Fluorophores emit white light
David L. Shenkenberg
Because intermolecular distances are small, Förster resonance energy transfer (FRET)
measurements must be precise to determine whether molecules interact. Using a fluorophore
that simultaneously emits two colors and averaging those signals can reduce the
error of the measurements. In living cells, new seminaphthofluorone (SNAFR) fluorophores
emit white light composed of red, green and violet-blue bands that may enable FRET
to be performed with three colors.
Robert M. Strongin and colleagues at Louisiana
State University in Baton Rouge created the fluorophores and evaluated their spectroscopic
properties. They started their synthesis with benzoxanthenes because they exhibit
two simultaneous emission bands, enabling two-color FRET. And because near-IR excitation
minimizes cellular autofluorescence, they followed another research group’s
prediction that variations of existing benzo-xanthenes would absorb near-IR radiation,
and the SNAFRs were produced.
Researchers created seminaphthofluorones, fluorophores that emit
white light and can be excited with various laser lines and viewed with common red,
green or blue filter sets. Reprinted with permission of the American Chemical Society.
The scientists confirmed that they
had created several fluorophores with naphthofluorone frameworks by x-ray crystallography
with Mo Kα radiation on a Bruker AXS diffractometer equipped with an Oxford
Cryosystems Ltd. cooling system. To study their properties, they collected UV-VIS
spectra with a Varian Inc. spectrometer and fluorescence spectra with one from Horiba
Jobin Yvon of Edison, N.J. Upon excitation with a 450-W xenon arc lamp, emission
passed through a dual monochromator and a Hamamatsu photomultiplier tube upon which
950 V had been applied.
When excited at varying pH, overlapping
points at which a prototypical SNAFR absorbs light occur at 484, 394, 327 and 304
nm, suggesting that it is suitable for FRET with common filter sets. It can be excited
from 260 up to 600 nm in buffer, a range of wavelengths that includes helium-neon,
argon-ion and helium-cadmium lasers. It exhibited two pH-sensitive, dual-emission
bands, as is the case with other benzoxanthenes. In methanol, it showed no noticeable
decrease in photostability for at least 3600 s.
These living cells were labeled with a seminaphthofluorone and viewed
through a Texas Red filter set.
The investigators examined the fluorophores
in live cells with an inverted microscope and digital camera, both from Carl Zeiss
of Hawthorne, N.Y., as well as with Chroma Technology Corp. filter sets.
They demonstrated that they could visualize
the fluorophore with ordinary red, blue and green filter sets in living cells. SNAFR
readily enters cells and labels the nucleus, possibly because it binds to DNA. It
also labels their lipophilic portions, including the endoplasmic reticulum and mitochondria,
because it is nonpolar at neutral pH.
Strongin hopes that the lipophilic
properties of naphthofluorones will enable the molecules to cross the blood-brain
barrier. He pointed out that the fluorophores will be readily conjugatable to amino
acids after appropriate analogs are synthesized, and that their ability to be used
with various filter sets and laser lines may make them useful for confocal microscopy
or flow cytometry. He envisions a future for multiplexing applications because they
are pH-sensitive and excitable over a broad range of wavelengths.
Journal of the American Chemical Society, Nov. 1, 2006, pp. 14081-14092.
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