Many naturally occurring materials are autofluorescent, a property that can reduce the discriminative ability of fluorescence methods, sometimes to the point where they cannot be usefully applied. Shifting from the spectral to the temporal domain, it is possible to discriminate fluorophores on the basis of their fluorescence decay lifetime. Luminophores with sufficiently long lifetimes can be discriminated from short-lived autofluorescence using time-gated luminescence (TGL). This technique relies upon the application of a brief excitation pulse followed by a resolving period to permit short-lived autofluorescence to decay, after which detection is enabled to capture persistent emission. In our studies, a high-power UV LED was mounted in the filter capsule of an Olympus BX51 microscope to serve as the excitation source. The microscope was fitted with an Andor DV885 electron-multiplying CCD (EM-CCD) camera with the trigger input synchronized to UV LED operation. Giardia lamblia cysts labeled with the europium chelate BHHST were analyzed against an autofluorescent background with the TGL microscope. The EM-CCD camera captured useful TGL images in real time with a single exposure cycle. With 4x frame averaging, images acquired in TGL mode showed a 30-fold improvement in SNR compared with conventional fluorescence microscopy.
This paper presents experimental and theoretical studies of time-gated discrimination of long-lived luminescence (lifetime: 1~2000μs) 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 μs LED excitation and ~60 μs gated detection. The spatial counting efficiency was evaluated by enumerating 5.5 μm diameter europium microspheres resulting in a counting accuracy approaching 100%.
Signal detection can present a major challenge for fluorescence based detection modalities when target is encountered together with intrinsically fluorescent (autofluorescent) components. Luminophores with long emission lifetimes (eg lanthanide chelates) afford a means to discriminate signal from short-lived autofluorescence through the use of Time-Gated Luminescence (TGL). We have recently synthesized a number of novel europium chelates and required an instrument to accurately compare luminescence lifetime, spectral output and emission intensity. The photophysical response of the chelates was captured using a lab-built time-gated luminescence analyser employing pulsed UV (360 nm) excitation from a high-power (~200 mW) LED. Chelate luminescence was detected using a R928 photomultiplier tube gated electronically into conduction shortly after the excitation pulse had ceased. The photomultiplier dynodes were configured in a novel switching arrangement using high-voltage field effect transistor (FET) devices driven by an optically isolated signal. Using this arrangement, the photomultiplier was gated off until LED excitation had fully extinguished whereon the tube was switched to full-gain within less than a microsecond. In the prototype instrument the strongly emitting europium line (5D0→7F2) was collected with high efficiency using epifluorescence optics. We used the instrument to compare two intensely luminescent europium chelates (BTOT and BHHT) using the analyser and report key photophysical parameters for both compounds.
The detection of the waterborne pathogens Giardia lamblia and Cryptosporidium parvum in environmental water bodies requires concentration of large volumes of water due to the low dose required for infection. The highly concentrated (10,000-fold) water sample is often rich in strongly autofluorescent algae, organic debris and mineral particles that can obscure immunofluorescently labeled (oo)cysts during analysis. Time-resolved fluorescence techniques exploit the long fluorescence lifetimes of lanthanide chelates (ms) to differentiate target fluorescence from background autofluorescence (ns). Relatively simple instrumentation can be used to enhance the signal-to-noise ratio (S/N) of labelled target. Time-resolved fluorescence techniques exploit the large difference in lifetime by briefly exciting fluorescence from the sample using a pulsed excitation source. Capture of the resulting fluorescence emission is delayed until the more rapidly decaying autofluorescence has faded beyond detection, whereon the much stronger and slower fading emission from labelled target is collected. BHHCT is a tetradentate beta-diketone chelate that is activated to bind with protein (antibody) as the chlorosulfonate. The high activity of this residue makes conjugations difficult to control and can lead to the formation of unstable immunoconjugates. To overcome these limitations a 5-atom hydrophylic molecular tether was attached to BHHCT via the chlorosulfonate and the BHHCT derivative was then activated to bind to proteins as the succinimide. The new compound (BHHST) could be prepared in high purity and was far more stable than the chlorosulfonate on storage. A high activity immunocojugate was prepared against Cryptosporidium that yielded an 8-fold increase in SNR using a lab-built time-resolved fluorescence microscope.
A capillary fluorometer was constructed using a 2 mW, 365 nm ultraviolet (UV) light emitting diode (LED) as the excitation source and a new-generation high-gain (3×108) channel photomultiplier tube. The use of a LED permitted rapid pulsing of the excitation source so that the instrument could be employed for time-resolved fluorescence (TRF) applications. A detection limit of ~2×108 molecules of BHHT (4,4’-bis (1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"yl)-o-tephenyl)-Eu (III) were resolved within a 1.25 nanoliter volume at a S/N ratio of 3:1. Ultimate sensitivity of the system was compromised due to visible luminescence emitted by the UV LED, centred around 550 nm extending to > 700 nm and 2nd-order exponentially decaying with lifetimes of 40 μs and 490 μs.
The ubiquity of naturally fluorescing components (autofluorophores) encountered in most biological samples hinders the detection and identification of labeled targets through fluorescence-based techniques. Time-resolved fluorescence (TRF) is a technique by which the effects of autofluorescence are reduced by using specific fluorescent labels with long fluorescence lifetimes (compared with autofluorophores) in conjunction with time-gated detection. A time-resolved fluorescence microscope (TRFM) is described that is based on a standard epifluorescence microscope modified by the addition of a pulsed excitation source and an image-intensified time-gateable CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flash lamp with rapid discharge characteristics was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, the flash output decayed with an approximate lifetime of 18 µs and the TRFM required a long-lived lanthanide chelate label to ensure that probe fluorescence was visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the waterborne parasite Giardia lamblia. For a 600-nm bandpass filter set and a gate delay of 60 µs, the TRFM provided an 11.3-fold improvement in the signal-to-noise ratio (S/N) of labeled Giardia over background. A smaller gain in an SNR of 9.69-fold was achieved with a 420-nm longpass filter set; however, the final contrast ratio between labeled cyst and background was higher (11.3 versus 8.5). Despite the decay characteristics of the light pulse, flash lamps have many practical advantages compared with optical chopper wheels and modulated lasers for applications in TRFM.
Water is routinely monitored for environmental pathogens such a Cryptosporidium and Giardia using immunofluorescence microscopy (IFM). Autofluorescence can greatly diminish an operators capacity to resolve labeled pathogens from non-specific background. Naturally fluorescing components (autofluorophores) encountered in biological samples typically have fluorescent lifetimes (τ) of less than 100 nanoseconds and their emissions may be excluded through use of time-resolved fluorescence microscopy (TRFM). TRFM relies on the large differences in τ between autofluorescent molecules and long-lived lanthanide chelates. In TRFM, targets labeled with a time-resolvable fluorescent immunoconjugate are excited by an intense (UV) light pulse. A short delay is imposed to permit the decay of autofluorescence before capture of luminescence from the excited chelate using an image intensified CCD camera. In our experience, autofluorescence can be reduced to insignificant levels with a consequent 30-fold increase in target visibility using TRFM techniques.
We report conjugation of a novel europium chelate to a monoclonal antibody specific for Giardia lamblia and use of the immunoconjugate for TRFM studies. Initial attempts to conjugate the same chelate to a monoclonal antibody directed against Cryptosporidium parvum led to poorly fluorescent constructs that were prone to denature and precipitate. We successfully conjugated BHHCT to anti-mouse polyvalent immunoglobulin and used this construct to overcome the difficulties in direct labeling of the anti-Cryptosporidium antibody. Both Giardia and Cryptosporidium were labeled using the anti-mouse protocol with a subsequent 20-fold and 6.6-fold suppression of autofluorescence respectively.
A rapid protocol for conjugating and purifying the immunoconjugate was found and methods of quantifying the fluorescence to protein ratio determined.
Performance of our TRFM was dependent on the quality and brightness of the immunoconjugate and optimization of the conjugation process is necessary to reap the full benefit of time-resolved techniques.
The abundance of naturally fluorescing components (autofluorophors) encountered in environmentally sourced samples can greatly hinder the detection and identification of fluorescently labeled target using fluorescence microscopy. Time-resolved fluorescence microscopy (TRFM) is a technique that reduces the effects of autofluorescence through precisely controlled time delays. Lanthanide chelates have fluorescence lifetimes many orders of magnitude greater than typical autofluorophors, and persist in their luminescence long after autofluorescence has ceased. An intense short pulse of (UV) light is used to excite fluorescence in the sample and after a short delay period the longer persisting fluorescence from the chelate is captured with an image-intensified CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flashlamp with a short pulse duration was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, flash output decays with an approximate lifetime of 18μs and the TRFM requires a long-lived chelate to ensure probe fluorescence is still visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a monoclonal antibody directed against the water-borne parasite Giardia lamblia. Fluorescence lifetime of the construct was determined to be 339μs ± 14μs and provided a 45-fold enhancement of labeled Giardia over background using a gate delay of 100μs. Despite the sub-optimal decay characteristics of the light pulse, flashlamps have many advantages compared to optical chopper wheels and modulated lasers. Their low cost, lack of vibration, ease of interface and small footprint are important factors to consider in TRFM design.
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