Single-molecule detection using confocal fluorescence detection: Assessment of optical probe volumes

We have constructed a simple component-based confocal detection system, that is capable of fluorescence detection at the single-molecule level. The component-based format maximises flexibility and reduces start-up costs. A new model for the optical probe volume is proposed, that is based on the 1/e(2) Gaussian intensity contour of a laser beam focused to the diffraction limit. Observation of the onset of single-molecule detection in our experimental system confirms that this model is more appropriate than a simple cylindrical approximation for a probe volume defined by a wide, tightly focused laser beam.