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 fluor...

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Detalles Bibliográficos
Publicado: 1998
Materias:
lanthanide
metal chelate
article
crystal structure
fluorescence microscopy
fluorescence polarization
luminescence
Acceso en línea:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00063495_v74_n5_p2210_Vereb
http://hdl.handle.net/20.500.12110/paper_00063495_v74_n5_p2210_Vereb
Aporte de:
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires
id paper:paper_00063495_v74_n5_p2210_Vereb
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spelling paper:paper_00063495_v74_n5_p2210_Vereb2023-06-08T14:31:16Z Temporally and spectrally resolved imaging microscopy of lanthanide chelates lanthanide metal chelate article crystal structure fluorescence microscopy fluorescence polarization luminescence 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. 1998 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00063495_v74_n5_p2210_Vereb http://hdl.handle.net/20.500.12110/paper_00063495_v74_n5_p2210_Vereb
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-134
collection Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic lanthanide
metal chelate
article
crystal structure
fluorescence microscopy
fluorescence polarization
luminescence
spellingShingle lanthanide
metal chelate
article
crystal structure
fluorescence microscopy
fluorescence polarization
luminescence
Temporally and spectrally resolved imaging microscopy of lanthanide chelates
topic_facet lanthanide
metal chelate
article
crystal structure
fluorescence microscopy
fluorescence polarization
luminescence
description 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.
title Temporally and spectrally resolved imaging microscopy of lanthanide chelates
title_short Temporally and spectrally resolved imaging microscopy of lanthanide chelates
title_full Temporally and spectrally resolved imaging microscopy of lanthanide chelates
title_fullStr Temporally and spectrally resolved imaging microscopy of lanthanide chelates
title_full_unstemmed Temporally and spectrally resolved imaging microscopy of lanthanide chelates
title_sort temporally and spectrally resolved imaging microscopy of lanthanide chelates
publishDate 1998
url https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00063495_v74_n5_p2210_Vereb
http://hdl.handle.net/20.500.12110/paper_00063495_v74_n5_p2210_Vereb
_version_ 1768544531248054272

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