METHOD AND APPARATUS FOR BACTERIAL ANALYSIS

Abstract

An apparatus (10) comprising: a light source (12); to cast light toward a substrate (20) defining a bacteria binding volume to create an evanescent field (22), the bacteria binding volume being within the evanescent field; a detector (32, 34) arranged to receive light from the bacteria binding volume and output data (36, 37); and a processor (38) arranged to determine vibration of bacteria (26) with the bacteria binding volume in three-dimensions from the data.

Claims

1. An apparatus comprising: a light source arranged to cast light toward a substrate defining a bacteria binding volume to create an evanescent field, the bacteria binding volume being within the evanescent field; a detector arranged to receive light from the bacteria binding volume and output data; and a processor arranged to determine a level of vibration of bacteria within the bacteria binding volume in three-dimensions from the data.

2. The apparatus of claim 1, further comprising a lens arranged to direct the light from the light source toward the substrate.

3. The apparatus of claim 2, wherein the lens has a numerical aperture of at least 1.4.

4. The apparatus of claim 2, wherein the lens is arranged to receive light from the bacteria binding volume.

5. The apparatus of claim 1, wherein the determined vibration is of a single bacterium.

6. The apparatus of claim 1, wherein the apparatus further comprises a coverslip arranged to support the bacteria binding volume, and wherein the evanescent field is created on the surface of the coverslip.

7. The apparatus of claim 1, wherein the detector is arranged to receive light scattered from the bacteria within the bacteria binding volume.

8. The apparatus of claim 7, wherein the apparatus further comprises a second detector, arranged to receive light from the light source having undergone total internal reflection, which is not scattered by the bacteria.

9. The apparatus of claim 6, further comprising a second light source, the second light source being arranged to direct light through the coverslip, the light from the second light source undergoing multiple total internal reflections within the coverslip.

10. The apparatus of claim 9, wherein the coverslip is formed from a dielectric material having a refractive index greater than 1.6.

11. The apparatus of claim 1, further comprising a second light source and an optical waveguide arranged to support the bacteria binding volume, the second light source being arranged to direct light through the optical -waveguide.

12. The apparatus of claim 11, wherein the optical waveguide is tapered at a point within the evanescent field.

13. A method comprising: arranging one or more bacteria on a surface of a coverslip; directing light to be totally internally reflected on the surface of the coverslip and thereby creating an evanescent field on the surface of the coverslip, the bacteria being within the evanescent field; detecting light from the evanescent field; and determining a level of vibration of the bacteria from the detected light.

14. The method of claim 13, wherein the determining comprises determining movement of the bacteria in three dimensions, and/or, wherein the determining comprises determining the level of vibration of individual bacteria.

15. (canceled)

16. The method of claim 13, wherein the light is directed toward the coverslip by a lens and the lens is also arranged to receive light from the coverslip.

17. The method of claim 13, wherein the detected light is light which hasve been scattered by the bacteria, optionally, further comprising detecting light from the light source which has been totally internally reflected and is not scattered by the bacteria.

18. (canceled)

19. The method of claim 13, further comprising directing light from a second light source through the coverslip, the light from the second light source undergoing multiple total internal reflections within the coverslip.

20. The method of claim 13, further comprising directing light from a second light source through an optical waveguide, the light from the second light source undergoing multiple total internal reflections within the optical waveguide.

21. The method of claim 20, wherein the optical waveguide is tapered at a point within the evanescent field.

22. The method of claim 13, further comprising obtaining and storing video footage showing the vibration of the bacteria from the detected light.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Embodiments of the invention will now be described with reference to the accompanying drawings, in which:

[0042] FIG. 1 shows an overall arrangement in accordance with an embodiment of the invention.

[0043] FIG. 2 shows a detailed drawing of a coverslip or waveguide arrangement in accordance with an alternative embodiment of the invention.

[0044] FIG. 3 shows a detailed drawing of a tapered waveguide arrangement in an alternative embodiment of the invention.

DETAILED DESCRIPTION OF EMBODIMENT(S)

[0045] FIG. 1 shows an overall system for observing vibrations of bacteria 26 in order to determine antimicrobial resistance.

[0046] In an antimicrobial detection system 10, there is a light source 12, which is preferably a laser light source, for example having a wavelength of 561 nanometres. The light source 12 produces a light beam 14 which is then refracted by a lens 16. The lens 16 may be a high numerical aperture lens, combined with immersion oil 40 which refracts the light beam 14 such that the light hits the top surface of a coverslip 20 at an angle greater than the critical angle and is totally internally reflected. It will be understood by a person skilled in the art that the critical angle is a function of the refractive indices of the coverslip 20 and the medium above the coverslip 20.

[0047] While the lens 16 having a numerical aperture equal to that of the medium above the lens 16 (approximately 1.33 if the medium is water) would result in total internal reflection and the creation of an evanescent field, the lens 16 having a numerical aperture of 1.4 or greater can produce an evanescent field of appropriate size for the analysis of bacteria.

[0048] The coverslip 20 can be a commercially available thin glass coverslip. The coverslip 20 may also be provided separately from the rest of the system 10 and exchangeable for different coverslips having different bacteria and different thicknesses as required by each user. While the coverslip 20 might not be part of the system 10, the system 10 includes a coverslip support 18. The system 10 can therefore be arranged such that the system 10 will function correctly when the coverslip 20 is positioned on the coverslip support 18, without the coverslip 20 being present at the time of the arranging.

[0049] The light 14 incident on the top surface of the coverslip 20 forms an evanescent field 22 which forms a circle on the coverslip surface with a Gaussian intensity distribution and a maximum decay length of approximately 200 nanometres. Other configurations are possible for the evanescent field 22; for example the field may be elliptical and have a non- Gaussian intensity distribution. The evanescent field 22 interacts with objects on the surface of the coverslip such as bacteria 26.

[0050] Bacteria 26 are elastically bound to the coverslip 20 using antibodies 24 such that the bacteria remain roughly in the centre of the evanescent field 22. The coverslip 20 can be prepared by first being cleaned using ethanol then distilled water and dried in a stream of nitrogen. The surface is then amino-functionalised using ethanolamine hydrochloride, which results in a covering of amino groups covalently bound to the glass to which antibodies 24 can bind. The antibodies 24 used should be those specifically targeted to bind the subject bacteria 26.

[0051] The coverslip 20 may form a substrate and the antibodies 24 on the coverslip 20 may form a bacteria binding volume for binding the bacteria 26 in place.

[0052] In use, light which has been scattered by the bacteria is then transmitted from the coverslip as scattered light 28. The light passes through the lens 16 and into a first detector 32, which may be a quadrant photodiode detector, which then produces data 36 to provide to a computer system 38.

[0053] A quadrant photodetector can monitor both light intensity and the position of peak intensity. Therefore, a quadrant photodetector can be used to determine the distance from the coverslip 22 to the bacteria 26 by the magnitude of the peak intensity of the light detected. The position of the bacteria 26 within the plane of the coverslip 22 can then be determined by the position of the peak intensity detected. Alternatively, an array of photodetectors such as CMOS or CCD may be used in order to determine the position of the bacteria.

[0054] Light which has been reflected by the top surface of the coverslip 20 is transmitted from the coverslip as un-scattered light beam 30, which, optionally or alternatively, also passes through the lens 16 and to a second detector 34. The second detector 34 produces second data 37 which are transferred to a computer system such as the computer system 38. As will be understood to the skilled person, either the first detector 32 or the second detector 34 can be omitted from the system or both may be used as required. The second detector 34 can be a quadrant photodetector identical to that of the first detector 32, or can be an array of photodetectors.

[0055] In order to direct the light from the light source 12 to the coverslip 20 and from the coverslip 20 to the photodetectors 32, 34, the system can comprise mirrors 39.

[0056] The computer system 38 can contain a non-transient computer readable medium and a processor, the non-transient computer readable medium containing instructions for the analysis of the data 36, 37 by the processor in order to provide results to a technician for analysis.

[0057] When using the system 10, the bacteria 26 can be arranged on the coverslip 20 using the antibodies 24 and the vibrations of the bacteria 26 can be recorded and stored by the computer system 38. An antimicrobial can then be added to the surface of the coverslip 20 and the subsequent vibration of the bacteria 26 on the coverslip 20 can be recorded by the computer system 38.

[0058] If the bacteria 26 are arranged in a liquid environment then the antimicrobial can be added to the liquid environment, either partially or totally. If the antimicrobial is added only to a part of the environment, then the bacteria 26 can be separated spatially on the coverslip 20 so that a group of bacteria 26 uncontacted by the antimicrobial and a group of bacteria 26 contacted by the antimicrobial can be analysed at the same time. Alternatively, a group of bacteria 26 uncontacted by the antimicrobial can be present before the addition of the antimicrobial and the same bacteria 26 can be observed after the addition of the antimicrobial.

[0059] If the two groups of bacteria 26 are separated in space (analysed at the same time), then a time saving can be made, whereas if the two groups are separated in time then a greater sample size may be used.

[0060] Once the data 36, 37 has been gathered by the computer system 38 it can be processed. In the processing, the level of activity of the bacteria 26 after the addition of the antimicrobial can be compared to the level of activity of the bacteria 26 before the addition of the antimicrobial.

[0061] If the level of activity of a bacterium has increased significantly, then it can be assumed that the bacterium has become unbound from the antibody 24 and the result can be discarded. If the level of activity has remained approximately constant, then the bacterium can be considered not to have been affected by the antimicrobial, and if the bacterium has significantly reduced in activity then the bacterium can be deemed to have been affected by the antimicrobial Antimicrobials can be bactericides or bacteriostats.

[0062] The determination of whether a change in a level of activity is significant may be determined by standard statistical testing, such as requiring the change to be greater than 5% to be significant and otherwise determining that the level is approximately unchanged.

[0063] The above-analysis can be repeated until a determination can be reached as to whether the bacteria exhibit antimicrobial resistance or not.

[0064] FIG. 2 shows a zoomed in image of a coverslip or waveguide arrangement 100 including a coverslip 116, which is functioning as an elongated waveguide. In order to function as a waveguide, the coverslip 116 can have a refractive index greater than 1.6, and optionally be a sapphire coverslip. The coverslip will then function as a waveguide in the apparatus 10 of FIG. 1, in particular in the presence of immersion oil 40.

[0065] A second light source 112 provides light 114 which enters coverslip 116 and undergoes multiple total internal reflections. When undergoing the multiple total internal reflections, light 114 creates evanescent field 118, which contains bacteria 122 bound to the coverslip by antibodies 120. As above, the activity of the bacteria 122 within the evanescent field 118 results in scattering of light 114. Light leaves the coverslip 116 as a light beam 115, which is affected by the bacteria 122. A light detector 124 receives the light beam 115.

[0066] The second light source 112 can be a laser light source the same as the first light source 12, and the light detector 124 can be a quadrant photodetector or one or more of CMOS OR CCD detectors, which may have the same properties as the first and second detectors 32, 34. Alternatively, a more simple detector measuring only light intensity but not position can be used.

[0067] The coverslip arrangement 100 shown in FIG. 2 can be combined with the system 10 of FIG. 1 or may be used on its own, as required. If the coverslip arrangement 100 of FIG. 2 is combined with the system 10 of FIG. 1, then light scattered by the bacteria 122 can be detected by the light detectors of FIG. 1.

[0068] In use, the evanescent field produced by the light beam 114 in the coverslip 116 of the coverslip arrangement 100 can encompass multiple bacteria such that light is scattered by more than a single bacterium. Hence, the light beam 115 which exits the coverslip 116 and is detected by light detector 124 contains data produced by interactions related to multiple bacteria 122.

[0069] A tapered optical waveguide 216 is shown in FIG. 3. The system 200 of FIG. 3 is similar to the system 100 of FIG. 2. In this system, a light source 212 provides a light beam 214 which undergoes multiple total internal reflections within the optical waveguide 216 and creates an evanescent field 218 containing bacteria 222 bound to the optical fibre 216 by antibodies 220. The vibrations of the bacteria 222 results in changes to the light beam 214, which results in a different light beam 215 exiting the optical fibre 216, which is detected by detector 224.

[0070] The tapered waveguide 216 differs from the coverslip 116 due to the tapered sides of the tapered waveguide 216. The tapering of the tapered waveguide 216 results in the waveguide being thinner in the middle, such that the overall system can be more sensitive to vibrations within the evanescent field 218. Although the tapering is shown on both sides of the tapered waveguide 216, the tapering may take place on only one side or on both sides. If the tapering takes place on only one side, then this side may be either the top side or the bottom side.

[0071] The waveguide 216 should not have any cladding on the outside, as this enables the evanescent field 218 to be exposed such that the bacteria 222 can be arranged within the evanescent field.

[0072] The processing for the coverslip arrangements 100 and 200 may be substantially similar and different from the processing for the system 10. In the processing of the data produced from the coverslip arrangements 100 and 200, each dataset contains details of multiple bacteria and aggregated vibrations. The analysis therefore takes a broader overview, with activity calculated for sets of bacteria rather than for individual bacteria. There is therefore less need for repeat readings with the data produced by the coverslip arrangements 100 and 200.

[0073] Where the word or appears this is to be construed to mean and/or such that items referred to are not necessarily mutually exclusive and may be used in any appropriate combination.

[0074] Although the invention has been described above with reference to one or more preferred embodiments, it will be appreciated that various changes or modifications may be made without departing from the scope of the invention as defined in the appended claims.