WO2010149770A1

WO2010149770A1 - Microscopic detection of objects in a fluid flow

Info

Publication number: WO2010149770A1
Application number: PCT/EP2010/059065
Authority: WO
Inventors: Christian Dietrich
Original assignee: Carl Zeiss Ag
Priority date: 2009-06-26
Filing date: 2010-06-25
Publication date: 2010-12-29
Also published as: DE102009030688A1

Abstract

A microscope for detecting objects in a fluid flow is described, which microscope has a channel (3) through which fluid containing the objects to be detected flows, and an optical imaging device (1) which comprises a lens (7) having a focal region (14) and images objects located in the focal region, wherein the focal region lies around a focal plane (14) which is located in the channel (3), wherein the focal plane (14) and the channel (3) are oriented with respect to one another in such a manner that the fluid flows towards the focal plane (14).

Description

Microscopic detection of objects in a fluid stream

The invention relates to a microscope for the detection of objects in a fluid flow, comprising: a channel through which fluid containing the objects to be detected flows and an optical imaging device comprising a lens having a focal region and imaging objects located in the focal region, wherein the focal region lies around a focal plane lying in the channel.

The invention further relates to a method for the microscopic detection of objects in a fluid flow, wherein the fluid flow is guided in a channel, an optical imaging device is used which comprises a lens having a focal region, wherein the focal region lies around a focal plane, which in the channel, and objects located in the focal area are optically imaged.

Microscopic detection of objects in a fluid stream is required in many applications. The objects may be, for example, molecules, particles or cells. Typical applications are cytometry, particle counting or protein extraction.

The prior art knows a variety of microscopes that are suitable or adapted for such applications. For example, reference may be made to US 6432630 or US 7214298, in which a microfluidic system is combined with an imaging microscope that optically images objects that flow into a fluid stream that is passed through the microfluidic system. A disadvantage of these optical images is that the channel wall, which lies in the optical imaging range, leads to aberrations. The fluid flow-related movement of the objects also causes unrecoverable image blurring. This movement is also due to the fact that the channel must be significantly larger than the objects in order to avoid blocking the channel. The invention is therefore the object of a microscope for the detection of objects in a fluid stream or a method for the microscopic detection of objects in a fluid flow in such a way that an improved image is achieved.

The invention solves this problem with a microscope of the type mentioned, in which the focal plane and the channel are aligned with each other so that the fluid flows to the focal plane.

The object is equally achieved by a method of the type mentioned, in which the focal plane is aligned with the channel so that the fluid flow to the focal plane or flows away.

The inventors recognized that significant problems of the prior art are caused by the fact that the flow direction is substantially along the focal plane. Now that the focal plane is aligned substantially perpendicular to the flow direction of the inflowing fluid, it is ensured that each object passes through the focal area and in particular the focal plane of the optical imaging device or the lens with one hundred percent certainty. At the same time, a parallel and simultaneous detection of several objects that simultaneously pass through the focal plane is possible. Movements of the objects through the fluid flow now proceed substantially along the optical axis of the objective. Since the resolution is lower in this direction, such movements are much weaker than when the movement is transverse to the optical axis, as is the case in the prior art. Thus, negative influences are significantly reduced by the fluid flow.

The inflow of the fluid to the focal plane causes the fluid to flow substantially along the optical axis of the objective through the focal region.

The orientation of the focal plane to the flow direction of the inflowing fluid automatically requires that the optical axis of the objective is substantially parallel to the direction of flow. The terms "vertical" and "parallel" are not to be understood in the strict geometric sense. Rather, in the context of this invention, the focal plane is then substantially perpendicular to the direction of flow of the influent fluid or the optical axis of the lens then parallel to the flow direction of the influent fluid when the image area covered by the lens covers the cross section of the incoming fluid stream, and the image area microscopically a detector can be imaged. Depending on the size of the image area which the imaging device images, an inflow of the fluid onto the focal plane at an oblique angle to the focal plane is thus also essentially perpendicular to the focal plane or essentially along the optical axis in the sense of this description. The advantages according to the invention and in particular the complete detection of the objects entrained in the fluid stream are or is achieved in that the focal region covers the cross-section of the inflowing fluid.

Due to the alignment according to the invention of the focal plane or focal region to the flow direction of the inflowing fluid and thus automatically to the channel through which the fluid flow passes, disturbances due to the channel walls are reduced since they are now oriented along the comparatively higher resolution axes of the optical imaging. They can therefore be easily recognized in the figure and, unlike the prior art, are not a source of artifacts.

Under a microscope is here any suitable magnifying imaging device understood, in particular a so-called reader.

In many applications, the fluid to be examined is supplied through a tube that realizes a feed channel. The optical arrangement of the lens and thus the focal plane to the channel is particularly simple if the channel comprises a feed channel through which the fluid flows, wherein the feed channel opens onto a cover glass over which the fluid runs laterally into at least one channel. Based on the fluid flow in the supply duct under the cover glass is the lens, the focal plane is either in the supply duct or in front of the mouth. The fluid flow is thus deflected from the supply channel via the cover glass, and the focal plane is in front of or in the region of this deflection.

An increased resolution of the optical imaging device is usually associated with a field of view reduction. For a particularly high-resolution imaging, it is therefore expedient to narrow the fluid flow to the smallest possible cross-section. For this purpose, an envelope flow channel can be provided through which an enveloping fluid flows, wherein the

Envelope flow channel surrounds the feed channel, also opens onto the cover glass and thus envelops the effluent from the feed channel fluid. The sheath flow focuses the incoming fluid flow in the lateral direction so that the required imaging range, i. H. the image field is smaller, which corresponds to a high-resolution image. So especially small objects can be detected well. Further, this arrangement is advantageous because reflections on

End of the supply duct no longer lie in the image plane. The alternative, one

Reducing the field of view by reducing the diameter of the feed channel involves the risk of sample clogging. Moreover, by the focused

Enveloping ensures that the fluid flows entirely through the focal plane, even if it is located in front of the mouth of and not in the inlet channel. - A -

The optical imaging can take place both in transmitted light mode and in incident light mode. For incident light measurement, the illumination in the area of the optical image is reflected in and applied through the objective. For selective excitation of a very thin boundary layer on the coverslip total reflection (TIRF) can be used, which is implemented with lenses with sufficient aperture. For a transmitted light image, a lighting cannula can be arranged parallel to the supply channel, is introduced through the transmitted light illumination. In a cylindrical supply channel, for example in the form of a tube, the illumination cannula can cause an annular illumination. When using a coaxial Hüllstromkanals the illumination cannula can be between supply and Hüllstromkanal. Other illumination systems are possible which irradiate a light sheet for fluorescence excitation or dark field illumination in the focus area of the lens. This can be done in particular by the supply duct, which can be used as a light guide and generates a local and homogeneous illumination and excitation light field with appropriate mirroring and shaping at the end.

The optical imaging by means of the objective can be used for sorting objects when the channel branches into at least two branches after the passage of the fluid flow through the focal plane and furthermore a device is provided for selectively blocking at least one of the branches. By operating this device depending on the image result, d. H. the detection of predetermined objects, can be made so that the predetermined objects are passed into one of the two branches.

The microscope according to the invention also allows a structure elucidation of the objects by mapping the objects in different rotational positions, if the channel has a

Supply passage and a return passage extending parallel thereto, through which the

Fluid flows. The feed channel opens onto a cover glass and this directs the fluid in the

Return channel around. The objective lies under the cover glass and its focal plane is located in the supply channel or in the region of the deflection of the fluid. Objects are then rotated relative to the imaging optics by the fluid flow deflection on the cover glass, so that they are imaged in different spatial positions.

Further structure elucidation is possible if the objects within the focus area in the focal plane are optically imaged several times in different axial positions. One then obtains a representation of the object in a different optical section through the object for each image. These sectional images can then be combined to form a 3D image of the object. The principle of an enveloping current can be modified compared with the already mentioned, coaxial with the supply current enveloping current when the supply channel opens through the coverslip in a transverse Hüllstromkanal, so that the enveloping current carries the fluid flowing from the supply channel to at least one outflow channel. If two outflow channels are used and the supply channel is stored in an elastic sleeve in such a way that it can be adjusted by displacing the supply channel, in which the outflow channels the enveloping fluid entrains the fluid flowing out from the supply channel, objects can be sorted by the displacement of the supply channel depending on the formation result is made.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the specified combinations but also in other combinations or alone, without departing from the scope of the present invention.

The invention will be explained in more detail for example with reference to the accompanying drawings, which also disclose characteristics essential to the invention. Show it:

1 is a schematic sectional view of a microscope for detecting objects in a fluid stream,

FIG. 2 shows a modification of the microscope of FIG. 1, FIG.

3a-d are schematic representations of images taken with the microscope of FIG. 1;

4 shows a further modification of the microscope of FIG. 1, FIG. 5 shows a further modification of the microscope of FIG. 1 with TIRF illumination,

Fig. 6 is a plan view of a fluid channel of a further modification of the microscope of Figure 1 and

Fig. 7 is a sectional view of the microscope of Figure 6 along the line A-A.

FIG. 1 schematically shows a microscope for the detection of objects which are carried in a fluid flow. The microscope includes an imaging assembly 1 that acquires images from a fluid stream flowing through a capillary system associated with the microscope. In this capillary system, the fluid flows in the direction of an arrow 2 through a channel 3, which comprises an inflow channel 3a and laterally leading away outflow channels 3b and 3c. The inflow channel 3a passes through a tube sealingly inserted into a fluidic chip 5. The inflow channel 3a formed by the tube 4 opens into an area above a cover glass 6 which is transparent to the imaging arrangement 1. The along the arrow 2 through the Inflow channel 3a incoming fluid flows through the tube 4 and exits from this, whereupon it is discharged in the direction of the arrows 10 and 1 1 in the drainage channels 3b and 3c.

For the sake of simplicity, only the focal plane 14 of the optical imaging arrangement 1 is shown in FIG. The same applies to the following figures 4, 5 and 7.

Relative to the direction of the arrow 2 under the coverslip 6 is the imaging assembly 1, which comprises an objective 7, which images an image on an image sensor 9 along an optical axis 8. To symbolize the imaged image area, a focus 12 of the objective 7 is shown in FIG. 1, which is located in a focal plane 14. The focal plane 14 is still in the inlet channel 3 a, so that flows in the inflow channel 3 a through the tube 4 in the direction of arrow 2 inflowing fluid to the focal plane 4 and passes through it. If, due to the hydrodynamic conditions, it is ensured that the inflowing fluid flows through the focal plane 4, this can also be outside the inflow channel 3a.

By means of a beam splitter 25 and a light source 19, the imaging arrangement 1 illuminates the fluid flowing out of the inlet channel 3 a in an incident light arrangement and images an image area lying in the focal plane 14 onto an image sensor 9. This figure allows objects carried in the fluid, e.g. As particles, proteins, etc., on the image sensor 9.

The optical imaging arrangement 1 has along the optical axis 8 a focus area which lies around the focal plane 14 and is essentially limited by the depth of field of the imaging arrangement 1.

The further drawn in Figure 1 a planes 22 to 24 are used for a special operation of the microscope and explained later with reference to FIG 3.

Figure 2 shows a modification of the microscope of Figure 1. Elements which correspond in their structure or function elements, which have already been explained with reference to Figure 1, carry in Figure 2 and in all other figures the same reference numerals, so that their repeated explanation is omitted ,

In the construction of Figure 2, the flowing through the tube 4 in the direction of arrow 2 through the inlet channel 3a fluid is enveloped by a coaxially surrounding sheath 17, which is fed in the direction of an arrow 16 in a tube 4 coaxially surrounding sheath channel 15. The sheath flow has the consequence that the fluid flowing through the inflow channel 3a is narrowed after the exit from the tube 4, that is focused. The image field size required within the focal plane 14 is thus lower, so that a Mapping arrangement can be used with higher resolution, which also allows to capture comparatively smaller objects. Also, the focal plane 14 can now be outside the feed channel 3a, which is favorable from the viewpoint of reduced optical interference.

FIG. 2 further shows a construction in which a transmitted-light illumination takes place in that radiation of the light source 19 is coupled in laterally of the tube 4 via an optical fiber bundle 18. This illumination occurs at the bottom of the tube 4, d. H. surrounding the fluid stream and causes transmitted light illumination of the image area to be imaged. Of course, this type of lighting can also be used in the construction of Figure 1 or one of the later to be explained construction methods.

Further, it is shown in Figure 2 that in the drain channels 3b and 3c check valves 20 and 21 are arranged, which allow to block these channels. Of course, such check valves may also be used in the construction of FIG. 1 or another of the variants described here. They allow, after evaluation of the image detected by the image sensor 9, to selectively block the drainage channels 3b and 3c. Thereby, it can be ensured that predetermined objects reach their detection by means of the imaging arrangement 1 in a certain one of the drainage channels 3b and 3c by the other drainage channel is blocked when a predetermined object was found in the image.

FIGS. 3a-3d show various images that can be obtained, for example, with the imaging arrangement of FIG. 1 or FIG. 2 (or another of the embodiments described here). Figure 26 Figure 3a was obtained in the plane 24 and does not allow further structural elucidation of the imaged object 27, which is to be illustrated by the dashed drawing of the object. However, this signal is used as a trigger, since at a known flow rate, the time is fixed and can be calculated, which requires an object to get from the level 24 to the focal plane. With very short illumination times, an image of the objects is repeated without reducing or stopping the flow. Each shot provides an image that represents a different optical section through the triggering particle. If high throughput rates are desired, a simple area detector can also be used for the trigger, which is independent of image acquisition and image evaluation via the detector 9 and is positioned in a plane which is conjugate to the plane 24. The separation of the light paths can be done analogous to reflected light illumination via a color or beam splitter. As soon as an object is found in the plane 24, this serves as a trigger for the sequential imaging in the focal plane 14. With this mode, a 3D image of the object can be taken very quickly, without the objective having a motorized z-axis. Drive to move. The plurality of images (28, 29, 32) are taken at times when the object has been transported differently across the fluid flow. From time intervals and flow velocity follows the axial transport path.

Thus, the images 28, 30 and 32 can be joined together and a three-dimensional image of an object in the focus area is thereby generated. An alternative is the procedure to bring the sample in the region of the focal plane 14 and to stop the flow. If no disturbing sedimentation then occurs, d. H. in the case of suitable samples, an image stack can then be recorded by moving the objective along the optical axis. If sedimentation is strong, it is necessary to wait until the sample has sedimented and then pick up the image stack.

FIG. 4 shows a further modification of the microscope of FIG. 1, in which, coaxially with the inflow channel 3 a, in which the fluid flows in the direction of the arrow 2, a return channel 3 b is formed, in which the fluid flows off in the direction of the arrow 10. The inflow channel 3a opens above the cover glass 6, resulting in a deflection 37 (symbolized by a dashed arrow) of the fluid flow in the region of the focal plane 14 results. Objects, e.g. B. particles that flow in the fluid flow are therefore rotated during the deflection, so that there is an image of the object in different rotational positions.

For the realization of the inflow channel 3a and the return channel 3b, a corresponding reflux tube 35 is arranged parallel to the tube 4, which has a rectangular cross-section, for example, like the tube 4. This cross-sectional shape is in principle for all channels, which are mentioned here in question.

FIG. 5 shows a refinement of the microscope of FIG. 1, in which the illumination from the light source 38 onto the cover glass 6 is radiated from the lower side at an angle 38 which lies below the total reflection angle. Thus, only an evanescent field can cause the illumination of the substances in the incoming fluid flow. This known per se TIRF illumination is in the embodiment of Figure 5 preferably (not necessarily) combined with the fact that the tube 4 protrudes at its mouth end 39 to the cover glass 6 so that only a narrow gap 40 remains, in the Magnitude of the propagation length of the evanescent field is. This ensures that all fluid flowing in the direction of arrow 2 is TIRF illuminated. This procedure can only be used for particles or molecules smaller than the penetration depth of the evanescent field (200 nm). For the small remaining gap, the flow is high if usual sample volumes of a few μl are to be analyzed in a reasonable time. If a 100% analysis of the sample flow is required, the sample flow must match the acquisition time of the sample Sensors (camera) are synchronized, with the dead times of the camera, the flow is stopped. This can be done either via check valves in discharge or inflow channels, or by appropriate control of the sample flow driving pumps.

FIG. 6 shows a modification of the fluid system for sorting predetermined objects in the fluid flow. In contrast to the previous figures, FIG. 6 is a plan view, that is to say seen in the direction of the arrow 2 of FIGS. 1, 2, 4 and 5. Through the tube 4, the fluid flows to. This tube 4, and thus the inflow channel 3a opens, as the figure 7, which shows a section along the line A-A of Figure 6, illustrates, substantially perpendicular to a Hüllstromkanal 41, in which a sheath flow along the arrow 16 flows. The sheath flow entrains the fluid flowing through the inflow channel 3a. The tube 4 is in an elastic sleeve 42 so that it can be displaced within the sleeve. Due to the position of the tube 4 is set whether the effluent through the drain channels 43 and 44 Höhlstrom entrains the supplied through the tube 4 fluid into the drain channel 43 or the drain channel 44.

If now by means of the optical arrangement 1 (not shown in FIG. 7) through the cover glass 6, the inflowing fluid flow can be sorted by displacing the tube 4 so that these all enter the same discharge channel, for example the discharge channel 43, in that each time a corresponding object is detected in the inflowing fluid, the tube 4 is set so that the sheath flow, which flows from the sheath flow channel 41, entrains the fluid flow from the inflow channel 3a into the outlet 43. On the other hand, as long as no predetermined object has been detected with the optical imaging arrangement 1, the tube 4 is displaced so that the enveloping flow from the sheath flow channel 41 directs the fluid flow supplied through the tube 4 to the outflow channel 44.

Claims

claims

1. A microscope for the detection of objects in a fluid stream, comprising: - a channel (3), through which fluid flows containing the objects to be detected, and an optical imaging device (1), which comprises a Fokalbereich having optics (7) and in the focal region, the focal region lying around a focal plane (14) which lies in the channel (3), characterized in that, - the focal plane and the channel are aligned with one another such that the fluid is directed towards the focal plane or away from it.

2. A microscope according to claim 1, characterized in that the channel (3) comprises a feed channel (3 a) through which the fluid flows, wherein the feed channel (3 a) on a cover glass (6) opens, via which the fluid laterally in at least a drainage channel (3b) runs and under which the optics (7) is located, and wherein the focal plane (14) in the supply channel (3a) or in the region of its mouth.

3. A microscope according to claim 2, characterized in that a Hüllstromkanal (17) is provided, through which flows an enveloping fluid, wherein the Hüllstromkanal (17) the

Supply channel (3a) encloses, also on the cover glass (6) opens and thus envelops the fluid flowing from the supply channel (3a).

4. A microscope according to claim 3, characterized in that for the transmitted light image between the feed channel (3 a) and the envelope flow channel (15), a lighting cannula (18) is arranged.

5. A microscope according to claim 1, characterized in that the channel (3) comprises a feed channel (3a) and a return passage extending parallel thereto (3b), through which the fluid flows, wherein the feed channel (3a) on a cover glass (6) flows and this deflects the fluid into the return duct (3b) and wherein the objective (7) lies under the cover glass (6) and the focal plane lies in the supply duct (3a) or in the area of the deflection (37) of the fluid.

6. Microscope according to one of the above claims, characterized in that the channel (3) branches after the passage of the fluid flow through the focal plane (14) in at least two branches (3b, 3c) and that means (20, 21) for selective blocking of at least one of the branches (3b, 3c) or a pump driving the fluid flow is provided for switching the flow on and off

7. A microscope according to claim 2, characterized in that an Hüllstromkanal (41) is provided, through which a Hüllfluid flows, wherein the feed channel (3a) above the cover glass (6) in the Hüllstromkanal (41) opens, wherein the enveloping current from the Supply passage (3a) outflowing fluid to at least one outflow channel (43, 44) entits.

8. A microscope according to claim 2, characterized in that the Hüllstromkanal (40) branches over the coverslip (6) in two outflow channels (3 b, 3 c) and the supply channel (3 a) in an elastic sleeve (42) is mounted in the the supply channel (3a) is displaceable, wherein the displacement position adjusts, in which the outflow channels (42, 43) of the enveloping current carries the fluid flowing from the supply channel (3a).

9. A microscope according to any one of claims 2 to 8, characterized in that a lighting device (19) is provided, which causes a TIRF illumination through the cover glass (6).

10. A method for the microscopic detection of objects in a fluid stream, wherein the fluid flow is guided in a channel (3), an optical imaging device (1) is used which comprises a focal area having optics (7), wherein the focal area about a focal plane (14) which lies in the channel (3) and objects in the focal region are optically imaged, characterized in that the focal plane (14) is aligned with the channel (3) so that the fluid flow onto the focal plane (14) flows to or from this.

11. The method according to claim 10, characterized in that the optical image for the detection of predetermined objects is evaluated and the fluid flow after passing through dividing the focal area into two effluent streams (3b, 3b, 42, 43), the division being made dependent on the evaluation to sort the predetermined objects.

12. The method according to claim 10 or 11, characterized in that at least one of the objects transported in the fluid stream is optically imaged multiple times within the focus area and results (26, 28, 20, 32) of the plurality of optical images to a 3D image of the object be joined together.