Solid state multi frequency fluorometric measurements system and method

A method of spectrographic measurement, comprising:

• (a) generating substantially incoherent excitation energy in the form of light energy using a solid state excitation source, said light energy being caused to fall on a sample to be assayed, causing said sample to output an output optical signal;
• (b) generating a plurality of modulation frequencies;
• (c) generating a plurality of heterodyne frequencies to form a set of heterodyne signals at said heterodyne frequencies, each of said heterodyne frequencies being associated with one of said modulation frequencies;
• (d) coupling said modulation frequencies to said excitation source, causing said excitation source to generate excitation energy modulated in intensity in proportion to said modulation frequencies;
• (e) sampling a portion of said substantially incoherent excitation energy to form a substantially incoherent reference excitation signal;
• (f) focusing said output optical signal as an image modulated with said plurality of modulation frequencies on an image intensifier;
• (g) intensifying said image to form an intensified image modulated with said plurality of modulation frequencies;
• (h) receiving said intensified image modulated with said plurality of modulation frequencies on a multielement optical detector;
• (i) generating a plurality of measurement signals using said multielement optical detector, each measurement signal associated with a single one of said elements;
• and
• (j) for each measurement signal associated with a single one of said elements of said multielement optical detector, comparing the output of said elements to a standard to generate an output signal indicating characteristics of said sample at the region on said sample associated with each of said elements.

Apparatus for performing fluorescence measurement, comprising:

• (a) a solid state light source generating substantially incoherent excitation energy in a range below 300 nm, oriented to illuminate a sample to be measured and cause said sample to emit fluorescent energy;
• (b) a frequency generator generating a plurality of modulation frequencies and a plurality of heterodyne frequencies, each of said heterodyne frequencies being associated with one of said modulation frequencies said frequency generator being coupled to said excitation source, whereby said source generates excitation energy modulated in intensity in proportion to said modulation frequencies;
• (c) an optical member positioned to receive said substantially incoherent excitation energy and divert a portion of said substantially incoherent excitation energy, said portion of said substantially incoherent excitation energy forming a substantially incoherent reference excitation signal;
• (d) focusing optics positioned to receive said fluorescent energy and form an image modulated with said plurality of modulation frequencies;
• (e) an image intensifier positioned to receive said image, said image intensifier having an output for outputting an intensified image modulated with said plurality of modulation frequencies;
• (f) a multielement optical detector positioned to receive said intensified image modulated with said plurality of modulation frequencies and generating response thereto a plurality of measurement signals, each associated with a single one of said elements;
• (g) a mixer coupled to receive each of said heterodyne frequencies and producing in response to said image and said heterodyne frequencies a plurality of low-frequency measurement modulation products, a low-frequency measurement modulation product being associated with each of said modulation frequencies and comprising the difference between a modulation frequency and its associated heterodyne frequency and having a measurement amplitude;
• and
• (h) a mixer coupled to said substantially incoherent reference excitation signals and said heterodyne frequencies to generate a plurality of low-frequency reference modulation products, a low-frequency reference modulation product being associated with each of said modulation frequencies and comprising the difference between a modulation frequency and its associated heterodyne frequency and having a reference amplitude and phase, each of said low frequency reference modulation products being associated with one of said measurement modulation products, each of said low-frequency measurement modulation products, and their associated low-frequency reference modulation products indicating phase and the modulation information.

Apparatus for performing fluorescence measurements, comprising:

• (a) a solid state low capacitance light source generating substantially incoherent excitation energy in the range below 300 nm, oriented to illuminate a sample to be measured and cause said sample to emit fluorescent energy;
• (b) a frequency generator generating a plurality of modulation frequencies and a plurality of heterodyne frequencies;
• each of said heterodyne frequencies being associated with one of said modulation frequencies said frequency generator being coupled to said excitation source, whereby said source generates excitation energy modulated in intensity in proportion to said modulation frequencies;
• (c) an optical member positioned to receive said substantially incoherent excitation energy and divert a portion of said substantially incoherent excitation energy, said portion of said substantially incoherent excitation energy forming a substantially incoherent reference excitation signal;
• (d) focusing optics positioned to receive said fluorescent energy and form an image modulated with said plurality of modulation frequencies;
• (e) an image intensifier positioned to receive said image, said image intensifier having an output for outputting an intensified image modulated with said plurality of modulation frequencies;
• (f) a multielement optical detector positioned to receive said intensified image modulated with said plurality of modulation frequencies and generating a response thereto a plurality of measurement signals, each associated with a one of said elements;
• and
• (g) a calculating device coupled to said measurement signals, said heterodyne signals and said substantially incoherent reference excitation signals and configured to extract phase and modulation information.

A method of fluorescence measurement comprising:

• (a) generating light energy in the form of substantially incoherent excitation energy in a range below 300 nm output by an excitation source, said substantially incoherent excitation energy being caused to fall on a sample to be measured and cause said sample to emit fluorescent energy;
• (b) generating a plurality of modulation frequencies;
• (c) generating a plurality of heterodyne frequencies to form a set of heterodyne signals at said heterodyne frequencies, each of said heterodyne frequencies being associated with one of said modulation frequencies;
• (d) coupling said modulation frequencies to said excitation source causing said source to generate excitation energy modulated in intensity in proportion to said modulation frequencies;
• (e) sampling a portion of said substantially incoherent excitation energy to form a substantially incoherent reference excitation signal;
• (f) focusing said fluorescent energy as an image modulated with said plurality of modulation frequencies on an image intensifier;
• (g) intensifying said image to form an intensified image modulated with said plurality of modulation frequencies;
• (h) receiving said intensified image modulated with said plurality of modulation frequencies on a multielement optical detector;
• (i) generating a plurality of measurement signals using said multielement optical detector, a signal being output from each of the elements of said multielement, optical detector, each measurement signal associated with one of said elements;
• (j) for each measurement signal associated with one of said elements of said multielement optical detector, said measurement signal being mixed with its respective one of said heterodyne frequencies to generate a plurality of low-frequency measurement modulation products, a low-frequency measurement modulation product being associated with each of said modulation frequencies and comprising the difference between a modulation frequency and its associated heterodyne frequency and having a measurement amplitude and phase;
• (k) mixing said substantially incoherent reference excitation signal with its respective one of said heterodyne frequencies to generate a plurality of low-frequency reference modulation products, a low-frequency reference modulation product being associated with each of said modulation frequencies and comprising the difference between a modulation frequency and its associated heterodyne frequency and having a reference amplitude and phase, each of said low-frequency reference modulation products being associated with one of said measurement modulation products;
• (l) for each of said plurality of low-frequency measurement modulation products, comparing said low-frequency measurement modulation product to its associated low-frequency reference modulation product to measure the relative phase and amplitude of said low-frequency measurement modulation product as compared to said low-frequency reference modulation product and generating a modulation data point and a phase data point;
• (m) for each element, fitting said modulation data points to a first curve;
• (n) for each element fitting said phase data points to a second curve;
• (o) comparing said first and second curves to a database to determine characteristics of said sample;
• and
• (p) displaying said characteristics;
• and generating a reference standard modulation data point and a reference standard phase data point using a zero lifetime standard by:
• (q) causing said generated light energy in the form of substantially incoherent excitation energy output by said excitation source, to fall on a zero lifetime standard causing said sample to output a reference standard optical signal;
• (r) generating a plurality of modulation frequencies;
• (s) generating a plurality of heterodyne frequencies to form a set of heterodyne signals at said heterodyne frequencies, each of said heterodyne frequencies being associated with one of said modulation frequencies;
• (t) coupling said modulation frequencies to said excitation source causing said source to generate excitation energy modulated in intensity in proportion to said modulation frequencies;
• (u) sampling a portion of said substantially incoherent excitation energy to form a reference substantially incoherent excitation signal;
• (v) focusing said reference standard optical signal as a standard image modulated with said plurality of modulation frequencies on said image intensifier;
• (w) intensifying said standard image to form an intensified standard image modulated with said plurality of modulation frequencies;
• (x) receiving said intensified standard image modulated with said plurality of modulation frequencies on said multielement optical detector;
• (y) generating a plurality of measurement signals using said multielement optical detector, a single signal being output from each of the elements of said multielement optical detector, each measurement signal associated with a single one of said elements;
• (z) for each measurement signal associated with one of said elements of said multielement optical detector, said measurement signal being mixed with said heterodyne signal to generate a plurality of low-frequency measurement modulation products, one low-frequency measurement modulation product being associated with each of said modulation frequencies and comprising the difference between a single modulation frequency and its associated heterodyne frequency and having a measurement amplitude and phase;
• (aa) mixing said substantially incoherent reference excitation signal with said heterodyne signal to generate a plurality of low-frequency reference modulation products, a low-frequency reference modulation product being associated with each of said modulation frequencies and comprising the difference between a modulation frequency and its associated heterodyne signal and having a reference amplitude and phase, each of said low-frequency reference modulation products being associated with one of said measurement modulation products;
• and
• (bb) for each of said plurality of low-frequency measurement modulation products, comparing said low-frequency measurement modulation product to its associated low-frequency reference modulation product to measure the relative phase and amplitude of said low-frequency measurement modulation product as compared to said low-frequency reference modulation product and generating a reference standard modulation data point used to generate phase and modulation data points and a reference standard phase data point.