Assessing blood brain barrier dynamics or identifying or measuring selected substances or toxins in a subject by analyzing Raman spectrum signals of selected regions in the eye

• US 6,574,501 B2
• Filed: 01/09/2001
• Issued: 06/03/2003
• Est. Priority Date: 07/13/1998
• Status: Expired due to Fees

• Current Assignee: Childrens Hospital Los Angeles
• Original Assignee: California Institute of Technology, Childrens Hospital Los Angeles
• Inventors: Borchert, Mark S., Lambert, James L.
• Primary Examiner(s): Lateef, Marvin M.
• Assistant Examiner(s): Shah, Devaang
• Application Number:
  US09/756,897
• Publication Number:
  US 20010034478A1
• Time in Patent Office: 875 Days
• Field of Search: 600/473, 600/475
• US Class Current: 600/473
• CPC Class Codes: A61B 5/14532 for measuring glucose, e.g. by tissue impedance measurement
• CPC Class Codes: A61B 5/14546 for measuring analytes not otherwise provided for, e.g. ions, cytochromes
• CPC Class Codes: A61B 5/1455 using optical sensors, e.g. spectral photometrical oximeters
• CPC Class Codes: A61B 5/4064 Evaluating the brain A61B5/031, A61B5/369, A61B5/14553 take precedence
• CPC Class Codes: A61B 5/411 Detecting or monitoring allergy or intolerance reactions to an allergenic agent or substance
• CPC Class Codes: G01J 3/027 Control of working procedures of a spectrometer; Failure detection; Bandwidth calculation
• CPC Class Codes: G01N 2021/656 Raman microprobe
• CPC Class Codes: G01N 21/65 Raman scattering

Abstract

A non-invasive method for analyzing the blood-brain barrier includes obtaining a Raman spectrum of a selected portion of the eye and monitoring the Raman spectrum to ascertain a change to the dynamics of the blood brain barrier.

Also, non-invasive methods for determining the brain or blood level of an analyte of interest, such as glucose, drugs, alcohol, poisons, and the like, comprises: generating an excitation laser beam (e.g., at a wavelength of 600 to 900 nanometers); focusing the excitation laser beam into the anterior chamber of an eye of the subject so that aqueous humor, vitreous humor, or one or more conjunctiva vessels in the eye is illuminated; detecting (preferably confocally detecting) a Raman spectrum from the illuminated portion of the eye; and then determining the blood level or brain level (intracranial or cerebral spinal fluid level) of an analyte of interest for the subject from the Raman spectrum. In certain embodiments, the detecting step may be followed by the step of subtracting a confounding fluorescence spectrum from the Raman spectrum to produce a difference spectrum; and determining the blood level and/or brain level of the analyte of interest for the subject from that difference spectrum, preferably using linear or nonlinear multivariate analysis such as partial least squares analysis. Apparatus for carrying out the foregoing methods are also disclosed.

Claims

1. An in vivo method for monitoring the blood brain barrier dynamics of a subject, comprising the step of:

obtaining a Raman spectrum of a selected region in the eye of the subject; and

monitoring the dynamics of the blood brain barrier of the subject based on the obtained Raman spectrum.

2. A method according to claim 1, further comprising the step of administering a non-specific marker to the subject selected for its normal reluctance to cross the blood brain barrier under a normal condition, and wherein said monitoring step comprises detecting the presence of the non-specific marker in the selected region of the eye of the subject after the administering step based on the obtained Raman spectrum.

3. A method according to claim 1, further comprising the steps of:

altering the dynamics of the blood brain barrier of the subject from a normal condition; and

administering a quantity of a selected therapeutic agent to a subject for treatment of condition in the brain or neurologic system after said altering step.

4. A method according to claim 3, further comprising the step of returning the blood brain barrier to a substantially normal state after a sufficient quantity of the therapeutic agent has been delivered to the brain.

5. A method according to claim 3, wherein said obtaining step is repeated a plurality of times.

6. A method according to claim 3, wherein said obtaining step is performed before said administering step to confirm that the dynamics of the brain have been successfully altered and subsequently to confirm that the blood brain barrier is returned to a substantially normal condition.

7. A method according to claim 3, wherein said method further comprises the step of assessing the dose amount of the therapeutic agent delivered to the brain.

8. A method according to claim 1, wherein the selected region of the eye comprises the aqueous humor.

9. A method according to claim 1, wherein the selected region of the eye comprises the vitreous humor.

10. A method according to claim 1, wherein the selected region of the eye comprises a conjunctive vessel.

11. A method according to claim 1, wherein the therapeutic agent is a cytotoxic agent suitable for treating a cancer in the brain.

12. A method according to claim 3, wherein said altering step is carried out by introducing a chemical to the subject to temporarily open the blood brain barrier which allows larger molecules to pass therethrough.

13. A method according to claim 3, wherein said altering step is carried out by increasing the intracranial pressure of the subject.

14. A method according to claim 3, wherein the therapeutic agent is directed to treating brain cancer.

15. A method of non-invasively monitoring the blood brain barrier of a subject, comprising the steps of:

generating an excitation beam;

focusing the excitation beam of said generating step into the eye of the subject so that a selected region is illuminated;

detecting a Raman spectrum corresponding to the illuminated region; and

monitoring the blood brain barrier dynamics during exposure to selected conditions based on said detecting step.

16. A method according to claim 15, wherein said monitoring step comprises assessing whether the dynamics of the blood brain barrier are altered sufficiently to allow at least one selected analyte, which would normally be inhibited from traveling through the blood brain barrier, to pass into the intracranial spinal fluid through the blood brain barrier.

17. A method according to claim 16, wherein said method further comprises the steps of:

administering a non-specific marker to the subject, the non-specific marker being selected for its normal reluctance to pass through the blood brain barrier;

comparing the Raman spectrum from said detecting step to predetermined reference spectrums corresponding to the non-specific marker; and

identifying the presence of the non-specific marker in the Raman spectrum of said detecting step to indicate that the dynamics of the blood brain barrier have changed.

18. A method according to claim 17, wherein said detecting step is carried out by illuminating the aqueous humor of the eye.

19. A method according to claim 17, wherein said detecting step is carried out by illuminating the vitreous humor of the eye.

20. A method according to claim 16, wherein said detecting step is carried out when the subject is exposed to elevated ambient pressures.

21. A method according to claim 17, further comprising the step of 30 introducing a cytotoxic agent to the subject after said identifying step.

22. A method according to claim 16, wherein said monitoring step is carried out when the subject is exposed to gravity deficient environments.

23. A method according to claim 16, wherein said method is carried out during drug trials to assess the impact of the drug on the dynamics of the blood brain barrier of the subject.

24. A method according to claim 17, wherein said method is carried out when the brain of the subject has been exposed to a trauma injury to determine whether there is an alteration of the dynamics of the blood brain barrier.

25. A method for identifying an alteration in the blood brain barrier of a biological subject, comprising the steps of:

non-invasively obtaining a first in vivo Raman spectrum of the aqueous humor of the subject;

non-invasively obtaining a second in vivo Raman spectrum of the aqueous humor of the subject; and

comparing the first and second Raman spectrums to detect an alteration in the function of the blood brain barrier.

26. A method for identifying an alteration or abnormality in the blood brain barrier of a biological subject, comprising the steps of:

non-invasively obtaining a first in vivo Raman spectrum of the aqueous humor of the subject;

obtaining at least one reference spectrum of an in vitro sample representing the aqueous humor and comprising at least one selected analyte;

comparing the in vivo Raman spectrum to the reference spectrum; and

identifying an abnormality in the blood brain barrier based on the detected presence of at least one selected analyte in the aqueous humor based on the comparison of the in vivo and reference Raman spectra.

27. A method according to claim 26, wherein said the least one analyte is a physiological compatible non-specific marker which is selected based on its natural reluctance to pass through the blood brain barrier.

28. A non-invasive method for determining the alcohol level in a subject, comprising the steps of:

generating an excitation beam;

focusing the excitation beam of said generating step into the anterior chamber of an eye of the subject so that aqueous humor in the anterior chamber is illuminated;

detecting a Raman spectrum corresponding to the illuminated aqueous humor;

comparing the Raman spectrum from said detecting step to reference spectrums corresponding to different concentrations of alcohol; and

assessing the brain alcohol level in the subject based on said detecting and comparing steps.

37. A method according to claim 28, wherein said identifying step identifies an elevated antibody level in the subject.

38. A method according to claim 28, wherein said identifying step detects the presence of a poison in the subject.

39. A method according to claim 28, wherein the reference spectra of selected analytes of interest are based on selected household chemicals.

40. A method according to claim 28, wherein the reference spectra of selected analytes of interest are of illegal narcotics.

41. A method according to claim 28, wherein said reference spectra for each analyte of interest comprises spectra generated from a Raman spectra training set comprising at least 10 in vitro aqueous humor samples for each analyte of interest with each of the at least 10 samples including different amounts of the analyte therein, and an analyte level corresponding to each of the samples.

47. An apparatus according to claim 39, wherein said excitation beam has a wavelength of from about 600 to 900 nanometers.

48. An apparatus according to claim 39, wherein said excitation beam is low energy.

49. An apparatus according to claim 39, wherein said excitation beam has a wavelength of about 633 nm.

50. An apparatus according to claim 39, wherein the computer program reference spectrums are produced with a training set of Raman spectra corresponding to at least 20 aqueous humor samples of varying concentrations of analytes therein for each of a plurality of different selected analytes and an identified blood level of said analyte of interest for each of said samples.

51. An apparatus according to claim 50, wherein said analyte of interest is alcohol.

57. An apparatus according to claim 40, wherein said housing is configured and sized to be a hand held device.

29. A non-invasive method for identifying an unknown poison in a subject, comprising the steps of:

generating an excitation beam;

focusing the excitation beam of said generating step into a selected region in the eye of the subject so that the selected region is illuminated;

detecting a Raman spectrum corresponding to the illuminated selected region;

comparing the Raman spectrum from said detecting step to reference spectra corresponding to different concentrations of a plurality of different substances; and

identifying the unknown substance in the subject based on said detecting and comparing steps, wherein the selected region of the eye is one of the aqueous humor and a conjunctiva vessel, said method further comprising the step of estimating the amount of the at least one analyte of interest in the cerebral spinal fluid of the subject based on said detecting, comparing, and identifying steps.

30. A method for non-invasively directly measuring the blood level of a substance of interest in a subject, comprising the steps of:

obtaining a plurality of reference Raman spectrums of varying concentrations of at least one analyte of interest;

generating an excitation beam;

focusing the excitation beam of said generating step into at least one conjunctiva vessel in the eye of the subject so that the vessel is illuminated;

detecting a Raman spectrum corresponding to the illuminated vessel;

comparing the Raman spectrum from said detecting step to the reference spectrums; and

determining the blood level of the at least one analyte based on said detecting and comparing steps.

31. A method according to claim 30, wherein the reference spectrums comprise a plurality of different Raman spectrums generated for each of the at least one analyte at different concentrations based on in vitro samples thereof.

32. A method according to claim 30, wherein said excitation beam pulse is low energy.

33. A method according to claim 30, wherein said at least one analyte is glucose.

34. A method according to claim 30, wherein said at least one analyte is a plurality of different selected analytes, and wherein said method further comprises, for a single detecting step, identifying the presence of at least one of the different plurality of substances in the subject.

35. A method according to claim 30, wherein said at least one analyte is alcohol.

36. A method according to claim 30, further comprising the step of subtracting a fluorescence spectrum from said vessel Raman spectrum to produce a difference spectrum.

43. An apparatus according to claim 35, wherein said system processor comprises means for subtracting a fluorescence spectrum for said Raman spectrum to produce a difference spectrum.

44. An apparatus according to claim 35, wherein said optical system is configured to illuminate the aqueous humor of the eye.

45. An apparatus according to claim 35, wherein said optical system is configured to illuminate the vitreous humor of the eye.

52. An apparatus according to claim 35, wherein said at least one analyte is cytotoxic and/or a poison.

54. An apparatus according to claim 35, wherein said at least one analyte is from a poisonous plant.

55. An apparatus according to claim 35, wherein said at least one analyte is a drug.

56. An apparatus according to claim 35, wherein said housing is configured and sized to fit onto and rest against the head of the subject during operation.

42. An apparatus for the non-invasive determination of a level of an analyte of interest in a subject, comprising:

a light source for generating an excitation beam;

an optical system operatively associated with said laser for directing said excitation beam into the eye of said subject so that a selected region in the eye is sufficiently illuminated to generate a detectable Raman spectrum thereof;

a detector operatively associated with said optical system and configured to detect a Raman spectrum from the selected region in the eye;

a system processor operably associated with said detector and including computer programs with computer program code for comparing the detected Raman spectrum signal to reference spectrums associated with at least one analyte of interest to identify the presence or absence of at least one analyte of interest in the subject, wherein said system processor comprises computer program code for determining the in vivo brain level of at least one analyte of interest for the subject based on the detected Raman spectrum;

a housing configured to overlie at least one eye of a subject during use and to house said optical system and said detector therein, so that, in position, the optical system is spaced apart from the eye of the subject; and

a focus adjustment unit including a visual display adapted to display visual indicia thereon, mounted in said housing so that, in operation, the visual indicia on said display is visible to a user to allow a user to look at a visual reference presented thereon to allow the visual indicia to be focused so that the excitation beam can be directed to the appropriate location in the eye of the subject and so that said detector can detect the generated Raman spectrum.

53. An apparatus according to claim 42, wherein said the poison comprises at least one of a household chemical, pesticide, herbicide, or petroleum product.

46. An apparatus for the non-invasive determination of a blood level of an analyte of interest in a subject, comprising:

a light source for generating an excitation beam;

an optical system operatively associated with said laser for directing said excitation beam into the eye of said subject so that a selected region in the eye is sufficiently illuminated to generate a detectable Raman spectrum thereof, wherein said optical system is configured to illuminate at least one blood vessel in the conjunctiva of the eye;

a detector operatively associated with said optical system and configured to detect a Raman spectrum from the at least one blood vessel in the conjunctiva of the eye;

a system processor operably associated with said detector and including computer programs with computer program code for comparing the detected Raman spectrum signal to reference spectrums associated with at least one analyte of interest to identify the presence or absence of at least one analyte of interest in the subject;

a housing configured to overlie at least one eye of a subject during use and to house said optical system and said detector therein, so that, in position, the optical system is spaced apart from the eye of the subject; and

a focus adjustment unit including a visual display adapted to display visual indicia thereon, mounted in said housing so that, in operation, the visual indicia on said display is visible to a user to allow a user to look at a visual reference presented thereon to allow the visual indicia to be focused so that the excitation beam can be directed to the appropriate location in the eye of the subject and so that said detector can detect the generated Raman spectrum.