Practical time-gated luminescence flow cytometry. II: experimental evaluation using UV LED excitation.

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 micros LED pulsed excitation and approximately 60 micros delay-gated detection. The performance of the TGL flow cytometer was evaluated by enumerating 5.7 microm Eu(3+) 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.     

Practical time-gated luminescence flow cytometry. I: concepts.

The method of time-gated detection of long-lifetime (1-2,000 micros) luminescence-labeled microorganisms following rapid excitation pulses has proved highly efficient in suppressing nontarget autofluorescence (<0.1 micros), 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-microm 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.     

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increasing lanthanide luminescence by use of the RETEL effect.

BACKGROUND: Luminescent lanthanide complexes produce emissions with the narrowest-known width at half maximum; however, their significant use in cytometry required an increase in luminescence intensity. The companion review, 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. METHODS: 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. RESULTS: With the RETEL effect, the luminescence of the EuMac-streptavidin conjugate increased at least six-fold upon drying. Nuclei of apoptotic cells were stained with DAPI and tailed with 5BrdUrd to which a EuMac-anti-5BrdU conjugate was subsequently attached. Time-gated images showed the long-lived EuMac luminescence but did not show the short-lived DAPI fluorescence. Imaging of DNA-synthesizing cells with an arc lamp showed that both S phase and apoptotic cells were labeled, and that their labeling patterns were different. The images of the luminescent EuMac and fluorescent DAPI were combined to produce a color image on a white background. This combination of simple chemistry, instrumentation, and presentation should make possible the inexpensive use of the lanthanide macrocycles, Quantum Dyes, as molecular diagnostics for cytological and histopathological microscopic imaging.     

Preparation and microscopic visualization of multicolor luminescent immunophosphors.

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.     

Lanthanide chelates as new fluorochrome labels for cytochemistry

Anti-rabbit IgG labeled with a new fluorescent europium chelate was used to localize rabbit IgG to human smooth muscle myosin in a histological section. The antibody labeled with the europium chelate could be viewed with a conventional fluorescence microscope with a steady-state light source. This result encourages the development of a time-resolved fluorescence microscope, because a significant improvement in the signal-to-noise ratio can be anticipated.     

Development of a novel neodymium compound for in vivo fluorescence imaging.

We developed a novel fluorescent probe that contains the neodymium(III) complex moiety and fluorescein moiety. This probe can emit long-lived near-infrared luminescence derived from a Nd ion through excitation of the fluorescein moiety with visible light (lambda(ex) = 488 nm, lambda(em) = 880 nm, lifetime = 2.3 micros). These results indicate the possibility of the probe as a candidate for in vivo fluorescence molecular imaging.     

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.     

STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.     

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.     

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.     

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5–25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument’s light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.     

Light-emitting diodes in modern microscopy--from David to Goliath?

Proper illumination is essential for light microscopy. Whereas in early years incandescent light was the only illumination, today, more and more specialized light sources, such as lasers or arc lamps are used. Because of the high efficiency and brightness that light-emitting diodes (LED) have reached today, they have become a serious alternative for almost all kinds of illumination in light microscopy. LED have a high durability, do not need expensive electronics, and they can be switched in nanoseconds. Besides this, they are available throughout the UV/Vis/NIR-spectrum with a narrow bandwidth. This makes them ideal light sources for fluorescence microscopy. The white LED, with a color temperature ranging from 2,600 up to 5,000 K is an excellent choice for bright-field illumination with the additional advantage of simple brightness adjustments without changing the spectrum. This review discusses the different LED types, their use in the fluorescence microscope, and discusses LED as specialized illumination sources for F?rster resonance energy transfer and fluorescent lifetime imaging microscopy.     

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.     

Synthesis of a coumarin-based europium complex for bioanalyte labeling

A coumarin-based europium chelate ready-to-use for analyte labeling and homogeneous time-resolved fluorescence measurements has been designed. Compound 1 displays three functional elements: an azide reactive spacer arm, a coumarin sensitizer, and a seven-coordinate europium complex. That complex can be excited at 370 nm by inexpensive UV-LEDs as a light excitation source.     

Discrimination of DNA and RNA in cells by a vital fluorescent probe: lifetime imaging of SYTO13 in healthy and apoptotic cells

BACKGROUND: Of the few vital DNA and RNA probes, the SYTO dyes are the most specific for nucleic acids. However, they show no spectral contrast upon DNA or RNA binding. We show that fluorescence lifetime imaging using two-photon excitation of SYTO13 allows differential and simultaneous imaging of DNA and RNA in living cells, as well as sequential and repetitive assessment of staining patterns. METHODS: Two-photon imaging of SYTO13 is combined with lifetime contrast, using time-gated detection. We focus on distinguishing DNA and RNA in healthy and apoptotic Chinese hamster ovary cells. RESULTS: In healthy cells, SYTO13 has a fluorescence lifetime of 3.4 +/- 0.2 ns when associated with nuclear DNA. Bound to RNA, its lifetime is 4.1 +/- 0.1 ns. After induction of apoptosis, clusters of SYTO13 with fluorescence lifetime of 3.4 +/- 0.2 ns become apparent in the cytoplasm. They are identified as mitochondrial DNA on the basis of colocalization experiments with the DNA-specific dye, DRAQ5, and the mitochondrial-specific dye, CMXRos. Upon progression of apoptosis, the lifetime of SYTO13 attached to DNA shortens significantly, which is indicative of changes in the molecular environment of the dye. CONCLUSIONS: We have characterized SYTO13 as a vital lifetime probe, allowing repetitive and differential imaging of DNA and RNA.     

Oligonucleotide probes for specific detection of Giardia lamblia cysts by fluorescent in situ hybridization

AIMS: Our study focused on the design of oligonucleotide probes and a suitable hybridization protocol that would allow rapid and specific identification of potentially viable cysts of the waterborne parasite Giardia lamblia. METHODS AND RESULTS: Comparative analysis of ribosomal RNA (rRNA) sequences of Giardia lamblia and a number of closely and more distantly related species identified six regions that appear to be specific for the G. lamblia 16S rRNA. Fluorescently labelled probes targeting these regions were produced and employed in fluorescent in situ hybridization (FISH) experiments. Two of the six probes tested successfully. CONCLUSION: Our study provides the first reported probes for specific FISH detection of G. lamblia. The method depends on sufficient amounts of intact rRNA in the target organism, which is unlikely to be present in nonviable cysts that have been exposed to the environment for a prolonged period. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, detection of G. lamblia cysts is largely based on immunofluorescence assays (IFA) targeting cyst wall surface antigens. These assays lack specificity and will detect species others than G. lamblia. Further, IFA will detect nonviable cysts and cyst wall fragments that do not pose a public health risk. In contrast, FISH probes allow specific detection and are likely to only detect viable, infectious cysts.     

Detection of respiratory enzyme activity in Giardia cysts and Cryptosporidium oocysts using redox dyes and immunofluorescence techniques.

The fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), combined with fluorescein-labeled antibodies, was tested for the simultaneous detection of the respiratory electron transport system (ETS) activity and enumeration of Giardia cysts and Cryptosporidium oocysts by spectral microfluorometry and epifluorescence microscopy. The reduction of CTC and p-iodonitrotetrazolium violet (INT), a non-fluorescent redox dye, was compared with propidium iodide (PI) and fluorescein diacetate (FDA) for the measurements of Giardia cyst viability over time. According to the PI and FDA staining techniques, nearly 60% of the cysts tested viable at the beginning of the observations; after 21 days their viability decreased to 5%. The redox dyes indicated that approximately 4-10% of the cysts were metabolically active 48 h after they were shed, followed by a decline in enzyme activity to near undetectable levels after 4 days. Spectral analysis on individual cysts indicated that the fluorescence emission of the reduced CTC and the fluorescein-labeled antibodies is distinctive for each compound and suitable for their simultaneous determination by microphotometry, flow cytometry and epifluorescence microscopy. The fluorescence signal remained without alteration when the cysts were transferred onto microscope slides coated with an optical embedding medium and stored at -20 degrees C. The fluorescence intensity of the reduced CTC, when properly standardized, can provide quantitative measurements of ETS activity of the cysts. This is the first report of a method to determine enzyme redox activity on intact cysts applicable to water, laboratory and animal samples.     

Quantification of nucleic acids on nitrocellulose membranes with time-resolved fluorometry.

We use a streptavidin-based macromolecular complex (SBMC) labelled with the europium chelate of 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as a staining reagent for biotinylated DNA present on nitrocellulose filters. The fluorescent spots or bands obtained can either be observed under UV illumination, photographed by instant camera photography or quantified by using a specially designed instrument working as a high resolution time-resolved fluorometric scanner. The detection limit is approximately 10 pg of target DNA. Various experiments with use of biotinylated DNA probes hybridized to Southern transferred targets have shown that the new procedure is a useful versatile non-isotopic methodology for staining DNA on solid supports.     

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.