Mitigative and remedial approaches to reduction of autofluorescence background noise are applied in analytical systems that rely upon sensitive measurement of fluorescent signals from arrays of fluorescent signal sources. Such systems are for particular use in fluorescence based sequencing by incorporation systems that rely upon small numbers or individual fluorescent molecules in detecting incorporation of nucleotides in primer extension reactions. Systems and methods for analyzing highly multiplexed sample arrays using highly multiplexed, high-density optical systems to illuminate high-density sample arrays and/or provide detection and preferably confocal detection off signals emanating from such high-density arrays. Systems and methods are applied in a variety of different analytical operations, including analysis of biological and biochemical reactions, including nucleic acid synthesis and derivation of sequence information from such synthesis.