Fluorescence detection is the basis of numerous measurements in the biological sciences, biotechnology, and medical diagnostics. While fluorescence is a highly sensitive method, there is always a need for increased sensitivity to detect smaller and smaller numbers of target molecules. Numerous approaches are used to obtain increased detectability, including amplified assays such as ELISA [1
] and PCR [2
], probe with multiple fluorophores such as the phycobiliproteins [3
], long wavelength excitation [5
], and/or gated detection to decrease the background emission [7
]. Several fundamental factors limit the sensitivity of fluorescence methods, typically photodestruction of the fluorophores and the extent of background fluorescence. However, sensitivity is also limited by the collection efficiency of the detection optics.
The importance of light collection efficiency can be seen by consideration of the requirements for single molecule detection [9
]. A typical fluorophore can undergo a finite number of excitation–relaxation cycles prior to photochemical destruction. For photostable molecules such as tetramethylrhodamine photodestruction occurs after about 105
cycles. However, the number of photons detectable from a single fluorophore is typically much smaller, near 103
photons. This difference is due to the isotropic distribution of fluorescence, which makes it difficult to capture more than small fraction of the total emission. According to Keller and co-workers [11
], even sensitive and efficient detection systems capture only about 1% of the total emission, and typically less. Some authors report higher efficiencies near 5%. Efficient collection of the emission is a promising approach to increased sensitivity.
In the present paper, we describe a novel method to efficiently collect about 50% of the total emission while simultaneously reducing the contribution of unwanted background signal. Our approach depends on localization of the probe chemistry near a thin silver film on a transparent substrate, typically 10–200 nm from the surface. At these distances the emission couples with the surface plasmon resonance of the silver and enters the transparent substrate at the surface plasmon angle [12
]. This coupling can be highly efficient to over 90% for molecules with the proper orientation and distance from the surface [13
]. Remarkably, directional emission occurs if the fluorophores are excited by using or not using the evanescent field due to plasmon resonance [14
]. This phenomenon of surface plasmon-coupled emission (SPCE) may be considered to be the reverse of surface plasmon resonance absorption in which the reflectivity is minimum at the angle of incidence for plasmon resonance [16
]. That is, instead of absorption of the incident light at its plasmon angle, SPCE occurs at the plasmon angle of the emission, and is highly directional.
In this initial report, we studied the angular dependence of the emission of fluorophores in polyvinyl alcohol (PVA) spin-coated on a continuous semi-transparent silver film. We found the emission to be sharply distributed around two angles symmetric from an axis normal to the glass–PVA interface. Additionally, we found that fluorophores that are more distant from the surface did not couple into the metal, allowing background rejection in assay formats with surface-localized chemistry. And finally, we found that fluorophores with different emission maxima emit at different angles, allowing spectral discrimination without additional dispersion optics.