Enhanced fluorescence cell imaging with metal-coated slides.
Biophysical Journal. 2007-03-01; 92(6): 2150-2161
DOI: 10.1529/biophysj.106.096750
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1. Biophys J. 2007 Mar 15;92(6):2150-61. Epub 2006 Dec 15.
Enhanced fluorescence cell imaging with metal-coated slides.
Moal EL(1), Fort E, Lévêque-Fort S, Cordelières FP, Fontaine-Aupart MP, Ricolleau
C.
Author information:
(1)Laboratoire Matériaux et Phénomènes Quantiques (UMR 7162, Paris VII), Paris
cedex 05, France.
Fluorescence labeling is the prevailing imaging technique in cell biology
research. When they involve statistical investigations on a large number of
cells, experimental studies require both low magnification to get a reliable
statistical population and high contrast to achieve accurate diagnosis on the
nature of the cells’ perturbation. Because microscope objectives of low
magnification generally yield low collection efficiency, such studies are limited
by the fluorescence signal weakness. To overcome this technological bottleneck,
we proposed a new method based on metal-coated substrates that enhance the
fluorescence process and improve collection efficiency in epifluorescence
observation and that can be directly used with a common microscope setup. We
developed a model based on the dipole approximation with the aim of simulating
the optical behavior of a fluorophore on such a substrate and revealing the
different mechanisms responsible for fluorescence enhancement. The presence of a
reflective surface modifies both excitation and emission processes and
additionally reshapes fluorescence emission lobes. From both theoretical and
experimental results, we found the fluorescence signal emitted by a molecular
cyanine 3 dye layer to be amplified by a factor approximately 30 when
fluorophores are separated by a proper distance from the substrate. We then
adapted our model to the case of homogeneously stained micrometer-sized objects
and demonstrated mean signal amplification by a factor approximately 4. Finally,
we applied our method to fluorescence imaging of dog kidney cells and verified
experimentally the simulated results.
DOI: 10.1529/biophysj.106.096750
PMCID: PMC1861783
PMID: 17172306 [Indexed for MEDLINE]