Method and a system for determination of particles in a liquid sample

• US 8,363,221 B2
• Filed: 04/03/2008
• Issued: 01/29/2013
• Est. Priority Date: 05/05/1997
• Status: Expired due to Fees

• Current Assignee: ChemoMetec A/S
• Original Assignee: ChemoMetec A/S
• Inventors: Hansen, Frans Ejner Ravn, Glensbjerg, Martin, Arnvidarson, Borkur, Jeppesen, Jesper Myron
• Primary Examiner(s): Toatley, Gregory J
• Assistant Examiner(s): MERLINO, AMANDA H
• Application Number:
  US12/062,298
• Publication Number:
  US 20080246946A1
• Time in Patent Office: 1,762 Days
• Field of Search: None
• US Class Current: 356/436
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/025 Methods for single or grouped particles
• CPC Class Codes: G01N 2015/1452 Adjustment of focus; Alignment
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2021/5957 using an image detector type detector, e.g. CCD
• CPC Class Codes: G01N 2021/6491 Measuring fluorescence and transmission; Correcting inner filter effect
• CPC Class Codes: G01N 21/31 Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry G01N21/72 takes precedence
• CPC Class Codes: G01N 21/59 Transmissivity G01N21/25 takes precedence
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 33/04 Dairy products

Abstract

The present invention relates to a method for the assessment of quantity and quality parameters of biological particles in a liquid analyte material. The method comprises applying a volume of a liquid sample to an exposing domain from which exposing domain electromagnetic signals from the sample in the domain can pass to the exterior, and exposing, onto an array of active detection elements such as CCD-elements, a spatial representation of electromagnetic signals having passed from the domain, the representation being detectable as an intensity by individual active detection elements, under conditions permitting processing of the intensities detected by the array of detection elements during the exposure in such a manner that representations of electromagnetic signals from the biological particles are identified as distinct from representations of electromagnetic signals from background signals. The size of the volume of the liquid sample is sufficiently large to permit the assessment of the quantity and quality parameters to fulfill a predetermined requirement to the statistical quality of the assessment based on substantially one exposure.

Claims

1. A method for the assessment of at least one quantity parameter and/or at least one quality parameter of biological particles in a liquid analyte material, comprising

applying a volume of a liquid sample representing the analyte material or particles isolated from a volume of liquid sample representing the analyte material, to an exposing domain from which exposing domain electromagnetic signals from the sample in the domain can pass to the exterior,

transmitting, onto the sample, light from a light emitting diode,

exposing, onto an array of active detection elements, an at least one-dimensional spatial representation of fluorescent signals having passed from the domain, the representation being one which is detectable as an intensity by individual active detection elements, under conditions which will permit processing of the intensities detected by the array of detection elements during the exposure in such a manner that representations of electromagnetic signals from the particles are identified as distinct from representations of electromagnetic signals from background signals,

processing the intensities detected by the detection elements in such a manner that signals from the particles are identified as distinct from background signals,

and correlating the results of the processing to the at least one quantity parameter and/or the at least one quality parameter of the liquid analyte material.

2. The method according to claim 1, wherein the volume is provided in a sample compartment having a wall part defining an exposing area, the wall part allowing electromagnetic signals from the sample in the compartment to pass through the wall and to be exposed to the exterior.

3. The method according to claim 1, wherein, at least a major part of the electromagnetic radiation from the light source has a direction which is transverse to the wall of the sample compartment or a plane defined by the domain.

4. The method according to claim 1, wherein the signal which is detected by the detecting elements originates from one or several types of molecules of types which bind to, are retained within, or interact with, the biological particles, such molecules being added to the sample or the isolated particles before or during exposure, the molecules being molecules giving rise to one or several of the following phenomena:

attenuation of electromagnetic radiation, photoluminescence when illuminated with electromagnetic radiation, scatter of electromagnetic radiation, or raman scatter.

5. The method according to claim 1, wherein the signal which is detected by the detecting elements originates from one or several types of molecules of types which bind to, are retained within, or interact with, the biological particles, such molecules being added to the sample or the isolated particles, the molecules causing, or enhancing the emission of electromagnetic radiation, when excited with radiation which is substantially higher in energy than the emitted photoluminescence.

6. The method according to claim 5, wherein an effective amount of one or more nucleic acid dyes and/or one or more potentiometric membrane dyes is added.

7. The method according to claim 2, wherein the interior of the sample compartment has an average thickness of between 20 μ

m and 2000 μ

m.

8. The method according to claim 7, wherein the interior of the sample compartment has an average thickness of between 20 μ

m and 1000 μ

m.

9. The method according to claim 8, wherein the interior of the sample compartment has an average thickness of between 20 μ

m and 200 μ

m.

10. The method according to claim 1, wherein the ratio of the image of a linear dimension on the array of detection elements to the original linear dimension in the exposing domain is at the most 10:

1.

11. The method according to claim 10, wherein the ratio is at the most 6:

1.

12. The method according to claim 11, wherein the ratio is smaller than 4:

1.

13. The method according to claim 1, wherein the ratio is in the range between 10:

1 and 1;

10.

14. The method according to claim 13, wherein the ratio is in the range between 6:

1 and 2;

1.

15. The method according to claim 10, wherein the particles the parameter or parameters of which is/are to be assessed are of a size between 3 μ

m and 100 μ

m, and the ratio of the image of a linear dimension on the array of detection elements to the original linear dimension in the exposing domain is in the range between 3;

1 and 1;

100.

16. The method according to claim 10, wherein the ratio is in the range between 2:

1 and 1;

100.

17. The method according to claim 16, wherein the ratio is in the range between 2:

1 and 1;

2.

18. The method according to claim 17, wherein the ratio is in the range between 1.4:

1 and 1;

100.

19. The method according to claim 18, wherein the ratio is in the range between 1:

1 and 1;

100.

20. The method according to claim 1, wherein the light source emits electromagnetic radiation of substantially the same wavelength.

21. The method according to claim 1, wherein the light sources are present in numbers of at least 2 or more.

22. The method according to claim 1, wherein at least two light sources have substantially different radiation properties in at least one waveband, the light sources being operated in such a way that:

all transmit substantially simultaneously or at least one source transmits while at least one source is not transmitting, or at least one source is transmitting at a time.

23. The method according to claim 1, wherein the spatial image of the volume is a two-dimensional image.

24. The method according to claim 1, wherein the array of detection elements is arranged in such a way that a series of detection elements form a substantially straight line.

25. The method according to claim 24, wherein the array of detection elements is arranged in two directions in such a way that the detection elements form a series of substantially parallel straight lines, the series forming a rectangle.

26. The method according to claim 1, wherein the exposure of the spatial image of the volume onto the array of detection elements is performed by focusing an image of electromagnetic signals from at least a part of the exposing domain onto the array of detection elements by means of a focusing means.

27. The method according to claim 26, wherein the focusing means is a lens consisting of one or several elements.

28. The method according to claim 1, wherein the individual particles the parameter or parameters of which is/are to be assessed are imaged on at the most 25 detection elements.

29. The method according to claim 28, wherein the individual particles the parameter or parameters of which is/are to be assessed are imaged on at the most 9 detection elements.

30. The method according to claim 29, wherein the individual particles the parameter or parameters of which is/are to be assessed are imaged on at the most 5 detection elements.

31. The method according to claim 1, wherein the sample compartment has dimensions, in a direction substantially parallel to the array of detection elements, in the range between 1 mm by 1 mm and 10 mm by 10 mm.

32. The method according to claim 1, wherein the particles the parameter or parameters of which is/are to be assessed are of a size of between 1/3 μ

m to 3 μ

m, and the volume of the liquid sample from which electromagnetic radiation is exposed onto the array is in the range between 0.01 μ

l and 1 μ

l.

33. The method according to claim 1, wherein the particles the parameter or parameters of which is/are to be assessed are of a size of between 3 μ

m to 100 μ

m, and the volume of the liquid sample from which electromagnetic radiation is exposed onto the array is in the range between 0.04 μ

l and 4 μ

l.

34. The method according to claim 1, wherein the parameter to be assessed is the number of the biological particles per volume of the liquid analyte material.

35. The method according to claim 1, wherein the parameter(s) to be assessed is the size and/or shape of the biological particles in the liquid analyte material.

36. The method according to claim 34, wherein the size of the volume of the liquid sample is sufficiently large to allow identification therein of at least two of the biological particles.

37. The method according to claim 36, wherein the size of the volume of the liquid sample is sufficiently large to allow identification therein of at least four of the biological particles.

38. The method according to claim 37, wherein the size of the volume of the liquid sample is sufficiently large to allow identification therein of at least 10 of the biological particles.

39. The method according to claim 38, wherein the size of the volume of the liquid sample is sufficiently large to allow identification therein of at least 50 of the biological particles.

40. The method according to claim 39, wherein the size of the volume of the liquid sample is sufficiently large to allow identification therein of at least 100 of the biological particles.

41. The method according to claim 40, wherein the size of the volume of the liquid sample is sufficiently large to allow identification therein of at least 1000 of the biological particles.

42. The method according to claim 1, wherein the parameter to be assessed is the presence or non-presence of a particular type of particles in the liquid analyte material.

43. The method according to claim 1, wherein the duration of the exposure is in the range from 100 milliseconds to 5 seconds.

44. The method according to claim 43, wherein the duration of the exposure is in the range of 0.5 to 3 seconds.

45. The method according to claim 43, wherein the exposure is performed as a single exposure.

46. The method according to claim 1, wherein the correlation comprises:

identifying and counting substantially all detection elements having intensities which are distinct from background signals,

adjusting the result of the counting by a predefined scaling value,

the scaling value being directly related to the number of detection elements representing a signal from a biological particle,

the result of the scaling being correlated to the number of particles represented exposure.

47. The method according to claim 46, where the measured intensities of the detection elements have been adjusted prior to counting, the adjustment comprising the steps of:

defining a range of a predetermined size in a co-ordinate system representing the intensity values of the detection elements, the size of the range being determined such that it is bigger than the representation of a biological particle having an average extension,

choosing a first detection element, the first detection element being one of which the intensity is subject to an adjustment,

positioning the range such that the detection element of which the intensity is to be adjusted is substantially in the centre of the range,

adjusting the intensity of the detection element in the centre of the range based on the result of an investigation of at least one gradient describing the variation of the signal intensities inside the range and around the centre of the range by considering intensities of detection elements describing the gradient, and repeating the step b) through c) until a predetermined number of detection elements has been adjusted a predetermined number of times.

48. The method according to claim 1, wherein the sample compartment is a removable sample compartment, which can be readily removed from the measuring system.

49. The method according to claim 48, wherein the sample compartment comprises one or more compartments, where chemical or physical components can be stored such that the chemical or physical components can be added to any sample present in the sample compartment one at a time or more than one at a time.

50. The method according to claim 1, wherein the identification of a particle present in the sample is done by comparing the level of signal from each detection element with a predefined level, or to a level which is estimated on the basis of neighbouring detection elements.

51. A system for the assessment of at least one quantity parameter and/or at least one quality parameter of biological particles in a liquid analyte material, comprising a sample compartment adapted to accommodate a volume of liquid sample of the liquid analyte material, a light emitting diode arranged to transmit light onto the sample,

an array of active detection elements onto which an at least one-dimensional spatial image of said volume is exposed, the image being one which is detectable as an intensity by individual active detection elements, under conditions which will permit processing of the intensities detected by the array of detection elements during the exposure in such a manner that images of electromagnetic signals from the biological particles are identified as distinct from images of electromagnetic signals from background signals, a lens consisting of one or several elements for focusing an image of electromagnetic signals from at least part of the exposing domain onto the array of detection elements,

wherein the system is adapted for receiving photoluminescence by having one or more means for separating electromagnetic radiation into substantially one or more wavelength components or wavebands,

a computer unit for processing the intensities detected by the detection elements in such a manner that signals from the biological particles are identified as distinct from background signals, and for correlating the results of the processing to the at least one quantity parameter and/or the at least one quality parameter of the liquid analyte material.

52. The system according to claim 51, wherein at least a major part of the electromagnetic radiation from the light source has a direction which is transverse to the wall of the sample compartment or a plane defined by the domain.

53. The system according to claim 51, wherein the photoluminescence is fluorescence or phosphorescence.

54. The system according to claim 51, wherein the means for separating electromagnetic radiation into substantially one or more wavelength components or wavebands is brought about by one or several of the following but not limited to:

interference filters, colored filters, an optical grating, a prism, or optically active crystals.

55. The system according to claim 51, wherein the system is adapted for receiving fluorescent signals by having at least 2 or more of the following but not limited to:

interference filters, colored filters, an optical grating, a prism, optically active crystals.

56. The system according to claim 51, wherein the electromagnetic radiation which is transmitted onto, or emitted from or transmitted through the sample is intensity modulated.

57. The system according to claim 51, wherein the electromagnetic radiation which is transmitted onto, or emitted from or transmitted through the sample is modulated by optically active crystals or interferometry.

58. The system according to claim 51, wherein the interior of the sample compartment has an average thickness of between 20 μ

m and 2000 μ

m.

59. The system according to claim 58, wherein the interior of the sample compartment has an average thickness of between 20 μ

m and 1000 μ

m.

60. The system according to claim 59, wherein the interior of the sample compartment has an average thickness of between 20 μ

m and 200 μ

m.

61. The system according to claim 51, wherein the ratio of the image of a linear dimension on the array of detection elements to the original linear dimension in the exposing domain is at the most 10:

1.

62. The system according to claim 61, wherein the ratio is at the most 6:

1.

63. The system according to claim 62, wherein the ratio is smaller than 4.1.

64. The system according to claim 51, wherein the ratio is in the range between 10:

1 and 1;

10.

65. The system according to claim 64, wherein the ratio is in the range between 6:

1 and 2;

1.

66. The system according to claim 51, wherein the ratio is in the range between 3:

1 and 1;

100.

67. The system according to claim 66, wherein the ratio is in the range between 2:

1 and 1;

100.

68. The system according to claim 67, wherein the ratio is in the range between 2:

1 and 1;

2.

69. The system according to claim 68, wherein the ratio is in the range between 1.4:

1 and 1;

100.

70. The system according to claim 69, wherein the ratio is in the range between 1:

1 and 1;

100.

71. The system according to claim 51, wherein the light source emits electromagnetic radiation of substantially the same wavelength.

72. The system according to claim 51, wherein the light sources are present in numbers of at least 2 or more.

73. The system according to claim 51, wherein at least two light sources have substantially different radiation properties in at least one waveband, the light sources being operated in such a way that:

all transmit substantially simultaneously or at least one source transmits while at least one source is not transmitting, or at least one source is transmitting at a time.

74. The system according to claim 51, comprising one or more valves which can control the flow of sample or any other component.

75. The system according to claim 51, adapted to keep the sample in the sample compartment at stand still during the exposure.

76. The system according to claim 51, wherein the array of detection elements is arranged in such a way that a series of detection elements form a substantially straight line.

77. The system according to claim 76, wherein the array of detection elements is arranged in two directions in such a way that the detection elements form a series of substantially parallel straight lines, the series forming a rectangle.

78. The system according to claim 51, wherein the sample compartment has dimensions, in a direction substantially parallel to the array of detection elements, in the range between 1 mm by 1 mm and 10 mm by 10 mm.

79. The system according to claim 51, wherein the sample compartment is a removable sample compartment, which can be readily removed from the measuring system.

80. The method according to claim 79, wherein the sample compartment comprises one or more compartments, where chemical or physical components can be stored such that the chemical or physical components can be added to any sample present in the sample compartment one at a time or more than one at a time.

81. The system according to claim 51, wherein the volume of the liquid sample from which electromagnetic radiation is exposed onto the array is in the range between 0.01 μ

l and 1 μ

l.

82. The system according to claim 51, wherein the volume of the liquid sample from which electromagnetic radiation is exposed onto the array is in the range between 0.04 μ

l and 4 μ

l.

83. The system according to claim 51, comprising a pump, said pump being situated either upstream to the sample compartment or downstream to the sample compartment.

84. The system according to claim 83, wherein the pump is one or several of the following:

peristaltic pump piston pump, membrane pump, centrifugal pump, hypodermic syringe.

85. The system according to claim 51, comprising means to assess the number of the biological particles per volume of the liquid analyte material.

86. The system according to claim 51, comprising means to assess the size and/or shape of the biological particles in the liquid analyte material.

87. The system according to claim 51, wherein the parameter to be assessed is the presence or non-presence of a particular type of particles in the liquid analyte material.

88. The system according to claim 51, wherein the duration of the exposure is in the range from 100 milliseconds to 5 seconds.

89. The system according to claim 88, wherein the duration of the exposure is in the range of 0.5 to 3 seconds.

90. The system according to claim 88, wherein the exposure is performed as a single exposure.

91. The system according to claim 51, where said array of detection elements is one or several of the following types:

full frame CCD, frame transfer CCD, interline transfer CCD, line scan CCD.

92. The system according to claim 51, where said array of detection elements is a CMOS image sensor.

93. The system according to claim 51, where the electromagnetic radiation transmitted onto the sample is focused by a focusing mean (102), the focusing mean (102) having the effect of substantially increasing the intensity of said electromagnetic radiation in or at said sample.

94. The system according to claim 51, where the electromagnetic radiation transmitted onto the sample is accomplished by two or more illuminating means, at least two of the illuminating means having substantially different radiation properties in at least one waveband, the illuminating means being operated in such a way that all transmit substantially simultaneously.

95. The system according to claim 51, wherein the identification of a particle present in the sample is done by comparing the level of signal from each detection element with a predefined level or by comparing to a level, which is estimated on the basis of the signals from neighbouring detection elements.

96. The system according to claim 51, further comprising a container for one or more chemical components, the container being connected to a flow system where the sample flows, at least a portion of the sample flowing through the chemical container and thus allowing the mixing of the chemical components with the sample.

97. The system according to claim 51, wherein the light source is a source of excitation light and the system further comprises an appropriate set of optical filters.