Opto-Fluidic Apparatus for Individual Interrogation of Organisms

Abstract

The present invention relates to an apparatus for the detection of optical emission from individual members of a sample of micro-organisms where the flow is focused in a specific region, comprising an optical device including a light source configured to emit light in a first direction to individual members of the sample and a detection device configured to detect in a field of view optical emission from the individual members emitted in a second direction opposite to the first direction and excited by the light emitted by the light source and a fluid device including a storage reservoir configured to store the sample of micro-organisms, a pumping device, in particular, a circulation pump capable of producing different pulsed pressures to affect the flow configured to circulate the sample of micro organisms in the storage reservoir and wherein a capillary channel is arranged in the storage reservoir configured to allow a flow of individual members of the sample such that one individual member only is present in the field of view of the detection device at a given time of detection of the optical emission.

Claims

1. An apparatus for detection of optical emission from individual members of a sample of micro-organisms, comprising an optical device including a light source configured to emit light in a first direction to individual members of the sample and a detection device configured to detect in a first field of view optical emission from the individual members emitted in a second direction opposite to the first direction and excited by the light emitted by the light source; and a fluid device including a storage tank configured to store the sample of micro-organisms, a pumping device configured to circulate the sample of micro-organisms in the storage tank and wherein a capillary channel is arranged in the storage tank configured to allow a flow of individual members of the sample such that one individual member only is present in the field of view of the detection device at a given time of detection of the optical emission.

2. The apparatus according to claim 1, wherein the pumping device is configured to produce pressure variations in the flow.

3. The apparatus according to claim 1, further comprising a data processing device and algorithm for extracting parameters of the detected optical emission, wherein a concurrent analysis of the extracted parameters allows for adjusting any control parameters of operation of the apparatus and reduction of false positive counts by means of artificial intelligence algorithms.

4. The apparatus according to claim 1, wherein the detection device is additionally configured to detect in a second field of view different from and larger than the first field of view other optical emission emitted in the second direction from a portion of the sample of micro-organisms stored in the storage tank.

5. The apparatus according to claim 4, wherein the optical device further comprises a first lens configured and arranged to focus light emitted by the light source into a particular region of the capillary channel and a second lens configured and arranged to collect light emitted by the micro-organisms of the storage tank different from the capillary channel.

6. The apparatus according to claim 1 wherein the optical device is configured to perform pulse-amplitude-modulation fluorometry or fast-repetition-rate pulse-amplitude-modulation fluorometry.

7. The apparatus according to claim 1, wherein the optical device further comprises a microscope objective arranged to guide the optical emission toward a sensing surface of the detection device.

8. The apparatus according to claim 1, further comprising a control means configured to control operation of the pumping device and wherein a) the geometry and dimensions of the capillary channel are selected and b) the control means is configured to control operation of the pumping device such that that a flow of the individual members through the capillary channel allows for the detection of biological signatures while one single member of the sample only is in the first field of view.

9. The apparatus according to claim 1, further comprising means configured to supply a dosage of visco-elastic polymers to the sample.

10. The apparatus according to claim 1, wherein the micro-organisms comprise chloroplasts, light emission modulated control and further comprising data processing means connected to the detection device and configured to determine an efficiency of a photosystem II of the chloroplasts based on data provided by the detection device.

11. A method of detecting optical emission from individual micro-organisms of a sample of micro-organisms, comprising the steps of: preparing the sample of micro-organisms; guiding a portion of the sample of micro-organisms through a capillary channel such that one single micro-organism only is present at a central portion of the capillary channel at a given time; radiating in a first direction excitation light to only the central portion of the capillary channel at the given time in order to excite optical emission from the one single micro-organism; and detecting the excited optical emission by a detection device, the optical emission being detected in a second direction opposite to the first direction.

12. The method according to claim 11, wherein the excitation light is radiated in a pulse-modulated manner.

13. The method according to claim 11, wherein the guiding of the portion of the sample of micro-organisms through the capillary channel comprises moving the portion of the sample of micro-organisms by means of a pumping device.

14. The method according to claim 13, further comprising storing the sample of micro-organisms in a storage tank, the storage tank comprising the capillary channel and being operatively connected to the pumping device.

15. The method according to claim 12, wherein the preparing of the sample of micro-organisms comprises adding a synthetic polymeric powder to the sample before storing the sample in a storage tank or adding the synthetic polymeric powder to the sample stored in the storage tank.

16. The method according to claim 14, further comprising radiating in the first direction another excitation light to a portion of the storage tank wherein the capillary channel is not arranged in order to excite other optical emission from a portion of the sample of micro-organisms, the portion comprising a plurality of micro-organisms; and detecting the other optical emission excited in the portion of the sample of micro-organisms.

17. The method according to claim 11, wherein a pumping device is operated to generate a pulsed flow of the portion of the sample of micro-organisms through the capillary channel.

18. The method according to claim 11, wherein the critical Womersley number for the flow of the portion of the sample of micro-organisms flowing through the capillary channel is larger than 1.

19. The method according to claim 11, wherein the micro-organisms comprise chloroplasts and further comprising determining the efficiency of the photosystem II of the chloroplasts based on data provided by the detection device.

20. The method according to claim 11, wherein the method steps are performed by an apparatus for detection of optical emission from individual members of the sample of micro-organisms, comprising an optical device including a light source configured to emit light in the first direction to individual members of the sample and a detection device configured to detect in a first field of view optical emission from the individual members emitted in the second direction opposite to the first direction and excited by the light emitted by the light source; and a fluid device including a storage tank configured to store the sample of micro-organisms, a pumping device configured to circulate the sample of micro-organisms in the storage tank and wherein the capillary channel is arranged in the storage tank configured to allow a flow of individual members of the sample such that one individual member only is present in the field of view of the detection device at a given time of detection of the optical emission.

21. The apparatus according to claim 1, wherein the pumping device is a circulation pump.

22. The method according to claim 12, wherein the pulse-modulated manner is a pulse-amplitude-modulated manner.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0033] FIG. 1 schematically illustrates a system for focusing flowing particles and optically interrogating individual members of a sample in accordance with an exemplary embodiment of the disclosure.

[0034] FIG. 2 is a pictorial depiction of the fluid flow velocity profiles for two different pulsed flowing conditions.

[0035] FIG. 3 is a collection of graphs showing experimental values of the detection of different sized polystyrene microspheres particles (histogram of the peak values of detected voltages) in a liquid solution. The bars indicate the frequencies of occurrence.

[0036] FIG. 4 illustrates a method of detecting optical emission from individual members of a sample of micro-organisms according to an embodiment of the present disclosure.

[0037] FIG. 5 schematically illustrates an apparatus comprising an optical device including a light source and a detection device and further comprising a fluid device including a sample storage tank wherein a capillary channel is formed according to an embodiment of the present disclosure.

[0038] FIGS. 6a-6b illustrate an investigation of photosystem II activity by a method according to an embodiment of the present disclosure.

DETAILED DESCRIPTION

[0039] The present disclosure provides an apparatus that allows for focusing micro-organisms for an accurate detection of optical emission (optical signals) from individual members of a sample. FIG. 1 illustrates an embodiment of such an apparatus 100 with a pulsed driving pressure (see 110 in FIG. 1) and an optical illumination/detection apparatus 120. In order to achieve hydrodynamic focusing using the pulsed regime α.sub.c has to be larger than 1 and at least one period of the pulsation has to be propagating inside the channel to largely avoid the generation of turbulence in the channel The period T of the pulses is equal to the linear flow speed divided by the pulsed frequency.

[0040] For example, in the apparatus 100 the channel 102 is about 50 mm in length and a squared cross-section has dimensions of 75 μm. A linear liquid flow 110 with a speed of 0.1 meters per second and a pulsed frequency of 10 Hz may be applied In this case, the Womersley number is 4.85, the critical Womersley is around 2.02 and the period of the propagating pulsation is 10 mm. Furthermore, the entry 104 to the capillary channel can have a conical shape to allow for reducing turbulence transition into the capillary channel

[0041] The applied flow pressure 110 allows for the fluid to have a positive net transport with a baseline pressure level indicated by A. At given pulsed frequency the pressure is rapidly reversed in direction indicated with a negative level B. However, over the entire pressure cycle a positive fluid flow momentum is maintained.

[0042] The generated flow velocity profile 106 is not a paraboloid but rather a depression is formed in the center of the velocity profile 102 in the capillary channel The velocity profile is further explained below. A single stream of micro-organisms is flowing in the center of the capillary channel which allows for individually detecting the micro-organisms by means of an optical technique. In an embodiment, the optical apparatus 120 has means to generate a narrow illumination and detection spot 122.

[0043] Hence, an oscillatory or pulsed flow regime (presumed to be laminar and incompressible) in a capillary channel allows for focusing the micro-organisms in the center of the channel capillary.

[0044] Specifically, in FIG. 2 the physical flowing phenomena are first explained for a non-focusing micro-organisms regime 200, where α.sub.c=1. The axial velocity profile for a liquid with no-slip boundary condition driven by a pressure difference in a channel is indicated by 202. The velocity profile 202 represents a Poiseuille paraboloid. The fluid at all times reaches a steady state before changes happen to the pressure. The velocity profile forces the particles to be located around 0.6 times the half height cross-section of the center of the channel. Then, in FIG. 2 the physical observations are illustrated for a focusing micro-organisms regime 210 where α.sub.c=2.5. The effect of inertia on the velocity profile is clearly observable for α.sub.c>1 by the deviation from the Poiseuille paraboloid 212. The diffusion length λ.sub.d=√{square root over (υ/ω)} (υ denotes the fluid velocity and ω denotes the pulse frequency) giving the width of the quasi-steady annular region is indicated. The fluid does not reach a steady state before the pressure changes and inertia becomes important. The velocity profile deviation from a paraboloid allows for particles being focused around the center of the channel.

[0045] For the purpose of explanation, FIG. 3 shows data measured experimentally and presented in the form of voltage histograms. The horizontal axes (the scaling is not representative) represent the voltage bins of the height of the detected optical pulses. The vertical axes represent the normalized frequencies of occurrence so that the most probable voltage values are clearly identified. Reference sign 310 designates a histogram for 50 μm particle beads in a pulsed flow regime obtained using apparatus 100. Reference sign 320 designates a histogram of 50 μm particle beads in a non-pulsed flow regime. Reference sign 330 designates a histogram of 10 μm particle beads in a pulsed flow regime obtained using apparatus 100. Reference sign 340 designates a histogram of 10 μm particle beads in a non-pulsed flow regime. Reference sign 350 designates a histogram of 2 μm particle beads in a pulsed flow regime obtained using apparatus 100. Reference sign 360 designates a histogram of 2 μm particle beads in a non-pulsed flow regime.

[0046] The apparatus 400 shown in FIG. 4 comprises an optical device 410 and a fluid device 420 for providing a sample S comprising micro-organisms M. The optical device 410 comprises a light source 412, for example, comprising a laser device. In general, wavelengths from the ultraviolet to the infrared range of the light spectrum depending on the excitation wavelength required for each specific application. Output powers of a few mW may be chosen.

[0047] Light emitted in a first direction by the light source 412 is collimated by a lens 414. Further, a detection device 418 is provided in order to detect fluorescence emission excited in the sample S. The detection device 418 is arranged to detect fluorescence emission or scattering emission radiated in a second direction that is opposite to the first direction. The detection device 418 may be a sensitive photodiode (APD, photomultiplier, multi-pixel photon counter), CCD sensor device or an image sensor device, and it may be equipped with an interferential filter or/and an absorption filter.

[0048] The fluid device 420 providing the sample S comprises a capillary channel 424 which is a small tube of a circular or square cross section and may comprise plastic, glass, or fused quartz (for UV light) material and may be designed to allow fluid to flow along the longitudinal axis of the channel The capillary channel 424 may have a relevant hydraulic height of about 100 μm, for example.

[0049] Due to the operation of the circulation pump 426, operating as in apparatus 100, a continuous or pulsed flow of the sample S for testing the same can be provided. It goes without saying that the circulation pump 426 may operate on a portion of an entire sample or on the entire sample.

[0050] The collimation lens 414 of the optical device 410 focuses the light emitted by the light source 412 of the optical device 410 into the capillary channel 424, for example, a central portion of the capillary channel 424. The capillary channel 424 may extend in a direction substantially perpendicularly to the optical axis of the first collimation lens 414. The central portion corresponds to the narrow field of view (for example, with a diameter of a few microns) of the detection device 418 of the optical device 410.

[0051] Moreover, the flow of micro-organisms M through the capillary channel 424 is controlled such that the one micro-organism that is detected at a given time in the field of view.stays long enough (for example, a few hundred μs) in the field of view in order to have the detection device 418 detecting desired biological signatures from the micro-organism (see also description below). Thus, the biological signatures of individual micro-organisms can be detected independently of the concentration of the micro-organisms M in the sample S and a plurality of parameters can be derived from the detection results given by the detection device 418 by means of some data processing means. The fluorescence emission from the micro-organism depends on the actual biological vitality. Examples of the parameters inter alia comprise reaction curves, photosynthetic activity and biological stress levels.

[0052] The optical device 410 of apparatus 400 can be configured to perform PAM fluorometry or FRR PMA fluorometry up to frequency of a few hundreds of MHz, for example. In this configuration, the apparatus 400 is suitable for the investigation of photosynthesis. By studying the fluorescence yield arising from phytoplankton chlorophyll, for example, one can determine many characteristics related to other internal processes of the chlorophyll cells. In the context of PAM fluorometric investigations on photosynthesis of chlorophyll cells (chloroplasts) the detection device 418 can gather information on two levels of light, namely, low and high levels.

[0053] FIG. 5 illustrates an apparatus of detecting optical emission from individual members of a sample of micro-organisms according to an embodiment of the present disclosure. A sample comprising a plurality of micro-organisms is prepared and it is stored in a reservoir 510. Preparation of the sample may partially be performed in that reservoir. Preparation of the sample of micro-organisms may comprise filtering and/or purifying a raw sample, for example, by means of concentration filters and an appropriate chemistry including, for example, acids, flocculators, etc.). Particulate concentrators in the form of disposable ultrafiltration devices with polymer membranes may be used for the concentration and/or purification of biological raw samples in order to prepare the sample.

[0054] A portion of the prepared sample is supplied to a capillary channel 520 formed in the reservoir. A portion of the capillary channel 520 is irradiated by light 530 from a light source (for example, a laser) emitting excitation light towards the capillary channel 520 in a first direction. The excitation light is focused to the portion of the capillary channel 520 by some collimation optics, for example, comprising a lens.

[0055] The excitation light causes optical emission from a micro-organism (thanks to its auto fluorescence behavior or because it has been labelled with a reagent having fluorochromes) being present in an excitation region of the capillary channel 520 that is irradiated by the light source. The capillary channel 520 and the flow through the capillary channel are designed such that only a single one of the plurality of micro-organisms is present in the portion of the capillary channel 520 that is irradiated by the light source at a given time of detection, i.e., the excitation region. At the given time of detection, the excited optical emission (optical signals) of the single micro-organism is detected by a detection device 540. The optical emission may be detected via some collimation optics, for example, a microscope objective. The optical emission detected by the detection device 540 is radiated in a second direction opposite to the first direction of emission of the excitation light. Subsequently, biological parameters can be derived from the detected optical emission.

[0056] As already mentioned, a portion of the prepared sample is supplied to the capillary channel 520 formed in the reservoir 510. The flow through the capillary channel 520 should be controlled such that only a single one of the plurality of micro-organisms is present in the portion of the capillary channel 520 that is irradiated by the light source at a given time of the detection device 540. The flow, in addition, has to be slow enough to allow for the detection of a meaningful biological signature from the micro-organism. To achieve such a flow a circulation pump is properly controlled to operate in order to supply a portion of the sample to the capillary channel 520.

[0057] In certain embodiments, measurements can be performed based on the PAM technique. Investigations on photosynthesis of chlorophyll cells, for example, can be carried out by the described operation. In this context FIGS. 6a-6b show an exemplary time sequence of optical interrogation by PAM measurements for two cases of detected biological signatures corresponding to F.sub.0 being the fluorescence yield in the dark, when all reaction centers are open (mainly photochemistry), and F.sub.m being the fluorescence yield when all reaction centers are closed (mainly fluorescence).

[0058] In operation of the apparatus 500 shown in FIG. 5, modulated excitation light (pump wave) can be radiated (see FIGS. 6a-6b) on a particular single micro-organism (chlorophyll cell) being in a target portion/excitation region (shown in FIGS. 6a-6b) of the capillary channel corresponding to a field of view of the detection device. Pump probing sequences with alternating F.sub.0 and F.sub.m are shown in FIGS. 6a-6b. In the shown example, the total optical probe sequence may have a duration of about 500 μs and the detection time needed for observing a biological signature concurrent with the illumination is also about 500 μs. Furthermore, the probing optical beam diameter and the field of view are about 50 μm. The linear flow speed of the individual micro-organisms through the capillary channel can suitably be adjusted to 0.1 m/s, for example. By using visco-elastic polymers it is made possible to focus, in the fluid, micro-organisms in the center of the capillary channel

[0059] The response/biological signature as detected from the excited fluorescence emission of the micro-organism (chlorophyll cell) is shown in the right column of FIGS. 6a-6b. In the case of a detected living organism a signature indicating a photochemical efficiency Φ.sub.II=1 is observed (FIG. 6a) whereas in the case of a detected dead organism a signature indicating a photochemical efficiency Φ.sub.II=0 (equal levels of F.sub.0 and F.sub.m) is observed (FIG. 6b). The organisms that are present in the sample can be counted individually when they are passing the above-mentioned portion of the capillary channel/field of view of the detection device.

[0060] All previously discussed embodiments are not intended as limitations but serve as examples illustrating features of the disclosure. It is to be understood that some or all of the above-described features can also be combined in different ways.