The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, would be enhanced by the availability of narrowband emitting, UV excited lanthanide calibration beads. 0.5-, 3-, and 5-µm beads containing a luminescent europium-complex are manufactured. The luminescence distribution of the 5-µm beads is measured with a time-delayed light-scatter-gated luminescence flow cytometer to have a 7.0% coefficient of variation (CV) The spacial distribution of the europium-complex in individual beads is determined to be homogeneous by confocal microscopy. Emission peaks are found at 592, 616 (width 9.9 nm), and 685 nm with a PARISS® spectrophotometer. The kinetics of the luminescence bleaching caused by UV irradiation of the 0.5- and 5-µm beads measured under LED excitation with a fluorescence microscope indicate that bleaching does not interfere with their imaging. The luminescence lifetimes in water and air were 340 and 460 µs, respectively. Thus, these 5-µm beads can be used for spectral calibration of microscopes equipped with a spectrograph, as test particles for time-delayed luminescence flow cytometers, and possibly as labels for macromolecules and cells.
The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy,
will be enhanced by the availability of narrow-band emitting lanthanide calibration beads. These beads can also be used to
characterize spectrographic instruments, including microscopes.
Methods: 0.5, 3, and 5 micron (µm) beads containing a luminescent europium-complex were manufactured and the luminescence
distribution of the 5 µm beads was measured with a time-delayed luminescence flow cytometer and a timedelayed
digital microscope. The distribution of the luminescence intensity from the europium-complex in individual
beads was determined on optical sections by confocal microscopy. The emission spectra of the beads under UV excitation
were determined with a PARISS® spectrophotometer. The kinetics of the luminescence bleaching caused by UV irradiation
were measured under LED excitation with a fluorescence microscope.
Results: The kinetics of UV bleaching were very similar for the 0.5, 3, and 5 µm beads. Emission peaks were found at
592, 616, and 685 nanometers (nm). The width of the principal peak at half-maximum (616 nm) was 9.9 nm. The luminescence
lifetimes in water and in air were 340 and 460 microseconds (µs), respectively. The distribution of the europium-
complex in the beads was homogeneous.
Conclusions: The 5 µm beads can be used for spectral calibration of microscopes equipped with a spectrograph, as test
particles for time-delayed luminescence flow cytometers, and possibly as labels for macromolecules and cells.
Improvements in the lanthanide enhanced luminescence (LEL) protocol have facilitated the use of the recently synthesized Eu(III)-macrocycle-mono-isothiocyanate, Quantum Dye, as a label. It was discovered that a homogeneous solution in ethanol or other solvent could be used to produce the lanthanide enhanced luminescence (LEL) effect, provided that the solution was permitted to evaporate. This protocol has been applied to the direct staining of cells in S phase, and was optimized for solid phase assays with Quantum Dye labeled streptavidin. Preliminary studies indicate that cells stained with the europium Quantum Dye can be observed both by conventional UV laser excitation and by infrared two-photon confocal microscopy. An enhancer has been found that enables the observation of simultaneous emissions from both the europium and terbium Quantum Dyes.
Conference Committee Involvement (2)
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XI
2 February 2013 | San Francisco, California, United States
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues X
21 January 2012 | San Francisco, California, United States
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