METHODS AND SYSTEMS FOR ANALYZING FLUORESCENT MATERIALS WITH REDUCED AUTOFLUORESCENCE
1 Assignment
0 Petitions
Accused Products
Abstract
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.
-
Citations
118 Claims
-
1-94. -94. (canceled)
-
95. 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. - View Dependent Claims (96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117)
-
-
118. 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.
-
Specification