Abstract
Optical detection and spectroscopy of single molecules has become an indispensable tool in biological imaging and sensing. Its success is based on fluorescence of organic dye molecules under carefully engineered laser illumination. In this paper we demonstrate optical detection of single molecules on a wide-field microscope with an illumination based on a commercially available, green light-emitting diode. The results are directly compared with laser illumination in the same experimental configuration. The setup and the limiting factors, such as light transfer to the sample, spectral filtering and the resulting signal-to-noise ratio are discussed. A theoretical and an experimental approach to estimate these parameters are presented. The results can be adapted to other single emitter and illumination schemes.
Highlights
The invention of the laser in the 1960s was a key evolution in the path towards optical single molecule detection
1980s M OERNER and K ADOR succeeded in the first optical detection of single pentacene molecules [2], but this new technique only became important for biology and sensing in the 1990s when the experiments were extended to work at room temperature
The sample for all further experiments and estimations consists of a doped thin crystalline film of p-terphenyl (Aldrich) which is spin coated on a microscopy coverslip [10]
Summary
The invention of the laser in the 1960s was a key evolution in the path towards optical single molecule detection. Using a laser to excite a fluorescently doped sample and detecting the spectrally filtered light on a photomultiplier, he was able to see the fluorescent fingerprint of a cluster of molecules. 1980s M OERNER and K ADOR succeeded in the first optical detection of single pentacene molecules [2], but this new technique only became important for biology and sensing in the 1990s when the experiments were extended to work at room temperature. These experiments rely on efficient discrimination of the excitation laser light from the molecule’s red shifted fluorescence [3]. In our experimental findings both parameters are not negligible and lead to a two times higher background intensity (see Figure 5(c))
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