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

• US 6,919,960 B2
• Filed: 12/04/2003
• Issued: 07/19/2005
• Est. Priority Date: 05/05/1997
• Status: Expired due to Term

• Current Assignee: ChemoMetec A/S
• Original Assignee: ChemoMetec A/S
• Inventors: Hansen, Frans Ejner Rvan, Jeppesen, Jesper Myron, Glensbjerg, Martin, Arnvidarson, Borkur
• Primary Examiner(s): Toatley, Jr., Gregory J.
• Assistant Examiner(s): MERLINO, AMANDA H
• Application Number:
  US10/727,832
• Publication Number:
  US 20040246483A1
• Time in Patent Office: 593 Days
• Field of Search: 356436-440, 356 39- 43
• 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 bacteria in a liquid analyte material, comprising

applying a volume of a liquid sample representing the analyte material, or bacteria 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,

exposing, onto an array of active detection elements, an at least one-dimensional spatial representation of electromagnetic signals having passed from the domain, the representation being one which is detectable as a 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 bacteria are identified as distinct from representations of electromagnetic signals from background signals, and wherein the spatial representation exposed onto the array of 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,

processing the intensities detected by the detection elements in such a manner that signals from the bacteria 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. A method according to claim 1, wherein the electromagnetic signals comprise chemoluminescence, photoluminscence, fluorescence or phosphorescence.

3. A method according to claim 1, wherein the sample compartment has 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.

4. A method according to claim 1, wherein the image of the electromagnetic signals is a one-dimensional image.

5. A method according to claim 1, wherein the image of the electromagnetic signals is a two-dimensional image.

6. A 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.

7. A method according to claim 6, 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 lies, the series forming a rectangle.

8. A method according to claim 1, wherein the exposure of the image of electromagnetic signals 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.

9. A method according to claim 8, wherein the focusing means is a lens consisting of one or several elements.

10. A method according to claim 1, wherein the individual bacteria the parameter or parameters of which is/are to be assessed are imaged on at the most 5 detection elements.

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

m and 200 μ

m.

12. 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 nm by 1 mm and 10 mm by 10 mm.

13. A method according to claim 1, wherein the volume of the liquid sample from which electromagnetic radiation is detected on the array is in the range between 0.01 μ

l and 20 μ

l.

14. A method according to claim 13, wherein the volume of the liquid sample from which electromagnetic radiation is detected on the array is in the range between 0.01 μ

l and 1 μ

l.

15. A method according to claim 1, wherein the sample in the sample compartment is at stand still during the exposure.

16. A method according to claim 1, wherein the sample in the sample compartment is moved through the sample compartment during the exposure, and wherein the exposure is performed over a sufficiently short period of time so substantially obtain stand still condition during the exposure.

17. 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 compartment.

18. A method according to claim 1, wherein the parameter to be assessed is the number of the bacteria per volume of the liquid analyte material.

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

20. A method according to claim 18, wherein the size of the volume of the liquid sample is sufficiently large to allow identification therein of at least 100 of the bacteria.

21. A method according to claim 1, comprising

applying a volume of between 0.01 μ

l and 20 μ

l of a liquid sample representing the liquid analyte material, or bacteria isolated from a volume of a liquid sample representing the liquid analyte material, to the sample compartment the sample in the sample compartment being at stand still during the exposure, and in the case where 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, then 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 compartment, and

the individual bacteria the parameter or parameters of which is/are to be assessed are imaged on at the most 25 detection elements of the array of detection elements.

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

23. A method according to claim 1, wherein bacteria isolated from a liquid sample representing the analyte are applied to the sample compartment or arranged in the sample compartment, the bacteria being retained on a bacterium retaining means selected from means chemically binding the bacteria, means capable of electronically or magnetically retaining the bacteria, and filtering means.

24. 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 bacteria, such molecules being added to the sample or the isolated bacteria 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.

25. A method according to claim 24, wherein an effective amount of one or more nucleic acid dyes and/or one or more potentiometric membrane dyes is added.

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

27. A method according to claim 26, wherein the duration of the exposure is in the range of 0.5 to 3 seconds.

28. A method according to claim 26, wherein the exposure is performed as a single exposure.

29. A method according to claim 1, wherein compression of information of the intensities representing distinct objects scattered over an area, an 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 direction(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 at 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 coined area.

30. 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 scaling value being directly related to the number of detection elements representing a signal from a bacterium,

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

31. A method according to claim 30, 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 coordinate 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 bacterium 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 centre of the range,

d) 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.

32. A method according to claim 1, wherein the sample compartment from which electromagnetic signals from the sample in the sample compartment can pass to the exterior is adapted to allow the assessment of substantially only one sample of liquid analyte material.

33. A method according to claim 32, wherein the sample compartment is connected with a reagent container, the reagent container containing one or several reagents component(s).

34. A method according to claim 33, wherein the reagent container contains one or several reagent component(s) in an amount substantially adequate for substantially only one assessment.