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Nature - A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging

: Super User; Reasearch and Development of Time-gated luminescence; 21 June 2016; Hits: 8921; Rating:

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Nima Sayyadi1,2,* , AndrewCare1,2,* , Russell E. Connally3, AndrewC.Try1, Peter L. Bergquist1,4 & Anwar Sunna1

Abstract

Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.d

Solid state time gated luminescence Grain

: Journal of Biomedical Optics 133; Hits: 6552; Rating:

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Solid state time gated luminescence microscope with UV LED

Russell Connally,* Dayong Jin, and James Piper

Abstract

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. © 2008 Society of Photo-Optical Instrumentation Engineers. DOI: 10.1117/1.2928169 K

Layout of the instrumentation used in the time-resolved £uorescence microscope.

: FEMS Microbiology Ecology; Hits: 7404; Rating:

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High resolution detection of Fuorescently labeled microorganisms in environmental samples using time-resolved Fluorescence microscopy

RUSSELL CONNALLY,1* DUNCAN VEAL,1 AND JAMES PIPER2

Abstract

The high level of discrimination offered by fluorescence microscopy has led to its widespread use for the analysis of individual microbial cells. The major limitation of fluorescence microscopy in microbial ecology is that many types of environmental samples contain autofluorescent material that can obscure emission from a fluorescent label. Time-resolved fluorescence microscopy (TRFM) is a technique that greatly reduces background autofluorescence whilst maintaining signal strength of the fluorescent target. TRFM differs from fluorescent microscopy in the use of fluorophores that are characterized by long-lived luminescence. Samples are briefly illuminated to excite fluorescence then capture of luminescence is delayed for a time interval sufficient to ensure autofluorescence has largely faded. TRFM has not been extensively used in microbiology because of the limitations and cost of available time-resolved microscopes and the lack of suitable long-lived fluorescent labels. Here we describe modification of a commercial fluorescence microscope for time-resolved operation through the addition of an image-intensified camera and low cost flashlamp. The TRFM was used in combination with a novel immunofluorophore for the specific detection of Giardia cysts in a water sample containing large amounts of autofluorescent material. A 60-Ws gate delay between excitation and detection resulted in a 30-fold increase in contrast of labeled parasites compared to conventional immunostaining. To our knowledge, this is the first report of the use of TRFM for the detection of microorganisms in environmental samples. 5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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Time-Resolved Fluorescence Microscopy Using an Improved Europium Chelate BHHST for the In Situ Detection of Cryptosporidium and Giardia

RUSSELL CONNALLY,1* DUNCAN VEAL,1 AND JAMES PIPER2

Abstract

Fluorescent immunoconjugates prepared with the europium chelate BHHCT (4,4bis(1,1,1,2,2,3,3-heptaﬂuoro-4,6-hexanedion-6-yl)-chlorosulfo-o-terphenyl) have previously been reported as suitable labels for time-resolved ﬂuorescence applications. BHHCT is limited by a tendency to destabilize immunoglobulins when covalently bound to the protein at moderate to high ﬂuorophore to protein ratios (F/P). We report a new derivative of BHHCT prepared by appending a short hydrophylic tether to the chlorosulfonate activating group on BHHCT. The new derivative, BHHST (4,4-bis-(1,1,1,2,2,3,3-heptaﬂuoro-4,6-hexanedion-6-yl)sulfonylaminopropyl-ester-N-succinimide-ester-o-terphenyl), was activated to bind at the tether terminus with a succinimide leaving group that displayed less aggressive coupling activity and improved storage stability. BHHST has been used to prepare a stable and useful immunoconjugate with the antiCryptosporidiummonoclonalantibodyCRY104.TheBHHSTimmunoconjugateprovidesmorethan a 10-fold enhancement in the signal to noise ratio (SNR) of labeled oocyst ﬂuorescence over background when observed using TRFM techniques. An immunoconjugate was also prepared with BHHST and (goat) anti-mouse that effectively labeled Giardia cysts in situ. Detection of cysts with the TRFM was achieved with an 11-fold increase in SNR when a gate-delay of 60 s was employed. The storage half-life of both immunoconjugates is extended more than 20-fold when compared to immunoconjugates prepared with BHHCT. Microsc. Res. Tech. 64:312–322, 2004. © 2004 Wiley-Liss, Inc.

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Flash lamp-excited time-resolved fluorescence microscope suppresses autofluorescence in water concentrates to deliver an 11-fold increase in signal-to-noise ratio

Russell Connally,* Duncan Veal, and James Piper

Abstract

t. 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 ms 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 ms, 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. © 2004 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.1756594]