Method in inspecting DNA and apparatus therefor

• US 20030087282A1
• Filed: 08/26/2002
• Published: 05/08/2003
• Est. Priority Date: 09/26/2001
• Status: Active Grant

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Yasuda, Kenji, Takahashi, Satoshi, Oshida, Yoshitada, Seino, Taisaku
• Granted Patent: US 7,309,568 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Abstract

A wide-dynamic range, high-resistivity and high-speed method of stably obtaining fluorescent images of two or more samples which may includes a fluorescent material, comprising the steps of: disposing each of said samples onto each of two or more compartments which an area on a substrate is split into; sequentially irradiating said compartments with an exciting light wherein each of said compartments is irradiated with said exciting light with the intensity changed; detecting one or more fluorescent lights as generated from each of said samples corresponding to said changed intensity of said exciting light every each of said samples; determining the true value of fluorescence as generated from each of said samples by using information on said one or more fluorescent lights as detected every each of said samples; and obtaining a fluorescent image of each of said samples by using said true value of fluorescence from each of said samples, and an apparatus for carrying out the same are provided.

Claims

1. A method of obtaining fluorescent images of two or more samples which may includes a fluorescent material, comprising the steps of:

(a) disposing each of said samples onto each of two or more compartments which an area on a substrate is split into;

(b) sequentially irradiating said compartments with an exciting light wherein each of said compartments is irradiated with said exciting light with the intensity changed;

(c) detecting one or more fluorescent lights as generated from each of said samples corresponding to said changed intensity of said exciting light every each of said samples; and

(d) determining the true value of fluorescence as generated from each of said samples by using information on said one or more fluorescent lights as detected every each of said samples.

2. A method of obtaining fluorescent images according to claim 1, wherein said exciting light consists of two or more beams, with which each of said two or more compartments is simultaneously irradiated.

3. A method of obtaining fluorescent images according to claim 1, wherein said determining step (d) includes the operation of obtaining one or more signals from fluorescence detection as generated from each of said samples with detection results as provided by the detecting step (c) so as to use information on any one proper signal from fluorescence detection corresponding to said changed intensity of said exciting light.

4. A method of determining the values of fluorescence as generated from samples which may include a fluorescent material, comprising the steps of:

(A) arraying said samples on an area on a substrate;

(B) sequentially irradiating said samples with one or more exciting lights wherein each of said samples is irradiated with said one or more exciting lights;

(C) obtaining two or more fluorescent images of each of said samples with two or more fluorescent lights different in intensity from one another as generated from each of said samples by the irradiation with said one or more exciting lights in said irradiating step (B); and

(D) treating said two or more fluorescent images of each of said samples as obtained in said obtaining step (C) so as to determine the true value of fluorescence as generated from each of said samples.

5. A method of determining the values of fluorescence according to claim 4, wherein said arranging step (A) includes the operation of splitting said area on said substrate into two or more compartments so as to dispose each of said samples into each of said compartments;

and said irradiating step (B) includes the operation of simultaneously irradiating two or more samples of said samples wherein each of said two or more samples is irradiated with one or more exciting lights; and

said obtaining step (C) includes the operation of simultaneously obtaining images of said two or more samples.

6. A method of determining the values of fluorescence according to claim 4, wherein said irradiating step (B) includes the operation of irradiating each of said samples with two or more exciting lights different in intensity from one another;

and said obtaining step (C) includes the operation of obtaining two or more images of each of said samples with two or more fluorescent lights different in intensity from one another as generated from each of said samples.

7. A method of determining the values of fluorescence according to claim 4, wherein the dynamic range of said true value of fluorescence as determined in said treating step (D) is larger than 1000.

8. An apparatus for obtaining fluorescent images of two or more samples which may include a fluorescent material, comprising:

(a) an exciting-lights generating means for generating two or more exciting lights different in intensity from one another;

(b) an exciting-lights irradiating means for sequentially irradiating each of said samples with said two or more exciting lights as generated by means of said exciting-lights generating means, with the intensity switched, so as to sequentially irradiate said samples with said two or more exciting lights;

(c) a fluorescent-lights detecting means for detecting two or more fluorescent lights as generated from each of said samples corresponding to said two or more exciting lights different in intensity from one another by sequentially irradiating each of said samples with said two or more exciting lights by means of said exciting-lights irradiating means;

(d) a treating means for treating said two or more fluorescent lights as generated from each of said samples corresponding to said two or more exciting lights different in intensity from one another so as to obtain information on said two or more fluorescent lights as generated from each of said samples; and

(e) an obtaining means for obtaining a fluorescent image of each of said samples by using said information on said two or more fluorescent lights.

9. An apparatus for obtaining fluorescent images according to claim 8, wherein said exciting-lights generating means is the one for branching each of said two or more exciting lights into two or more beams so as to simultaneously irradiating each of said compartments with each of said two or more beams.

10. An apparatus for obtaining fluorescent images according to claim 8, wherein said fluorescent-lights detecting means is the one for receiving two or more signals from fluorescence detection from each of said samples by detecting said two or more fluorescent lights as generated from each of said samples corresponding to said two or more exciting lights different in intensity;

said treating means is the one for obtaining information on a more proper signal from fluorescence detection corresponding to any one of said exciting lights different in intensity from one another; and

said obtaining means is the one for obtaining a fluorescent image of each of said samples by using information on said more proper signal from fluorescence detection.

11. A method of inspecting DNAs, comprising the steps of:

(a) irradiating each of p of DNA-samples (wherein p represents an integer, p≧

2) labelled with a fluorescence, which are each disposed upon each of p′

(wherein p′

represents an integer, p′

≧

p≧

22) of compartments which an area on a substrate is divided into, with one of q (wherein q represents an integer, q≧

1) of exciting lights, wherein with each of said q of exciting lights switched into r (wherein r represents an integer, r≧

2) of beams different in intensity from one another, each of said p of DNA-samples is sequentially irradiated with said r of beams;

(b) detecting r of fluorescent lights as generated from each of said p of DNA-samples in said irradiating step (a), corresponding to said r of beams different in intensity from one another; and

(c) inspecting a DNA in each of said p of DNA-samples by using information upon said r of fluorescent lights as detected in said detecting step (b).

12. A method according to claim 11, wherein s (wherein s represents an integer, p≧

s≧

2) of exciting lights are used; and

said irradiating step (c) includes the operation of simultaneously irradiating s of said DNA-samples with said s of exciting lights.

13. A method of inspecting DNAs according to claim 11, wherein each of said p (wherein p represents an integer, p≧

2) of DNA-samples is attached to the surface of each of p of said beads.

14. A method of inspecting DNAs, comprising the steps of:

(a) irradiating each of p of DNA-samples (wherein p represents an integer, p≧

2) labelled with a fluorescence, which are each disposed upon each of p′

(wherein p′

represents an integer, p′

≧

p≧

2) of compartments which an area on a substrate is divided into, with one of q (wherein q represents an integer, q≧

1) of exciting lights, wherein with each of said q of exciting lights switched into two beams different in intensity from each other, each of said p of DNA-samples is sequentially irradiated with said two beams;

(b) converging two fluorescent lights as generated from each of said p of DNA-samples in said irradiating step (a), corresponding to said two beams different in intensity from each other;

(c) branching said two fluorescent lights as converged in said converging step (b) into two fluorescent lights different in intensity from each other so as to detect each of said branched two fluorescent lights; and

(d) inspecting a DNA in each of said p of DNA-samples by using information upon each of said two fluorescent lights as detected in said detecting step (c).

15. A method of inspecting DNAs according to claim 14, wherein s (wherein s represents an integer, p≧

s≧

2) of exciting lights are used; and

said irradiating step (c) includes the operation of simultaneously irradiating s of said DNA-samples with said s of exciting lights.

16. A method of inspecting DNAs according to claim 14, wherein the dynamic range of a fluorescence image of each of said p (wherein p represents an integer, p≧

2) of DNA-samples is 1000 or more.

17. A method of inspecting DNAs according to any one of claims 14, wherein each of said p (wherein p represents an integer, p≧

2) of DNA-samples is attached to the surface of each of p of said beads.

18. A method of inspecting DNAs, comprising the steps of:

(a) continuously moving, in one direction, p of DNA-samples (wherein p represents an integer, p≧

2) labelled with a fluorescence, which are each attached to the surface of each of p′

(wherein p′

represents an integer, p′

≧

p≧

2) of beads or dots as arrayed at a certain fixed pitch on a bead- or dot-array on a substrate;

(b) irradiating each of said p′

of beads or dots with one spot of a spot exciting-light comprising q (wherein q represents an integer, q≧

1) of spots, each of said q of spots having the diameter of a beam approximately equal to the diameter of each of said beads or dots, wherein said p′

of beads or dots which are continuously travelling in one direction are irradiated with said spot exciting-light so that said spot exciting-light can track said beads or dots, so that a period of time as required for moving each spot by said certain fixed pitch can be almost used for irradiating each of said beads or dots;

(c) separating fluorescence as generated from each of said p of DNA-samples from said spot exciting-light so as to detect said fluorescence; and

(d) inspecting said DNAs by using information as obtained in said detecting step (c).

19. A method of inspecting DNAs according to claim 18, wherein said beads or dots are two-dimensionally arrayed;

and said spot exciting-light comprising q′

(wherein q′

represents an integer, q′

≧

2) of spots.

20. A method of inspecting DNAs according to claim 18, wherein the operation wherein said spot exciting-light tracks said beads or dots is carried out by synchronizing said spot exciting-light with the continuous movement of said p (wherein p represents an integer, p≧

2) of DNA-samples into one direction by means of a light deflector.

21. A method of inspecting DNAs according to claim 18, wherein said detecting step (c) is carried out according to a photon counting detection method.

22. A method of inspecting DNAs, comprising the steps of:

(a) moving p (wherein p represents an integer, p≧

2) of DNA-samples labelled with a fluorescence which are each attached to the surface of each of p′

(wherein p′

represents an integer, p′

≧

p≧

2) of beads as arrayed at a certain fixed pitch on a substrate, in one direction, pitch by pitch of said p′

of beads;

(b) irradiating each of said p′

of beads with one spot of q (wherein q represents an integer, q>

1) of exciting-lights, each of said q of exciting-lights having the diameter of a beam approximately equal to the diameter of each of said beads, wherein q (wherein p≧

q≧

1) of said beads are irradiated with said q of exciting-lights while said p′

of beads which step-travel are stopping;

(c) separating fluorescence as generated from each of said p of DNA-samples from said q of exciting-lights so as to detect said fluorescence; and

(d) inspecting said DNAs by using information as obtained in said detecting step (c).

23. A method of inspecting DNAs according to claim 22, wherein said beads are two-dimensionally arrayed;

and said spot exciting-light comprises q′

(wherein q′

represents an integer, q′

≧

2) of spots.

24. A method of inspecting DNAs according to claim 22, wherein said detecting step (c) is carried out according to a photon counting detection method.

25. An apparatus for inspecting DNAs, comprising:

(A) an exciting-light generating means for generating one or more exciting lights;

(B) an exciting-light irradiating means for sequentially irradiating each of two or more DNA-samples with each of said two or more exciting lights as generated by means of said exciting light generating means, with two or more beams switched different in intensity from one another, wherein a fluorescent label is added to each of said two or more DNA-samples, and each of said two or more DNA-samples is disposed on each of two or more compartments upon a substrate; and

said DNA-samples being sequentially irradiated with said one or more exciting lights;

(C) a fluorescent-lights detecting means for detecting two or more fluorescent lights as generated from each of said DNA-samples, corresponding to said two or more beams switched different in intensity from one another by sequentially irradiating each of said DNA-samples with each of said one or more exciting lights, with two or more beams switched different in intensity from one another; and

(D) a treating means for treating two or more signals for detecting fluorescence as detected according to said two or more beams different in intensity from one another, switched by said fluorescence detecting means so that information upon fluorescence as generated from each of said two or more beams can be obtained.

26. An apparatus for inspecting DNAs according to claim 25, wherein said exciting-light generating means (A) is the one for generating two or more exciting lights;

and said exciting-light irradiating means (B) is the one for simultaneously irradiating each of two or more samples of said DNA-samples with each of said two or more exciting lights.

27. An apparatus for inspecting DNAs according to claim 25, wherein said fluorescent-lights detecting means (C) is the one for obtaining two or more signals for detecting fluorescence of each of said DNA-samples by detecting two or more fluorescent lights as generated from each of said DNA-samples, corresponding to said two or more exciting lights with two or more beams switched different in intensity from one another;

and said treating means (D) is the one for obtaining the fluorescent image of each of said DNA-samples by using a signal for detecting fluorescence corresponding to a beam with any one proper intensity.

28. An apparatus for inspecting DNAs according to claim 25, wherein said fluorescent-lights detecting means (C) comprises a photon pulses counting detector.

29. An apparatus for inspecting DNAs according to claim 25, wherein said apparatus further comprises a table means which can travel with said two or more DNA-samples mounted;

and said exciting light irradiating means is the one for irradiating each of said DNA-samples with said two or more beams switched different in intensity from one another, while tracking each of said DNA-samples which continuously travels by means of said table means.

30. An apparatus for inspecting DNAs, comprising:

(A) an exciting-light generating means for generating q (wherein q represents an integer, q≧

1) of exciting lights;

(B) an exciting-light irradiating means for irradiating each of p (wherein p represents an integer, p≧

2) of DNA-samples with one of said q of exciting lights, wherein a fluorescent label is added to each of said p of DNA-samples, and each of said p of DNA-samples is disposed on each of p′

(wherein p′

represents an integer, p′

≧

p≧

2) of compartments upon a substrate;

(C) a fluorescent-lights converging and branching means for converging r (wherein r represents an integer, r≧

1) of fluorescent lights as generated from each of said p of DNA-samples by the irradiation with said exciting-light irradiating means, and branching said converged fluorescent lights into s (wherein s represents an integer, s≧

2) of fluorescent lights;

(D) a fluorescent-lights detecting means for detecting each of said branched s of fluorescent lights; and

(E) a fluorescent-image obtaining means for obtaining fluorescent images of said detected s of fluorescent lights; and

(F) a treating means for treating said fluorescent images as obtained by said fluorescent-image obtaining means (E) so as to obtain information upon fluorescence as generated from each of said p of DNA-samples.

31. An apparatus for inspecting DNAs according to claim 30, wherein said exciting-light generating means is the one for generating q′

(wherein q′

≧

2) of exciting lights; and

said exciting light irradiating means (B) is the one for simultaneously irradiating q′

of said DNA-samples with q′

of said exciting lights.

32. An apparatus for inspecting DNAs according to claim 30, wherein said fluorescent-image obtaining means (E) is the one for simultaneously obtaining the fluorescent images of said q′

(wherein q′

≧

2) of said DNA-samples.

33. An apparatus for inspecting DNAs according to claim 30, wherein said fluorescence detecting means (D) comprises a photon pulses counting detector.

34. An apparatus for inspecting DNAs according to claim 30, wherein said apparatus further comprises a table means which can be moved in plane with said p of DNA-samples (wherein p≧

2) disposed; and

said exciting-light irradiating means (B) is the one for sequentially irradiating said p of DNA-samples with said q (wherein q≧

1) of exciting lights by synchronizing said q of exciting lights with the movement of said table means.