Enhanced fluorescence cell imaging with metal-coated slides.
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.
Moal
E Le
EL
Laboratoire Matériaux et Phénomènes Quantiques (UMR 7162, Paris VII), Paris cedex 05, France.
Fort
E
E
Lévêque-Fort
S
S
Cordelières
F P
FP
Fontaine-Aupart
M-P
MP
Ricolleau
C
C
D018929:Cell Culture Techniques / Q000379:methods*; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D007089:Image Enhancement / Q000379:methods*; D008670:Metals / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010783:Photometry / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D020099:Coated Materials, Biocompatible; D008670:Metals