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

• US 7,068,365 B2
• Filed: 05/17/2005
• Issued: 06/27/2006
• Est. Priority Date: 05/05/1997
• Status: Expired due to Term

• Current Assignee: ChemoMetec A/S
• Original Assignee: ChemoMetec A/S
• Inventors: Jeppesen, Jesper Myron, Glensbjerg, Martin, Arnvidarson, Borkur, Hansen, Frans
• Primary Examiner(s): Toatley, Jr., Gregory J.
• Assistant Examiner(s): MERLINO, AMANDA H
• Application Number:
  US11/130,717
• Publication Number:
  US 20050225766A1
• Time in Patent Office: 406 Days
• Field of Search: 356 39- 43, 356436-440
• US Class Current: 356/246
• 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

providing a volume of between 0.01 μ

l and 20 μ

l of a liquid sample comprising the liquid analyte material,

exposing, onto an array of active detection elements, an at least one-dimensional spatial image of said volume, 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, and wherein the spatial image exposed onto the array of active detection elements is subject to such a linear enlargement that 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 smaller than 10;

1,

the sample being at stand still daring the exposure,

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

3. A method according to claim 1, wherein at least a major part of the electromagnetic radiation emitted from the sample during exposure originates from or is caused by electromagnetic radiation supplied to the sample from a light source, at least a major part of the radiation from the light source having a direction which is transverse to the wall of the sample compartment or a plane defined by the domain.

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

5. A method according to claim 1, wherein the array of detection elements is arranged in such a way tat the a series of detection elements from a substantially straight line.

6. A method according to claim 5, 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.

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

8. A method according to claim 1, wherein the ratio is at the most 6:

1.

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

1 and 1;

10.

10. A method according to claim 8, wherein the particles the parameter or parameters of which is/are to be assessed are eta size between 3 μ

m and 100 μ

m and the ratio is in the range between 3;

1 and 1;

100.

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

12. A method according to claim 1, wherein the interior of the sample compartment has an average thickness of between 20 μ

m and 2000 μ

m.

13. A 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.

14. A 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 ⅓

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.

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

m to 100 μ

m, ant 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.

16. A 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.

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

18. A 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, raman scatter.

19. A 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 to the isolated particles, the molecules causing, or enhancing the emission of electromagnetic radiation such as chemiluminescence or photoluminescence, such as fluorescence or photoluminescence, such as fluorescence or phosphorescence, when excited with radiation which is substantially higher in energy than the emitted photoluminescence.

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

21. A method according to claim 20, wherein the exposure is performed as a single exposure.

22. A method according to claim 1, wherein compression of information of the intensities representing distinct objects scattered over an area, and object being represented by a variation in the intensity information

said information existing in the form of varying degrees of measurable intensity of a physical property distributed over a confined area divided into sub-areas, each of which sub-areas having assigned thereto an index uniquely identifying the sub-area, the method comprising

determining the intensity of the physical property,

a) defining a sub-area of interest situated in a group of sub-areas comprising of at least 2×

2 sub-areas situated adjacent to each other,

b) evaluating in said sub-area of interest at least one directional derivative(s) of the measurable intensity in the sub-area of interest with respect to predetermined geometrical directions(s) in the plane of the confined area, the directional derivative(s) is (are) based on measurable intensities in sub-areas situated adjacent to or in proximity of the group of sub-area,

c) based on the evaluation of the at least one directional derivative an attribute is assigned to the value assigned to said sub-area of interest;

the attribute represent an adjusted measurable intensity and/or information(s) related to a predetermined strategy for adjustment of the measurable intensity in the sub-area of interest or sub-areas situated adjacent to or in proximity to the sub-area of interest,

d) repeating the step a)–

c) for substantially all sub-areas of the confined area.

23. A 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 sealing 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.

24. A method according to claim 23, where the measured intensities of the detection elements have been adjusted prior to counting, the adjustment comprising the steps of:

a) 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 an biological particle having an average extension,

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

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

d) adjusting the intensity of the detection element in the center 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 center of the range by considering intensifies 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.

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

26. A method according to claim 25, wherein the sample compartment comprises one or more compartments, where chemical or physical components can be stored such tat 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.

27. A 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 neighboring detection elements.

28. 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 defining a volume of between 0.01 μ

l and 20 μ

l of a liquid sample,

an array of active detection elements, onto witch an at lease 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 such tat 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 smaller than 10;

1,

the system being adapted to keep the sample in the sample compartment at stand still dining the exposure,

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.

29. A system according to claim 28, wherein at least a major parrot the electromagnetic radiation emitted from the sample during exposure originates from or is caused by electromagnetic radiation supplied to the sample from a light source, at least a major part of the radiation from the light source having a direction which is transverse to the wall of the sample compartment or a plane defined by the domain.

30. A system according to claim 28, comprising of one or more valves which can control the flow of sample or any other component.

31. A system according to claim 28, wherein the array of detection elements is arranged in such a way that the a series of detection elements form a substantially straight lime.

32. A system according to claim 31, 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.

33. A system according to claim 28, wherein the ratio is at the most 6:

1.

34. A system according to claim 28, wherein the ratio is in the range between 10:

1 and 1;

10.

35. A system according to claim 28, wherein the ratio is in the range between 3:

1 and 1;

100.

36. A system according to claim 28, wherein the interior of the sample compartment has an average thickness of between 20 μ

m and 2000 μ

m.

37. A system according to claim 28, 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.

38. The system according to claim 28, comprising a pump, said pump being situated either upstream to the sample compartment or downstream to the sample compartment.

39. A system according to claim 28, comprising means to assess the number of the biological particles per volume of the liquid analyte material.

40. A system according to claim 28, comprising means to assess the size and/or shape of the biological particles in the liquid analyte material.

41. A system according to claim 28, wherein the duration of the exposure is in the range from 100 milliseconds to 5 seconds.

42. A system according to claim 28, 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.

43. A system according to claim 28, where said array of detection elements is a CMOS image sensor, preferably a CMOS image sensor with on-chip integrated signal condition and/or signal processing, more preferably a CMOS image sensor with on-chip integrated computing means capable of performing image processing.

44. A system according to claim 28, where the transmission of electromagnetic radiation onto the sample is accomplished by the use of illuminating means.

45. A system according to claim 44, where the electromagnetic radiation transmitted onto the sample is focused by a focusing mean, the focusing mean having the effect of substantially increasing the intensity of said electromagnetic radiation in or at said sample.

46. A system according to claim 28, 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 neighboring detection elements.

47. A system according to claim 28, 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 trough the chemical container and thus allowing the mixing of the chemical components with the sample.

48. A system according to claim 28, further comprising a source of excitation light and an appropriate set of optical filters.