GB2508906A

GB2508906A - Frame element for sample injection with optical control means

Info

Publication number: GB2508906A
Application number: GB201222626A
Authority: GB
Inventors: Jan De Sonneville
Original assignee: LIFE SCIENCE METHODS BV
Current assignee: LIFE SCIENCE METHODS BV
Priority date: 2012-12-14
Filing date: 2012-12-14
Publication date: 2014-06-18
Also published as: GB201222626D0; WO2014090415A1

Abstract

An injection apparatus (10) comprises a frame element (11) defining a support (12) for a sample (13); an injection needle (10) capable of penetrating a sample (13) supported by the support (12); an optical image generator (14) that images the sample (13), on a second side (11 b) of the frame element (11) that differs from the first side (11a); one or more motors (17) that move the frame element (11) and the needle (16) in x-, y- and z-directions to effect the relative positioning of the support (12) and the needle (16); a recess (23) formed in the frame element (11) adjacent the support (12) and being optically detectable on the second side (11b) of the frame element (11); a further optical image generator (24), located adjacent the recess (23); and a processor (27) capable of transmitting commands to the motor or motors (17) based on images from the optical image generators (14, 24).

Description

INJECTION APPARATUSES AND METHODS OF CALIBRATING INJECTION

APPARATUSES

The invention relates to injection apparatuses, and to methods of calibrating injection apparatuses. The invention also relates to methods of using injection apparatuses.

Various forms of injection apparatus are known in science and industry. The invention is of particular utility in relation to so-called microinjection apparatuses, that are used primarily in scientific (e.g. bioscience) laboratories for the purpose of studying, treating and modifying samples in the form of small living particles, such as cells, cellular organisms, eukaryotes, spores, tissues, zygotes, eggs, oocytes or embryos; or parts thereof such as nuclei, enzymes or proteins. The term "sample" as used herein is to be interpreted in line with the foregoing guidance. The invention may also find utility in other types of injection device.

Several examples of areas in which the apparatuses and methods of the invention may be useful are described in W020121131000 Al, the entire content of which is incorporated herein by reference.

Generally microinjection systems include some means of supporting such samples together with means for observing them and a needle for injecting a further, flowable substance into the sample or into a medium surrounding the sample. The flowable substance may be a liquid, such as a pharmaceutical composition, a powder, suspension or train of particles; and it may include or consist of one or more small living particles as exemplified above, or parts thereof. The invention is applicable in all such situations.

It is furthermore known to use of a microinjection or similar apparatus in a substance removal mode in which an injection probe such as a needle removes a liquid or other flowable substance from a target particle that may be of the kinds listed above. A so researcher may use a microinjection apparatus in this way for example by exploiting capillary action of the needle, or by switching on a suction pump attached to the needle of the apparatus, following penetration of the particle by the needle.

The means for observing the samples typically includes a microscope that includes an optical image generator in the form of a camera, such that a user may observe the samples at typically a sub-cellular scale.

Some of the sample types listed above are essentially spherical or spheroidal in shape, and are very small. Often it is necessary to effect injection into a nucleus forming part of the sample, the nucleus being surrounded by a spheroidal further substance such as cytoplasm. The nucleus typically represents a very small injection target indeed, and furthermore it may not be consistently positioned from one sample to the next owing to the variability of living matter. In the case of an egg, the nucleus may represent or may indeed be the yolk.

Positioning of the needle for the purpose of injecting a sample, or a nucleus within a sample, requires very high levels of precision-Known microinjection apparatuses for this reason usually include highly accurately addressable motors for moving either a support for the samples, or the needle itself, in three mutually orthogonal directions referred to herein by the conventional nomenclature of x-, y-and z-directions. The motors are commanded using instructions generated in a processor such as a laptop or desktop computer (or a dedicated processor forming part of a control section of the apparatus) to position the needle and the sample relative to one another such as to permit injection of the sample or when desired the nucleus (or another small region of a sample) to take place.

In some microinjection apparatuses the motors act on the needle or a support that supports the needle, such that the needle moves as desired relative to a fixed support for the sample. In other apparatuses the support is moveable under the influence of the motors and the needle fixed. In yet further apparatuses both the support and the needle are moveable under the influence of appropriately positioned and connected motors.

A problem however with prior art microinjection devices is that it is necessary to calibrate the position of the needle relative to the sample or at least the support for the sample before an injection operation can take place. In the absence of calibration accurate injecting of samples becomes more or less a matter of chance, so this is an important aspect of operation of microinjection devices.

When injecting cells into a gel, for example as described in W02012/131000 Al, it is beneficial if the injection height (he. the height of the in-use needle tip) is defined with respect to the bottom of a well supporting a multicellular spheroid. This allows for automated imaging to take place at exactly the height of the injection, and reduces the total volume to be imaged, saving imaging time and reducing computer storage space.

These considerations give rise to a need for accurate setting up of microinjection apparatuses.

Furthermore, microinjection needles currently are difficult to clean or re-use. Therefore, in many injection system designs a new needle has to be installed and calibrated before each use.

Also multiple needles are sometimes used to perform multiple injections into a target.

This arises for example when multiple cell types are provided in e.g. a volume of gel such that each cell type is injected individually with the result that a scientist can study the effects of injections on the various cell types starting from the same initial condition in each case. In such an experiment each needle used in turn needs accurate calibration to allow for well-defined spacings between the sequentially created injection sites in the volume of gel. Currently, such an accurate calibration is not possible with the single viewing angle provided by a solitary camera as described in W020l2f131000 Al. The invention as explained below provides advantages in relation to such injection regimes, which therefore lie within the scope of the invention as defined hereinbelow.

In the context of the invention "calibration" includes defining an initial position of the needle and sample relative to one another. Such an initial position can be represented in terms of co-ordinates in x-, y-and z-planes the concept of which will be familiar to technologists.

This type of calibration must be completed with high accuracy not least because in nearly all microinjection devices a large number of samples may be processed at the same time, or in a short period by reason of adopting a batch processing approach. These approaches in turn are made possible through the use of a usually horizontal array of supports for the samples, for example in a per se familiar titre pFate or a similar means of presenting a large number of samples in one and the same plane.

It follows that any calibration of a needle in a microinjection apparatus may have to remain accurate with respect to multiple, sequential positionings of a needle to inject a large number of samples in turn; or with respect to the positioning of a large number of needles based on the calibration for example of one or a limited number of them.

Errors in the calibration process tend to be additive in such situations.

Partly for this reason the prior art microinjection apparatuses have included complicated sub-systems for measuring and recording the position of one or more needles relative to a support for a sample.

Such sub-systems have included multiple cameras that must be operated sequentially in order to obtain calibration positioning information in respect of each of the x-, y-and z-co-ordinate axes mentioned.

This approach to calibration however is undesirable firstly because the need for multiple cameras significantly increases the costs of manufacturing the apparatuses. The positions of these cameras with respect to the sample furthermore need to be accurately known, or need to be calibrated as well, thereby adding to the complexity of building or operating the system.

is Furthermore the need to activate three cameras sequentially means that the calibration action is time-consuming. Since laboratory time may be costed in thousands or even tens of thousands of Euros per day clearly it is undesirable for microinjection apparatuses to be responsible for delay.

In some types of microinjection system the injection needle is arranged to approach the sample at an oblique angle so that the elongate axis of the needle is not aligned with the viewing axis of a camera, microscope or other optical device that is used for monitoring or assessment of the experiment being conducted.

In such cases it is possible to calibrate the needle position by causing the needle to advance to touch a surface of a frame element that defines a support for the sample.

When such contact occurs a physical change such as an increase in the output of a strain gauge attached to the needle, or an increase in the current in an electric motor responsible for effecting movement of the needle, may be used to signify that the needle has reached a datum position. It then is possible to zero co-ordinate registers corresponding to the x-, y-and z-axis positions of the needle relative to the frame element; and thereafter accurately position the needle using the zeroed registers as datum values. W020061034249 Al for example describes a method in which initial contact of a needle tip with the surface of a sample holder is detected; and then a a5 computer vision system used to assess movement in a direction perpendicular to the approach of the needle tip to the surface and thereby establish a so-called Thome" position for the needle.

Such approaches however are associated with several disadvantages.

Firstly the technique of causing the needle to contact a surface of the frame element may damage or completely break the needle.

Furthermore it has been found that the accuracy of experiment observation and data gathering in microinjection apparatuses may be significantly improved if the optical axis of any optical image generator such as a camera or microscope is aligned with the elongate axis of the injection needle forming part of the apparatus. This desirable condition does not arise in the designs of microinjection apparatuses that include obliquely orientated needles.

Approaches of causing contact of the needle with a surface as part of a calibration or setting-up process are not suitable in apparatuses in which the needles extend perpendicular to the adjacent surface of the frame element. In such cases an unacceptably high rate of breakage of the needles would occur. It is believed to be for this reason that W02008/034249 Al illustrates needles that obliquely approach and contact a surface.

Overall there is a need to improve the calibration or setting-up of needles in microinjection apparatuses and injection apparatuses.

According to the invention in a broad aspect there is provided a frame element for use with a microinjection apparatus, the frame element defining a support for a sample and/or a support for a sample-holder; and the frame element comprising a recess formed in at least a first side of the frame element adjacent the support and being optically detectable on the first side of the frame element or on a second side of the frame element that is distinct from the first side; and at least one optical device, located adjacent the aperture, that is capable of generating an optical image of the recess and/or an object received in it.

Such a frame element advantageously addresses the problems of the prior art arrangements, in ways described herein.

According to a second aspect of the invention there is provided injection apparatus comprising a frame element defining a support for a sample; an injection needle having an elongate axis and that is capable of penetrating a sample supported by the support and injecting thereinto from a first side of the frame element a flowable substance; an optical image generator that is capable of generating an optical image of the sample, on a second side of the frame element that differs from the first side; one or more motors for causing movement between the frame element and the needle in x-, y-and z-directions so as to effect relative positioning of the support and the needle; a recess formed in the frame element adjacent the support so as to open on the first side of the frame element and being optically detectable on the second side of the frame element; a further optical device, located adjacent the recess, that is capable of generating a further optical image; and a processor that is capable of transmitting commands to the motor or motors based on images generated by the optical image generator and the further optical device, or data derived from such images, the motor or motors being capable of manoeuvring the frame element and the needle relative to one another such that the needle penetrates the recess; and the optical image generator and the further optical device being capable of generating optical images representing x-, y-and z-positioning of the needle relative to the recess when penetrated by the needle.

This injection apparatus may be of any of the types outlined hereinabove. It is particularly suitable when the elongate axis of the injection needle is aligned with the optical axis of the optical image generator.

In this regard, it has been found advantageously that forces acting on a sample during injection cause minimal movement of the sample when the injection axis is perpendicular to the sample support. This minimizes possible off-axis displacement of the needle by the sample, and increases the reproducibility of the injection,.

The arrangement of the invention however may also be used in apparatuses in which the needle axis and the optical axis are not aligned with one another.

The arrangement of the invention advantageously minimises the number of optical image generators needed for calibration operations; it provides for reliable position zeroing or calibration; the process of calibration is quick and easy to perform using the apparatus; and the risk of needle damage is reduced effectively to zero.

A further advantage of the apparatus of the invention, that cannot arise in any of the described prior art arrangements, is that the presence of the recess permits ready optical inspection of the tip of the needle for example while an experiment is taking place.

Thus for example the needle tip may be manoeuvred to penetrate the recess following injection of a sample. The optical image generator then can be activated to generate an image of the needle tip in or protruding from or positioned in the recess and indicating whether the needle tip is clogged or damaged. Such a check can be performed rapidly and reliably following initial calibration of the needle position, and indeed could be automated by way of the programming of commands to the motor(s).

Additionally, a test injection in the recess can show formation of a droplet. This can be used to inspect the droplet size and hence provide for example confirmation that the needle is in good condition.

In some embodiments an additional sample holder is positioned next to the sample support. Such an additional sample holder is viewable from the in-use underneath of the frame element, and may contain a sample liquid in which one or more test injections can be made. Such test injections can be used to calibrate the size of droplet produced by the needle. Examples of such an additional sample holder include petri-dishes or tubes such as Eppendorf tubes.

Injections into a second sample holder can also be used to culture one or more droplets containing cells to permit counting or monitoring of the droplet cell density.

Further, preferred features of the apparatus of the invention are set out in the claims hereof depending from Claims 1 and 3.

According to a further aspect of the invention there is provided a method of calibrating injection apparatus as defined herein the method comprising the steps of, before injecting a sample supported by the support, operating the or at least one said motor to position the elongate axis of the needle in register with the recess; generating one or more optical images or signals derived from optical images, using the optical image generator, corresponding to x-and y-axis positioning of the needle relative to the recess, the said x-and y-axes being defined with reference to a plane including a surface of the frame element; operating the or at least one said motor to cause the needle to penetrate the recess until the further optical device generates an optical image or a signal derived from an optical image corresponding to z-axis positioning of the needle relative to the recess, the said z-axis being defined with reference to the said plane including a surface of the frame element; and recording the optical images, or the signals derived therefrom, as calibration signals. Optionally the method includes the step of operating the or at least one said motor to cause withdrawal of the needle from the aperture.

The x-and y-axis optical images or signals can be used to position the needle within the focal plane of the further optical device.

Further optional features of the method of the invention are set out in the claims depending from Claim 19.

According to yet a further aspect of the invention there is provided a method of using injection apparatus as defined herein, the method comprising the steps of, one or more times, operating the or at least one said motor to position the elongate axis of the needle in register with the recess; operating the or at least one said motor to cause the needle to penetrate the recess; and generating an image of the needle using the optical image generator and/or the further optical device.

According to yet another aspect of the invention there is provided a method of using injection apparatus according to any of Claims 1 to 18 or calibrated according to any of Claims 19 to 22, the method including a step selected from the list comprising: (a) sequentially injecting at least two samples at a predetermined spacing from one another into a gel or other medium; (b) injecting at least two samples in contact with one another into a gel or other medium; (c) injecting into a first sample a second sample; (d) simultaneously injecting a plurality of samples into a gel or other medium so that they adopt a chosen pattern; (e) injecting one or more samples so as to lie on a surface of a well, a gel or another medium; or (f) injecting one or more samples into a gel or another medium; and injecting one or more samples onto a surface of a gel or other medium.

There now follows a description of preferred embodiments of the invention, by way of non-limiting example, with reference being made to the accompanying drawings in which: Figure 1 is a part-cross sectional, part-schematic view of one form of apparatus according to the invention; Figure 2 shows a practical form of frame element of the kind illustrated in principle in Figure 1, in which a plurality of supports for samples is arranged in an array; Figures 3A -3D show one method, within the scope of the invention, of calibrating injection apparatus; Figure 4 shows another form of apparatus according to the invention; Figures 5A -51-I show in schematic plan and vertically sectioned views some sample injection patterns within the scope of the invention; Figures 6A -6D are schematic plan and vertically sectioned views of further injection patterns within the scope of the invention; and Figure 7 is a schematic, vertically sectioned view of yet a further injection pattern within the scope of the invention.

Referring to the drawings there is shown an injection apparatus 10 that in the embodiment shown is a microinjection apparatus although as stated the invention may readily be embodied in other forms of injection apparatus.

Apparatus 10 includes a frame element 11 that in the embodiments illustrated resembles a titre plate having at least one sample support 12a, 12b, 12c for supporting a sample 13.

The sample 13 is shown as a cell having a nucleus that is surrounded by cytoplasm and that is intended to be injected using a needle 16 described in more detail below. As indicated however the sample 13 may be for example a cellular organism, eukaryote, spore, zygote, oocyte, egg, or embryo; or part thereof such as but not limited to a nucleus, enzyme or protein. The sample moreover does not necessarily need to comprise living matter, although it is envisaged that in many uses of the apparatus of the invention this will be the case. As will be appreciated there is a requirement for very high accuracy in the injecting of all types of samples 13.

The support 12 is constituted in the embodiment illustrated by one or more flanking walls 12b, 12c that extend upwardly from an in-use upwardly facing, fiat surface ha of the frame element 11. The flanking walls 12b, 12c (and further flanking walls if present: the external shape of the support 12 is designed according to manufacturing and usage expediency) define a cup-like recess 12c that in the embodiment shown is part-spherical and open at its in-use upper side.

Such a support design has been found to be highly useful for supporting a wide range of types of sample 13, many of which are spherical or spheroidal and hence readily accommodated in the shape of the support illustrated.

This advantage of the part-spherical support arrangement notwithstanding, numerous other designs of support are possible within the scope of the invention. One option in this regard is for the support itself to support a further holder or other supporting arrangement for a sample.

An elongate injection needle 16, that is hollow and in the majority of embodiments of the invention is a microinjection needle, is mounted so as to permit injection of a sample supported from underneath in the support 12. The needle 16 may be made from, for example, glass or a range of metals. The manner of constructing the needle 16 will be familiar to those of skill in the art.

Needle 16 has an elongate axis that extends parallel to the optical axis of a camera 14 or other image generating device described in more detail below. As stated above however the invention is applicable in arrangements in which the needle axis of elongation is not aligned with the optical axis of an optical image generator in this way.

The needle 16 is disposed on a first side of the frame element 11 that one may define as being that including the surface ha. Thus injection of a flowable medium (such as a liquid, powder, gas or mixture of substances) into the sample 13 takes place in a direction towards the plane containing surface 1 Ia.

The camera 14 (or another optical image generator) is of a type that is capable of generating an optical image of parts of the apparatus 10. Camera 14 is located on a second side of the frame element 11 that in the embodiment shown is the opposite side llb to surface ha. In other embodiments of the invention however the optical image generator may be located on a side of the frame element 11 that is not the opposite of surface 1 Ia. Such an arrangement may be of benefit for example if the needle 16 is obliquely mounted in one of the ways described herein.

The primary purpose of the camera 14 is to capture one or more images of the sample 13. This may in the embodiment shown be achieved for instance through the provision of a transparent section of the frame element defining the in-use underside of the support 12; or in a range of other ways. The material of the frame element 11 and support 12 may be such as to transmit light or other detectable energy in a range of wavelengths that the camera may capture, without the frame element appearing transparent to the human eye. Alternatively a window that in some embodiments of the invention may be a through-going aperture could be provided.

The camera 14 or other optical image generator may be such as to produce a digital output that can be processed directly by a personal computer; or the signal may be in some other form, including but not limited to analogue electrical signals, optical signals or acoustic signals.

The camera, etc., may be arranged if desired to capture multiple images of the sample 13 for example as a sequence recording the period before, during and after injection It may alternatively be arranged to capture continuous motion in the form of a video sequence.

The apparatus 10 includes one or more motors, represented schematically by numeral 17. These are capable of effecting relative movement between the needle 16 and frame element 11 in each of mutually orthogonal x-, y-and z-directions as referred to hereinabove and as indicated schematically in Figure 1 In Figure 1 the motor 17 is an electric motor (although numerous other motor types are possible and depending on the exact usage of the apparatus 10 may be positively preferable over an electric motor) having a rotatable output shaft 18 having secured thereto a rotatable, toothed pinion 19.

The teeth of the pinion 19 are drivingly engaged with the teeth 21 of a toothed rack 22 that in the embodiment shown is secured to the frame element 11. In the embodiment illustrated the rack 22 extends parallel to the x-direction defined Operation of the motor 17 to cause rotation of the pinion 19 on the shaft 18 thus causes movement of the frame element in the x-direction, with the direction of rotation of the shaft 18 determining whether the frame element 11 moves to the left or the right in the figure.

The rotational direction of the shaft 18 is selectable depending on the movement requirement of the frame element 11.

Similar motor, pinion and rack arrangements may be provided to cause movement of the frame element respectively in the y-and z-directions.

In other arrangements within the scope of the invention however the frame element 11 may be fixed and the motor(s) may effect movement of the needle; and in yet further embodiments relative motion between the frame element 11 and the needle 16 may arise as a combination of moveability of the frame element 11 and the needle 16. One example of this would be an arrangement in which two motor and rack combinations cause movement of the frame element in x-and y-direcUons; and a further motor causes movement of the needle in the z-direction. Numerous other ways of effecting relative movement between the frame element 11 and the needle 16 also are possible within the scope of the invention.

Frame element 11 includes formed therein a recess in the form of an aperture 23 that in the embodiment shown is through-going and extends from surface ha of frame element 11 to surface 11 b (although in other arrangements conceivably the aperture does not need to pass all the way through the frame element and therefore may be formed as a closed-ended recess that has an opening on an in-use upper (first) side of the frame element).

The aperture 23 is formed adjacent the support 12.

In Figure 1 the camera 14 is shown aligned with the open end of the aperture 23 such as to be able to capture one or more images of it. The positioning of the camera 14 relative to the support 12 however as described above is variable depending on the operation of the motor(s) 17, such that at other times the camera may capture one or more images of the sample 13 rather than the aperture 23, the relative positioning of frame element 11, needle 16 and camera 14 in Figure 1 being essentially to illustrate the range of possible movement options. In normal use of the apparatus of the invention when as shown the needle 16 is penetrating a sample 13 the camera 14 is likely to be positioned so as to capture images of the sample rather than the aperture.

A further optical device 24 is positioned adjacent the aperture 23, on surface llb of frame element 11.

Further optical device 24 is shown schematically and may be for example a prism or a mirror (or any of several other optical devices such as a further camera, a diffraction grating or a lens). While camera 14 is arranged to obtain an end-on view of the aperture 23 the further optical device 24 is arranged to capture a side view of the opening of the aperture 23.

In one particularly useful arrangement of the invention the optical device is used in combination with the further optical device to generate a side view of the needle. In this arrangement the further optical device comprises a passive optical element such as a mirror or prism which feeds a side-view of the needle as an optical signal into the optical device. To use this arrangement the stage (frame element 11) has to be moved to align the optical axis of the further optical device with that of the optical device, and the needle has to be positioned in the resulting transformed/bent focal plane of the optical device.

In this arrangement only one active (i.e. powered) optical device is required. This further reduces the costs and complexity of the apparatus.

The camera or other optical image generator 14 and the further optical device 24 in some other embodiments of the invention are both such as to generate images that may be conveyed as signals. In the case of an essentially passive optical device 24 such as a prism, mirror or lens it may be necessary in this regard to provide a converter 26 such as a waveguide or other element (also shown schematically in Figure 1) that converts any light signal into an electronic form of a similar kind to the preferred output of the optical image generator 14. Many variants on how the optical image generator and the further optical device 24 generate useable outputs are possible within the scope of the invention.

The outputs of the two optical devices 14 and 24 are fed in the example shown to a processor in the form of a personal computer or a dedicated control section of the apparatus 10, represented by numeral 27. The processor 27 is capable of generating instructions for the motor(s) 17 based on the thus-fed image data or signals derived therefrom.

The basic operation of the apparatus as described to calibrate the position of the needle 16 before injection operations commence involves operating the motor(s) 17 under command of the processor 27 in order to cause the tip 16a of needle 16 to enter the upper end of aperture 23. To this end the diameter of aperture 23, which diameter preferably is constant along the length of the aperture, is greater than the largest dimension of at least the tip portion 1 6a of the needle 16 so that positioning of the needle in register with the opening of the aperture 16 may be facilitated. In the embodiment shown the tip 16a is columnar and tapered, but other designs of the tip are of course possible within the scope of the invention. The shape and size of the aperture or recess 23 are chosen to allow insertion of the needle tip at least partially into it.

The motor(s) 17 then may be operated to cause the needle 16 to advance inside the aperture until the tip 16a protrudes from surface llb. Such protuberance of the needle may be detected through the generation of an optical image in the further optical device 24 and hence the generation of a signal that is fed to the processor 27.

At this point the operator of the apparatus 10 would know that the position of the needle relative to the remainder of the parts of the apparatus is known. The optical devices 14, 24 may then generate images andlor signals (depending on their precise nature) that in turn can establish datum values for the needle position. One way, of many, in which this can be achieved is through the zeroing of registers in the processor 27 once the position of the needle 16 becomes established in the manner described above.

Thereafter the datum information may be used to control the position of the needle relative to a sample supported by the support 12 such that injection of e.g. an internal nucleus may be achieved with high accuracy.

It is not necessary however that the tip ISa protrudes from the aperture 23 and indeed as indicated it is not essential that the aperture extends all the way through the frame element 11. On the contrary it is believed that the further optical element for example could operate by detecting a change in the colour, hue or brightness of a translucent part of the frame element 11 adjacent the aperture 23, or in a range of other ways that do not require protrusion of the needle from the surface 11 b.

Furthermore "aperture" as used herein is to be construed broadly, and need not necessarily mean a physical hole extending through the material of the frame element 11. On the contrary, the aperture may be a somewhat notional one defined for example by a pair of spaced marks formed on an edge of the surface 11 a of element 11. During calibration of the needle position it then becomes necessary to cause the needle to pass by the edge of the element 11 such that the marks lie to either side of it. When the further optical device 24 detects protrusion of the tip iSa of the needle beyond the surface llb the optical image generator 14 and the further optical device 24 may be activated to capture images and/or generate data that are useful as described as datum co-ordinates of the position of the needle 16.

In a practical version of the apparatus of the invention a large number of the supports 12 would be provided, preferably supported in an array such as that shown in Figure 2.

An arrangement of this general kind is illustrated in Figures 3A -3D. These figures schematically show apparatus 10' including a frame element 11 in the form of a clamp or retainer for a sample holder that adopts the form of a titre plate 28. As is well known a titre plate includes an array of wells 29 that are suitable for retaining a sample and that unless closed by a cover are open on the in-use upwardly facing side of the titre plate.

In the arrangements shown in Figures 3A -3D the wells 29 are transparent at their lowermost edges for the purpose of allowing camera 14 to capture images of the insides of the wells 29.

Frame element 11 is moveable and in the example shown may be driven to move in at least x-and y-directions by one or more motor 17, pinion 19 and rack 22 combinations of the general kind described above. As in the case of the embodiment of Figure 1 the frame element 11 also may be capable of controlled movement in a z-direction; or it may be fixed in the z-cI.irection. Needle 16, that can be as described hereinabove or that may be of a different design, in the latter case would be controlledly moveable in the a-direction relative to the frame element 11.

In Figure 3A the camera 14 is shown focussing on a sample 13 in one of the wells 29. A needle 16 forming part of the apparatus 10' at this stage is not present in the vicinity of the titre plate 28.

Such focussing may be required in order to set up the camera 14 for the remainder of the calibration procedure described below, since otherwise the camera may not be able correctly to identify the small tip of the needle 16.

In Figure 3B the initial stage of calibration of the apparatus is shown. In this figure the needle 16 and the camera 14 are manoeuvred relative to one another in the x-and y-directions, until the optical axis of the camera coincides with the centre of circular aperture 23 that perforates the frame element 11 to one side of an edge of the titre plate 28. This may be achieved for example by repeatedly digitising the distance from a datum point defined in the field of view of the camera 14 to the edge, until the distances in the x-and y-directions are equal.

The focal plane of camera 14 typically will be different when focussing respectively on the samples 13 and and the aperture 23 due to refraction of light by any medium (gel, or another medium) in the wells 29. One should correct for this difference, to allow for visual inspection during the injection process. The correction distance between the two focal planes referred to can be determined for a given amount and type of medium in a well by changing the injection height (as explained herein) during a series of test injections. One can then use the injection height which offers the best ability to inspect the droplet formation.

The frame element 11 and the needle 16 may then be manoeuvred relative to one another, preferably but not necessarily using techniques as described herein, until the elongate axis of the needle 16 coincides with the optical axis of the camera 14. Centring of the needle tip in this way also may employ techniques of assessing the x-and y-direction distances of the needle tip from the edge of the aperture 23. During the process of manoeuvring the frame element 11 and the needle 16 relative to one another for this purpose the position of the camera 14 relative to the frame element 11 is maintained fixed. This may be achieved e.g. through appropriate software control of the various motors and other drive components, or perhaps in some embodiments of the invention by temporarily clamping the camera 14 and the frame element 11 to one another.

Once calibration of the needle position in the x-and y-directions is achieved in the manner described, z-direction calibration takes place. This is shown in Figure 3C.

In this figure the frame element 11 has been moved relative to the camera 14 such that the latter focuses on the light output of a prism 31 forming a further optical device as defined herein.

Prism 31 is mounted on the frame element 11 so as to capture light emitted from the needle in the aperture in the x-y plane. The prism 31 therefore is capable of detecting the z-direction position of the needle 16.

As shown in Figure 3C when the frame element 11 and the needle 16 are manoeuvred relative to one another in the z-direction an image of the needle tip will be incident on the prism 31. Assuming the latter is correctly orientated the image may then be rotated through 90 degrees owing to the optical characteristics of the prism 31, such that it may then be readily viewed by the camera 14. Such an arrangement and method therefore may readily identify when the tip of the needle 16 is in a datum position in the z-direction, for example by reason of the image of the tip attaining a particular position in the field of view of the camera 14.

Subsequent operation of the apparatus 10' is as illustrated in Figure 3D, in which the camera 14 records the results of injection or fluid removal operations carried out on samples 13 in the wells 29 of the titre plate 28.

S It will be appreciated that instead of the prism 31 a mirror could be employed to cause rotation of the needle tip image out of the x-y plane for viewing by a camera aligned in the i-direction. The prism or mirror is absent from Figures 3A, 3B and 3D for clarity only.

Figure 4 illustrates in schematic form an embodiment of the invention that is a variant on the arrangement of Figures 3A -3D.

In Figure 4 the frame element 11 differs from that of Figures 3A -3D in that the recess 23 does not extend all the way through the frame element; and the camera 14 is positioned on the first side 1 la of the frame element. The optical axis of the camera 14 in this case lies parallel to the x-y plane.

A further optical device in the form of a beam splitter or half-silvered mirror 32 is positioned in the optical path between (a) the location in the recess 23 at which calibration of the needle position occurs and (b) the objective lens 14' of the camera 14.

This permits the camera 14 to view an image generated in the x-y plane of the position of the tip of the needle 16 and thereby provide for i-direction position calibration in a manner as outlined herein.

When the beam splitter is present the apparatus 10" includes in line with the optical axis of the beam splitter that is parallel to the i-direction a prism 31. Prism 31 is capable of transmitting an image of the tip of the needle 16 when the latter lies directly above the prism 31 as a result of manoeuvring of the frame element and the needle 16 relative to one another in ways as described herein. The prism 31 rotates this image through 90 degrees so it can be captured by the camera 14 and x-y position calibration of the needle carried out in ways similar to those described above.

If the further optical device 32 is a half-silvered mirror the prism is not separately required as the mirror is capable of transmitting images in both the x-y plane and the z-direction, without any need for an additional optical component.

Advantages of the arrangement of Figure 4 include that of compactness, since there is no need to position any parts of the apparatus 10" below the upper edge of the frame element 11. Also pre-focussing of the camera 14 is not required.

Of course the apparatus 10" of Figure 4 cannot provide observations from underneath the wells 29 unless a second camera is provided; and possibly the mirror and prism, being located on an upper side of the apparatus, may acquire dust and debris more readily than their counterparts in Figures 1 -3 that lie beneath the frame element 11.

Despite these minor disadvantages the embodiment of Figure 4 is expected to be of considerable utility in some situations.

In all the described embodiments of the invention there may exist a single needle 16 that is moved to inject samples in the respective supports 12 seriatim; or there may exist an array of needles corresponding in part or entirely with the pattern of supports 12. There may furthermore be provided a single aperture or recess 23 that may be used for calibration of one or more needles for the purpose of permitting subsequent injection operations; or there may be provided a respective such aperture associated with and adjacent each support 12 or equivalent feature. Vet a further possibility is for an aperture to be provided in respect of each of several groups of the supports 12 making up the array.

One mode of operation of the apparatus 10 of the invention involves periodically (for example after each injection, or after each tenth injection, or at some other interval) causing the needle 16 to penetrate the aperture 23 by generating commands to the motor(s) 17 in the processor 27 based on the co-ordinate data generated during the calibration steps described above. Thereafter it is readily possible to inspect the condition of the tip ISa of the needle, for example using the optical image generator 14, in order to check for clogging or damage.

This mode of operation is not available in any prior art injection apparatus known to the inventors.

More generally the apparatus of the invention confers great versatility on the injection regimes that, owing to the needle positioning accuracy resulting from the superior calibration available, may be practised. Figures 5 to 7 provide examples of injection regimes within the scope of the invention.

Figures 5A and SB show the result of two consecutively completed injections of e.g. spheroids 36, 37, each containing one or more cells in a body of gel 38 (or another medium that supports and sustains the cells 36, 37) in a well 39.

The depths to which the spheroids 36, 37 are injected are accurately controlled as signified by the arrows in Figure SB, as are (a) the spacings between the spheroids and (b) the distances of the spheroids 36, 37 from the wall of the well.

Such an injection regime may be suitable for e.g. stimulating chemotaxis, studying cell migration and studying cell interactions (for instance between bacteria and immune cells).

The apparatus and methods of the invention are particularly useful at such times both because the task of observing the cells in 36, 37 is made easier through precise initial positioning; and also because the meniscus 38' of the gel may influence needle cannula back-pressure during injection and thereby the injected volume (and such influence can be taken account of through accurately determining the injection locations).

Furthermore if the well 39 is one of a series the ability accurately to determine the injection locations improves the repeatability of experiments from one well to the next.

Yet a further advantage of the method and apparatus of the invention arises in the example of Figures SA and SB, in which it might be necessary to use two different needles to inject the respective spheroids 36, 37. The ability to calibrate the needles to the same calibration point using the features of the invention means that such a multiple calibration requirement does not have a deleterious effect on the quality of the experiment.

The principles underlying the example of Figures 5A and 5B may be extended to situations in which more than two spheroids 36, 37 (or other sample types) are injected into one and the same body of gel 38. An instance of this type of injection is described below in relation to Figures 5G and 5H.

In Figures 5C and SD a similar arrangement to that of Figures 5A and 5B is shown, except that the spheroids 36, 37 are placed closely adjacent, or if desired even in contact with, one another.

In Figures 5E and 5F a first spheroid 41, which may be e.g. a spheroid containing one or more cells, is modified by creating a second spheroid 42 partly or wholly inside it.

s The spheroid 42 could be created separately from the first spheroid 41 and then injected into the latter using apparatus according to the invention. Alternatively the second spheroid 42 could be created by other techniques such as but not limited to the printing technique described in publication no. US2OI1/0250688A1.

Figures 5G and 5H show the result of using multiple injections (preferably to the same controlled depth and all taking the same amount of time) to create a pattern determined by the amount of back-pressure of the gel 38 influenced by the distance from the wall of the well 39. Such an arrangement as illustrated is rotationally symmetrical. This can be advantageous when creating multiple essentially similar spheroids of the same cell-type, using similar distances from the wall of well 39. Such injection sites have essentially the same gel back-pressure such that the same injection settings (e.g. injection pressure, injection time and back-pressure) can be used. The accuracy of the point of symmetry affects the reproducibility of the spheroid formation within such an arrangement. Precise needle calibration, as results from use of the apparatuses and methods of the invention, is required.

Figures 6A and 6B show how the apparatus and methods of the invention may be used to position e.g. spheroids 36, 37 or other samples as exemplified herein onto the surface of e.g. a second gel or medium 43 in a layer contained within the gel or other medium 38 shown in Figures 5A to 5K.

In order to achieve this result two needles 14A, 14B (that are individually calibrated using the principles of the invention) may be employed with confidence. This is because the researcher will have certainty that the positioning of the spheroids 36, 37 would be accomplished accurately.

This technique allows for the resultant creation of islands 36', 37' or patterns of cells on the gel 43 or, if gel layer 43 is absent, the wall of well 39. This situation is represented schematically in Figures 6C and 6D.

W02007/l24023 describes a technique of inject-printing of cells but this requires a matrix in order to guide the printed cells. The principles of the invention aflow to dispense with the matrix (which may interfere with an experiment in numerous ways).

The technique of Figures 5A and 6B is highly advantageous because the resulting, controlled ability of the cells to form a 3D cell culture and/or spread out mimics the conditions cells encounter in viva.

US2007/l72944 Al describes ways in which cell co-cultures may be viable. The technique of Figures 6A to 6D among other things improves the ability of such co-cultures to be experimentally useful.

Some cell types that may be co-cultivated may require differing gels or other media. In such cases a sequential injection technique, in which injections are interspersed with changes in the gel 38 once each injected cell type has become established, is possible within the scope of the invention.

Another possibility involves combining techniques described in 1JS2007/l 72944 Al with microinjection and micro-patterning (injecting just above the gel) to reduce the number of wells required for to embody the ideas set out in US2007/l72944 Al, for example by using fewer wells than cell-types. This reduces the required number of wells and the medium volume above the wells (thus allowing for more compounds per plate to be tested than in the prior art). As well as using injection in a gel, in a well plate as explained in US2007/172944 Al containing for example cancer cells, viruses or bacteria, testing not only the toxicity but simultaneously also the effectiveness of compounds becomes possible.

Figure 7 shows a combination of the techniques of e.g. Figures 5A I 5B on the one hand and Figures 6A to 6D on the other. The result is a pair of cells (or other samples) 36, 37 in a lower gel layer 43 the upper surface of which supports cell islands or patterns 36', 37'. The islands or patterns 36', 37' are maintained by an upper gel or other medium layer 38.

The islands / patterns 36', 37' could be organ cells growing in a 2D layer; and the injected cells 36, 37 could be e.g. fibroblasts, immune cells, cancer cells or bacteria the effect of which on the organ cells is to be studied.

One could also mix the gel with cells such as fibroblasts to create a particular microenvironment. However mixing is a chance process and may not be very reproducible. Injecting tiny droplets of fibroblasts at fixed locations may be better, and thus preferred at the cost of more time required to set up the apparatus. It would also allow exactly to observe the change upon adding such types of cells to an existing cell culture, i.e. the time measured from the injection in and/or on top of the gel can be used as a parameter in experiments.

The immediate ability of cells to adhere due to the injection technique allows better use of multiple cell-types because it constrains the time available for one cell-type to overgrow the other.

In other systems when more time is needed to create a stable 3D tissue (such as in hanging drop culture, or when mixing cells with a gel), the use of more than one cell-type is more dependent on possible differences between cell-types of growth, division rates, migration speed and so on.

Overall the apparatus and methods of the invention are highly practical and advantageous. The method of calibrating the needle position may be effected rapidly since once the position of the needle is established by reason of its penetrating the aperture 23 the capturing of data andfor images useful for calibration purposes occurs almost instantaneously. This is turn is because it is not necessary to activate more than one optical device (i.e. camera 14).

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Claims

CLAIMS1. A frame element for use with a micro-injection apparatus, the frame element defining a support for a sample and/or a support for a sample-holder and the frame element comprising a recess formed in at least a first side of the frame element adjacent the support and being optically detectable on the first side of the frame element or on a second side of the frame element that is distinct from the first side; and at least one optical device, located adjacent the aperture, that is capable of generating an optical image of the recess and/or an object received in it.
2. A frame element according to Claim 1 wherein the recess is located and dimensioned to receive and permit viewing from the first side of the frame element a needle tip that is moveable from a first side of the frame towards (a) a first focal position corresponding, with respect to the frame element, to an in-use location of a sample supported by the support or by a sample-holder, and (b) a second focal position between the tip of the needle when in the first focal position and the further optical device, as considered along a surface forming part of the frame.
3. Injection apparatus comprising a frame element defining a support for a sample; an injection needle having an elongate axis and that is capable of penetrating a sample supported by the support and injecting thereinto from a first side of the frame element a flowable substance; an optical image generator that is capable of generating an optical image of the sample, on a second side of the frame element that differs from the first side; one or more motors for causing movement between the frame element and the needle in x-, y-and z-directions so as to effect relative positioning of the support and the needle; a recess formed in the frame element adjacent the support so as to open on the first side of the frame element and being optically detectable on the second side of the frame element; a further optical device, located adjacent the recess, that is capable of generating a further optical image; and a processor that is capable of transmitting commands to the motor or motors based on images generated by the optical image generator and the further optical device, or data derived from such images, the motor or motors being capable of manoeuvring the frame element and the needle relative to one another such that the needle penetrates the recess; and the optical image generator and the further optical device being capable of generating optical images representing x-, y-and z-positioning of the needle relative to the recess when penetrated by the needle.
4. Injection apparatus according to Claim 3 wherein the injection needle is a hollow microinjection needle.
5. Injection apparatus according to Claim 3 or Claim 4 wherein the support includes a hollow open sided recess defined in the frame element for supporting from below a sample selected from the list comprising a cell, cellular organism, eukaryote, spore, zygote, oocyte, egg, embryo, nucleus, protein, enzyme a gel or a hydrogel.
6. Injection apparatus according to Claim 5 wherein when the support supports a gel or a hydrogel the gel or hydrogel is selected from the list comprising collagen, agarose, cellulose, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate and chitosan.
7. Injection apparatus according to Claim 5 or Claim 6 wherein the support includes a part-spherical recess having an in-use upwardly directed opening.
8. Injection apparatus according to Claim 7 wherein the second side of the frame element is opposite the first side.
9. Injection apparatus according to any of Claims 3 to 8 wherein the frame element is optically transmissive in the vicinity of the support whereby the optical image generator may generate an optical image of the sample on the said second side of the frame element.
10. Injection apparatus according to Claim 7 or any preceding claim depending therefrom wherein the frame element is transparent in the vicinity of the support.
11. Injection apparatus according to Claim 7 or any preceding claim depending therefrom wherein the frame element is perforated in the vicinity of the support. :30
12. Injection apparatus according to any of Claims 3 to 11 wherein the optical image generator is or includes a camera that is capable of generating electrical signals and transmitting them to the processor.
13. Injection apparatus according to any of Claims 3 to 12 wherein the recess penetrates the frame element such that the needle is capable of protruding on the second side of the frame element when the recess is penetrated by the needle.
14. Injection apparatus according to any of Claims 3 to 13 wherein the needle includes a columnar tip; and wherein the recess Ls of circular cross section of a diameter greater than that of the columnar tip.
15. Injection apparatus according to any of Claims 3 to 14 wherein the further optical device is or includes one or more elements selected from the list comprising a prism and a mirror.
16. Injection apparatus according to Claim 15 including a converter for converting optical signals generated by the further optical device into electrical signals and transmitting them to the processor and/or to the optical image generator.
17. Injection apparatus according to any of Claims 3 to 16 including a plurality of needles and one or more recesses arranged to permit calibration of a plurality of needles.
18. Injection apparatus according to Claim 17 including a plurality of supports and wherein the plurality of recesses and the plurality of supports are arranged in an array defined on or by the frame element.
19. A method of calibrating injection apparatus according to any preceding claim, the method comprising the steps of, before injecting a sample supported by the support, operating the or at least one said motor to position the elongate axis of the needle in register with the recess; generating one or more optical images or signals derived from optical images, using the optical image generator, corresponding to x-and y-axis positioning of the needle relative to the recess, the said x-and y-axes being defined with reference to a plane including a surface of the frame element; operating the or at least one said motor to cause the needle to penetrate the recess until the further optical device generates an optical image or a signal derived from an optical image corresponding to z-axis positioning of the needle relative to the recess, the said z-axis being defined with reference to the said plane including a surface of the frame element; and recording the optical images, or the signals derived therefrom, as calibration signals.
20. A method according to Claim 19 including the step of operating the or at least one said motor to cause withdrawal of the needle from the recess after recording of the optical images or signals derived therefrom.
21. A method according to Claim 20 further including the steps of operating the or at least one said motor to position the needle for injecting a sample supported by the support based on the calibration signals.
22. A method according to any of Claims 19 to 21 including the step of completing a plurality of test injections into one or more samples, the injection height (as defined herein) of the test injections varying from one said test injection to another, the method further including selecting a said injection height that is optimal from the standpoint of sample image quality, and using the selected injection height as a parameter indicative of the focal plane of the optical device when capturing images of samples.
23. A method of using injection apparatus according to any of Claims 1 to 18 or calibrated according to any of Claims 19 to 22, the method comprising the steps of, one or more times, operating the or at least one said motor to position the elongate axis of the needle in register with the recess; operating the or at least one said motor to cause the needle to penetrate the recess; and generating an image of the needle using the optical image generator and/or the further optical device.
24. A method of using injection apparatus according to any of Claims 1 to 18 or calibrated according to any of Claims 19 to 22, the method including a step selected from the list comprising: (a) sequentially injecting at least two samples at a predetermined spacing from one another into a gel or other medium; (b) injecting at least two samples in contact with one another into a gel or other medium; (c) injecting into a first sample a second sample; (d) simultaneously injecting a plurality of samples into a gel or other medium so that they adopt a chosen pattern; 3o (e) injecting one or more samples so as to lie on a surface of a well, a gel or another medium; or (fl injecting one or more samples into a gel or another medium; and injecting one or more samples onto a surface of a gel or other medium.
25. Injection apparatuses generally as herein described, with reference to and/or as illustrated in the accompanying drawings.
26. Methods generally as herein described, with reference to and/or as illustrated in the accompanying drawings.