Evaluation of a time-resolved fluorescence microscope using a phosphorescent Pt-porphine model system

Hennink, E.J.; Haas, R. de; Verwoerd, N. P.; Tanke, Hans J.

doi:10.1002/(sici)1097-0320(19960801)24:4<312::aid-cyto2>3.0.co;2-h

Cited by 34 publications

(19 citation statements)

References 29 publications

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“…Unreacted porphyrins remain at the top of the column because they bind strongly to the Sephadex beads (Hennink et al 1996), whereas the proteinporphyrin conjugate elutes in the void volume.…”

Section: Conjugation Of Ptp-n-nhs and Ptcp-n-nhs To Avidin Streptavimentioning

confidence: 99%

“…At present, the ultimate detection sensitivity in fluorescence microscopy is mainly determined by the nonspecific background signals caused by autofluorescence of the biological objects, fixative-induced fluorescence, and the natural fluorescence of components such as microscope objectives, immersion oil, and optical filters (Aubin 1979;Jovin et al 1989). These fast-decaying types of autofluorescence can be suppressed by application of time-resolved microscopy (Zotikov and Polyakov 1977;Seveus et al 1992;Marriott et al 1991Marriott et al ,1994Verwoerd et al 1994;Hennink et al 1996) using a delayed luminescent label (Hemmilä et al 1982;Seveus et al 1992;Hennink et al 1996) as reporter molecule bound to immunospecific reagents or nucleic acid probes (Diamandis et al 1988;Savitski et al 1989;Beverloo et al 1990;Marriott et al 1991Marriott et al ,1994Seveus et al 1992; de Hennink et al 1996). Most of the applications of time-resolved microscopy reported thus far use europium chelates.…”

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confidence: 99%

“…In addition to lanthanide chelates, metalloporphyrins form another class of potentially interesting labels for time-resolved microscopy (Eastwood and Gouterman 1970;Savitski et al 1989;Vinogradov and Wilson 1995;Hennink et al 1996). Their luminescence properties depend on the metal substitution.…”

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confidence: 99%

“…The palladium (Pd)-and platinum (Pt)-substituted complexes have a high phosphorescence quantum yield even in an aqueous environment at room temperature (RT). In particular, the Pt porphyrin complexes are of interest because they exhibit a higher quantum yield and shorter lifetime under aqueous conditions at RT than the Pd complexes, enabling a higher excitation and emission duty cycle when used for time-resolved microscopy (Hennink et al 1996).…”

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confidence: 99%

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Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy

Haas

Gijlswijk

Tol

et al. 1997

J Histochem Cytochem.

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SUMMARYWe investigated phosphorescent metalloporphyrins as potential labels for time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coefficient, decay times) the best candidates were selected. Next, we synthesized antibody and avidin metalloporphyrin conjugates. The optimal F/P ratio with respect to quantum yield, decay time, and retention of biological activity of these immunoreagents was determined. The reagents were then evaluated by in situ hybridization and immunocytochemical procedures for demonstration of haptenlabeled DNA probes, membrane antigens (CD type), and 28S rRNA. All stained samples exhibited bright phosphorescence that could be selectively detected using time-resolved microscopy, especially when glucose/glucose oxidase was added to the embedding medium to deplete oxygen. Applications of time-resolved detection of phosphorescent porphyrins in strongly autofluorescent material (histological sections) are discussed.

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Section: Conjugation Of Ptp-n-nhs and Ptcp-n-nhs To Avidin Streptavimentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy

Haas

Gijlswijk

Tol

et al. 1997

J Histochem Cytochem.

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“…Heliumcadmium (HeCd) lasers are relatively economical continuous wave sources of UV that can be controlled by quartz acousto-optical modulation (AOM) to generate the required short UV pulses for lanthanide chelate TRF studies. Platinum and palladium porphyrin fluorophores have shorter lifetimes (s < 100 ls) and they can be excited in the blue or violet spectral region, permitting the use of readily available argon-ion lasers (488-nm line) gated with an AOM (19). Gas-discharge lasers require substantial electrical power input and generate significant heat that must be dissipated.…”

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confidence: 99%

High intensity solid‐state UV source for time‐gated luminescence microscopy

2006

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Background: The unique discriminative ability of immunofluorescent probes can be severely compromised when probe emission competes against naturally occurring, intrinsically fluorescent substances (autofluorophores). Luminescence microscopes that operate in the timedomain can selectively resolve probes with long fluorescence lifetimes (s > 100 ls) against short-lived fluorescence to deliver greatly improved signal-to-noise ratio (SNR). A novel time-gated luminescence microscope design is reported that employs an ultraviolet (UV) light emitting diode (LED) to excite fluorescence from a europium chelate immunoconjugate with a long fluorescence lifetime. Methods: A commercial Zeiss epifluorescence microscope was adapted for TGL operation by fitting with a time-gated image-intensified CCD camera and a highpower (100 mW) UV LED. Capture of the luminescence was delayed for a precise interval following excitation so that autofluorescence was suppressed. Giardia cysts were labeled in situ with antibody conjugated to a europium chelate (BHHST) with a fluorescence lifetime >500 ls. Results: BHHST-labeled Giardia cysts emit at 617 nm when excited in the UV and were difficult to locate within the matrix of fluorescent algae using conventional fluorescence microscopy, and the SNR of probe to autofluorescent background was 0.51:1. However in time-gated luminescence mode with a gate-delay of 5 ls, the SNR was improved to 12.8:1, a 25-fold improvement. Conclusion: In comparison to xenon flashlamps, UV LEDs are inexpensive, easily powered, and extinguish quickly. Furthermore, the spiked emission of the LED enabled removal of spectral filters from the microscope to significantly improve efficiency of fluorescence excitation and capture. q

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Fluorescence Imaging Microscopy

Lemasters

2000

Encyclopedia of Analytical Chemistry

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In fluorescence microscopy, an optical microscope creates a magnified image of the fluorescence and phosphorescence of specimens excited by light. Autofluorescence is the intrinsic fluorescence of a specimen, which is often weak. To enhance fluorescence, tags are added, such as fluorescently labeled antibodies which precisely localize specific molecular components. Living cells and tissues may be labeled with a variety of parameter‐indicating fluorophores to measure ion concentrations, electrical potentials and specific chemical species. Thick living specimens create problems of superimposition and haze from out‐of‐focus fluorescence, but advances in confocal fluorescence microscopy and multiphoton fluorescence microscopy now permit successful fluorescence imaging of thickliving specimens with submicrometer three‐dimensional resolution.

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Evaluation of a time-resolved fluorescence microscope using a phosphorescent Pt-porphine model system

Cited by 34 publications

References 29 publications

Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy

Platinum Porphyrins as Phosphorescent Label for Time-resolved Microscopy

High intensity solid‐state UV source for time‐gated luminescence microscopy

Fluorescence Imaging Microscopy

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