Mirror slides enhance fluorescence signal
Standard fluorophores do not luminesce brightly enough for some applications. Instead of improving fluorophores, researchers in France have developed metal-coated microscope slides that behave like a mirror. The slides reflect fluorescence toward a microscope’s objective lens and significantly boost the fluorescence signal.
The researchers set out to develop the mirror coating for nanometer-scale applications such as building biosensors, particularly for labeling DNA biochips. However, they suspected that the coating might improve the fluorescence signal on a microscopic scale as well. So they tested whether mirror slides enhanced the fluorescence signal during live-cell imaging with a wide-field fluorescence microscope. The investigators were affiliated with the Laboratoire Matériaux et Phénomènes Quantiques and the Laboratoire de Physique du Solide in Paris, as well as the Laboratoire de Photophysique Moléculaire and the Institut Curie in Orsay.
The scientists coated the slides with silver because it reflects visible light well. They also added an aluminum oxide layer. Aluminum oxide eliminated metal toxicity and prevented the silver from tarnishing, although the researchers remained concerned that salts could accumulate on the slides after long-term use and thereby damage the silver layer.
To determine the ability of the mirror slides to boost the fluorescence signal at the molecular level, the researchers employed a Leica microscope and a Roper Scientific interline CCD camera to examine a mirror slide coated with the blue fluorophore, indocarbocyanine. They found that the mirror slide increased the fluorescence signal by approximately 30 times at specific mirror-fluorophore distances.
The investigators used the same microscope and camera to view living dog kidney cells expressing a red fluorescent protein. Dog kidney cells are commonly used models for epithelial cells, especially in cancer research. The scientists also performed immunofluorescence labeling on those cells with either of two Invitrogen dyes with respective emission wavelengths of 599 nm (blue) and 647 nm (green). For all fluorophores, the mirror slides boosted the fluorescence signal by approximately four times and increased the signal-to-noise ratio by about 1.5 times.
A member of the research team, Sandrine Lévêque-Fort, said that the scientists have most recently employed the slides to examine neurons, GFP-labeled bacteria and HEK cells to study membrane properties and cell adhesion. They also have used their mirror slides for viewing biopsied tissue, to showcase their utility for the medical field.
Lévêque-Fort said that the mirror slides enable sufficient signal sensitivity to be achieved for many biomedical microscopy applications using a low numerical-aperture objective. With the slides, one can simultaneously have a large field of view and a high depth of field, which are essential for routine analysis and high-throughput screening.
Lévêque-Fort also said that the researchers are working to improve the slides. For example, they may further functionalize the slides by making them wettable, and they may take more steps to ensure that the silver is protected from long-term degradation.
Toward that goal, they are working with other cover layers such as polymers, and they are exploring metals that will allow the slides to reflect UV light.
Biophysical Journal, online publication, Dec. 15, 2006, doi: 10.1529/biophysj. 106.096750.
- An instrument consisting essentially of a tube 160 mm long, with an objective lens at the distant end and an eyepiece at the near end. The objective forms a real aerial image of the object in the focal plane of the eyepiece where it is observed by the eye. The overall magnifying power is equal to the linear magnification of the objective multiplied by the magnifying power of the eyepiece. The eyepiece can be replaced by a film to photograph the primary image, or a positive or negative relay...
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