A system and method for fluorescence excitation and detection having distinct optical paths is disclosed. A system for detecting fluorescence comprises a light source that emits an excitation light into an illumination tube; a plurality of collection optics located around an aperture in the illumination tube for collecting fluorescence; and a detector for determining the amount of fluorescence. A method for detecting fluorescence comprises emitting an excitation light from a light source into an illumination tube; directing the excitation light to an excitation filter; illuminating a sample with the excitation light to generate an emission light; and detecting the optical characteristics of the emission light using a plurality of collection optics located around the illumination tube.