal tissue autofluorescence and reduced unspecific fluorescence background, requisites that become more critical when direct immunofluorescence studies are concerned. To control autofluorescence, different reagents and treatments can be used. Until now, the efficacy of the processes described depended on the tissue type and on the processing technique, no general recipe for the control of autofluorescence being available. Using paraffin sections of archival formalin-fixed murine liver, kidney and pancreas, we have found that previously described techniques were not able to reduce autofluorescence to levels that allowed direct immunofluorescence labelling. In this work, we aimed at improving currently described methodologies so that they would allow reduction of the autofluorescent background without affecting tissue integrity or direct immunofluorescence labelling. We have found that the combination of short-duration, high-intensity UV irradiation and Sudan Black B was the best approach to reduce autofluorescence in highly vascularised, high lipofuscins' content tissues, such as murine liver and kidney, and poorly vascularised, low lipofuscins' content tissues such as the pancreas. In addition, we herein show that this methodology is highly effective in reducing autofluorescent background to levels that allow detection of specific signals by direct immunofluorescence.     

Dual-laser, differential fluorescence correction method for reducing cellular background autofluorescence

A method has been developed for reducing the intrinsic autofluorescence background component in cells labeled with fluorescent antibodies, thus permitting low levels of antibody-binding on highly autofluorescent cells to be quantified. The method is based on the broad autofluorescent excitation spectra compared to the well-defined spectra of the fluorescent label. Two laser wavelengths were used, one optimally to excite the fluorescent label plus autofluorescence and the second to excite only the autofluorescence. Two fluorescence measurements were made in the same wavelength region and the signals were subtracted on a cell-by-cell basis using a difference amplifier to zero the autofluorescence and amplify the signal from the fluorescent label. Test results on unlabeled autofluorescent macrophages showed that the autofluorescence component was reduced by balancing the signal inputs to the difference amplifier. When labeled macrophages were analyzed, the autofluorescence was reduced and the fluorescent-labeled antibody-binding component was amplified. The method was also able to resolve labeled lymphocytes from unlabeled autofluorescent macrophages.     

Convenient synthesis of maleimido-derivatized lanthanide(III) chelates and their use in mercapto group conjugation

Simple synthesis of luminescent europium(III) and terbium(III) chelates tethered to a maleimido function (7, 8) is described. The method is based on the following: (i) synthesis of protected ligands tethered to a maleimido function and their purification on silica gel; (ii) deprotection by acidolysis; (iii) conversion of the deprotected ligands to the corresponding lanthanide(III) chelates by passing them through a column of strong cation exchange resin loaded with the appropriate lanthanide(III) ions. According to this procedure, large quantities of mercapto-selective biomolecule-labeling reactants of high purity can be prepared.     

Temporally and spectrally resolved imaging microscopy of lanthanide chelates.

The combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal. Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and rotational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope.     

Pontamine sky blue: a counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry

The stain pontamine sky blue (PSB) has been shown to reduce background autofluorescence in catecholamine fluorescence and immunofluorescence histochemical preparations. Using PSB as a counterstain on whole-mount stretch preparations of human mesenteric blood vessels, a medium dense noradrenergic nerve plexus is clearly revealed, which previously had been only partially visible because of background autofluorescence. Image analysis of nerve densities in whole-mount stretch preparations of guinea-pig arteries containing noradrenergic, substance P-, and vasoactive intestinal polypeptide (VIP)-positive nerve plexuses shows that PSB staining does not alter the specific neuronal fluorescence and that it improves image definition.     

Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis.

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 time-resolved 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.     

Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts

In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real‐time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa‐660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co‐expressing melanin and green fluorescent protein (GFP), C8161‐GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near‐infrared iRFP protein, and quantum dot‐carbon nanotube conjugates. Negative contrast flow cytometry provided label‐free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time‐resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Background suppression in frequency-domain fluorometry

Gated detection is often used in time-domain measurements of long-lived fluorophores for suppression of interfering short-lived autofluorescence. However, no direct method has been available for gated detection and background suppression when using frequency-domain fluorometry. We describe a direct method for real-time suppression of autofluorescence in frequency-domain fluorometry. The method uses a gated detector and the sample is excited by a pulsed train. The detector is gated on following each excitation pulse after a suitable time delay for decay of the prompt autofluorescence. Under the same experimental conditions a detectable reference signal is obtained by using a long lifetime standard with a known decay time. Because the sample and reference signals are measured under identical excitation, gating and instrumental conditions, the data can be analyzed as usual for frequency-domain data without further processing. We show by simulations that this method can be used to resolve single and multiexponential decays in the presence of short lifetime autofluorescence.