System, method, and product for dynamic noise reduction in scanning of biological materials

• US 6,490,533 B2
• Filed: 12/03/2001
• Issued: 12/03/2002
• Est. Priority Date: 04/26/2001
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Honkanen, Peter D., Weiner, Nathan K., Woolaver, Timothy J.
• Primary Examiner(s): Bui, Bryan
• Application Number:
  US09/683,216
• Publication Number:
  US 20020161529A1
• Time in Patent Office: 365 Days
• Field of Search: 702/27, 702/28, 702/32, 702/19, 356/317, 356/300-303, 356/344, 356/614, 356/622, 250/458.1, 250/459.1, 250/461.1, 250/461.2, 435/6, 435/91.1, 435/91.2, 204/461, 204/456
• US Class Current: 702/27
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00533 essentially rectangular
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 35/00 Methods or apparatus for measurement or analysis of nanostructures
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: G01N 2035/00158 Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2035/0494 Detecting or compensating piositioning errors
• CPC Class Codes: G01N 21/276 with alternation of sample and standard in optical path
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 27/44717 : Arrangements for investigating the separated zones, e.g. localising zones
• CPC Class Codes: G01N 27/44721 : by optical means
• CPC Class Codes: G16B 25/00 : ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

Systems and methods are described for processing an emission signal, such as a fluorescent signal, to compensate for noise in an excitation beam, such as a laser beam. As one example, a scanning system is described that includes an excitation signal generator that provides an excitation signal having one or more representative excitation values representative of an excitation beam; an excitation reference provider that provides at least one excitation reference value; a normalization factor generator that compares the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and a comparison processor that adjusts at least one emission value corresponding to the at least one representative excitation value based, at least in part, on the normalization factor.

Claims

1. A method for analyzing molecules, comprising the steps of:

(1) directing an excitation beam to a plurality of pixel locations on a surface having a plurality of probe locations, each probe location including one or more probe molecules;

(2) determining one or more representative excitation values, each related to a value of the excitation beam as directed to at least one of the plurality of pixel locations;

(3) detecting an emission signal having one or more emission values;

(4) correlating each of the one or more emission values with one or more of the representative excitation values;

(5) providing at least one excitation reference value;

(6) comparing the excitation reference value to at least one representative excitation value, thereby generating a normalization factor;

(7) adjusting at least one emission value based, at least in part, on the normalization factor; and

(8) analyzing at least one probe location based, at least in part, on the at least one adjusted emission value.

2. A method comprising the steps of:

(1) directing an excitation beam to a plurality of pixel locations on a substrate;

(2) determining one or more representative excitation values, each related to a value of the excitation beam as directed to at least one of the plurality of pixel locations;

(3) detecting an emission signal having one or more emission values;

(4) correlating each of the one or more emission values with one or more of the representative excitation values;

(5) providing at least one excitation reference value;

(6) comparing the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and

(7) adjusting at least one emission value based, at least in part, on the normalization factor.

3. The method of claim 2, wherein:

one or more probes of a biological microarray are disposed in relation to the substrate.

4. The method of claim 3, wherein:

the one or more probes are disposed at different probe locations on a surface of the substrate.

5. The method of claim 2, wherein:

the substrate comprises a plurality of different polymer sequences coupled to a surface of the substrate.

6. The method of claim 5, wherein:

the plurality of different polymer sequences comprises a plurality of different oligonucleotide sequences, wherein each of the different polymer sequences is coupled in a different probe location of the surface.

7. The method of claim 6, wherein:

each of the probe locations has an area of one-hundredth of a square centimeter or less.

8. The method of claim 2, wherein:

the substrate comprises more than one thousand different ligands of known sequence collectively occupying an area of less than one square centimeter, the different ligands occupying different known locations within the area.

9. The method of claim 2, wherein:

the substrate has a surface comprising more than ten different nucleic acids of known sequences at a plurality of probe locations on the surface of the substrate, each of the probe locations having an area of one-hundredth of a square centimeter or less, at least one of the nucleic acids being bound to a labeled receptor.

10. The method of claim 2, wherein:

the excitation beam is a laser beam.

11. The method of claim 2, wherein:

the plurality of pixel locations comprises one or more scan lines.

12. The method of claim 2, wherein:

the representative excitation values are each related to a power of the excitation beam.

13. The method of claim 2, wherein:

step (2) includes the steps of

(a) directing the excitation beam to a dichroic mirror, and

(b) determining the representative excitation values based on a partial excitation beam that passes through the dichroic mirror.

14. The method of claim 2, wherein:

the emission signal arises from the direction of the excitation beam to the plurality of pixel locations.

15. The method of claim 14, wherein:

the emission signal comprises a fluorescent signal.

16. The method of claim 15, wherein:

the substrate has a surface comprising a plurality of different probes at a plurality of probe locations on the surface of the substrate, at least one of the probes at a first probe location being bound to a fluorescently labeled receptor, wherein at least one of the emission values corresponds to an emission from the fluorescently labeled receptor responsive to the excitation beam being directed to the first probe location.

17. The method of claim 16, wherein:

the probes comprise a plurality of different nucleic acids of known sequences.

18. The method of claim 2, wherein:

step (4) includes spatially correlating the emission values with the representative excitation values.

19. The method of claim 18, wherein:

step (4) includes providing that each emission value is correlated with at least one representative excitation value that is related to a value of the excitation beam that gave rise to the emission value.

20. The method of claim 18, wherein:

step (2) includes determining a first representative excitation value related to a power of the excitation beam as directed to a first of the plurality of pixel locations;

step (3) includes detecting an emission value arising from the direction of the excitation beam to the first pixel location; and

step (4) includes correlating the first emission value with the first representative excitation value.

21. The method of claim 2, wherein:

the excitation reference value is based, at least in part, on at least one of the one or more representative excitation values.

22. The method of claim 2, wherein:

the excitation reference value is based, at least in part, on a plurality of representative excitation values related to values of the excitation beam as directed to pixel locations in one or more scan lines.

23. The method of claim 2, further comprising the step of:

(8) filtering the representative excitation values to provide one or more filtered representative excitation values; and

wherein

the excitation reference value is based, at least in part, on at least one of the one or more filtered representative excitation values.

24. The method of claim 2, wherein:

the excitation reference value is based, at least in part, on a measured calibration value.

25. The method of claim 2, wherein:

the excitation reference value is based, at least in part, on a predetermined specification value.

26. The method of claim 2, wherein:

step (6) includes determining a ratio between the excitation reference value and the at least one excitation value.

27. The method of claim 2, wherein:

step (7) includes multiplying or dividing the emission value by the normalization factor.

28. A system for processing an emission signal having one or more emission values, comprising:

an excitation signal generator constructed and arranged to provide an excitation signal having one or more representative excitation values representative of an excitation beam;

an excitation reference provider constructed and arranged to provide at least one excitation reference value;

a normalization factor generator constructed and arranged to compare the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and

a comparison processor constructed and arranged to adjust at least one emission value corresponding to the at least one representative excitation value based, at least in part, on the normalization factor.

29. The system of claim 28, wherein:

the excitation reference value is determined based at least in part on a low-frequency component of the excitation signal.

30. The system of claim 28, wherein:

the at least one representative excitation value comprises an instantaneous analog value.

31. The system of claim 28, wherein:

the at least one representative excitation value comprises a sampled digital value.

32. The system of claim 28, wherein:

the comparison processor adjusts the at least one emission value corresponding to the at least one representative excitation value based on multiplying or dividing the emission value by the normalization factor.

33. The system of claim 28, wherein:

the excitation signal comprises a laser signal and the emission signal comprises a fluorescent signal resulting from excitation of a fluorophore by the laser signal.

34. A method for processing an emission signal having one or more emission values, comprising the steps of:

providing at least one excitation reference value;

comparing the excitation reference value to at least one excitation value, thereby generating a normalization factor; and

adjusting at least one emission value corresponding to the at least one excitation value based, at least in part, on the normalization factor.

35. A scanning system, comprising:

one or more excitation sources constructed and arranged to generate one or more excitation beams;

an excitation signal generator constructed and arranged to provide an excitation signal having one or more representative excitation values representative of the excitation beam;

an excitation reference provider constructed and arranged to provide at least one excitation reference value;

a normalization factor generator constructed and arranged to compare the excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and

a comparison processor constructed and arranged to adjust at least one emission value corresponding to the at least one representative excitation value based, at least in part, on the normalization factor.

36. The scanning system of claim 35, further comprising:

a processor and a memory unit, wherein the normalization factor generator includes a set of normalization factor generating instructions stored in the memory unit and executed in cooperation with the processor.

37. The scanning system of claim 36, wherein:

the comparison processor includes a set of comparison processing instructions stored in the memory unit and executed in cooperation with the processor.

38. A computer program product for use with a computer having a processor and a memory unit, comprising:

a set of normalization factor generating instructions stored in the memory unit and executed in cooperation with the processor, constructed and arranged to compare at least one excitation reference value to at least one representative excitation value, thereby generating a normalization factor; and

a set of comparison processing instructions stored in the memory unit and executed in cooperation with the processor, constructed and arranged to adjust at least one emission value corresponding to the at least one representative excitation value based, at least in part, on the normalization factor;

wherein the at least one emission value results from excitation of a labeled receptor at a probe location of a probe array.