Methods and systems for analyzing fluorescent materials with reduced autofluorescence

A system for monitoring a plurality of discrete fluorescent signals from a plurality of discrete fluorescent signal sources, the system comprising:

• a substrate in a first focal plane having the plurality of discrete signal sources disposed thereon;
• a first excitation illumination source providing light having a first spectrum;
• a detector for detecting the plurality of discrete fluorescent signals from the plurality of discrete fluorescent signal sources;
• and,
• an optical train positioned to simultaneously direct excitation illumination from the first excitation illumination source to each of the plurality of discrete fluorescent signal sources on the substrate and to direct the discrete fluorescent signals from the plurality of discrete fluorescent signal sources to the detector, wherein the optical train comprises:
• an objective lens in the first focal plane, which objective lens is focused at the substrate and collects the discrete fluorescent signals from the plurality of discrete fluorescent signal sources on the substrate,
• a first pair of tunable lenses that are adjustable relative to each other, and
• a first diffractive optical element (DOE) configured to convert a single originating illumination beam from the first excitation illumination source into a plurality of discrete illumination beams, each beam being directed at a different one of the plurality of discrete fluorescent signal sources on the substrate.

A method of reducing fluorescence background noise in monitoring fluorescent signals from at least one fluorescent signal source, the method comprising:

• providing an excitation illumination source, a substrate having the at least one fluorescent signal source disposed thereon, and an optical train comprising optical components, which optical train is positioned to direct excitation illumination from the illumination source to the at least first fluorescent signal source and transmit fluorescent signals from the at least first fluorescent signal source to a detector;
• photobleaching at least one of the optical components to reduce an amount of autofluorescence background noise produced by the at least one optical component in response to the excitation illumination;
• directing excitation illumination through the at least one optical component and at the at least one fluorescent signal source;
• and,
• detecting the fluorescent signals from the at least first fluorescent signal source.