Reagent delivery apparatus and methods

Apparatus for dispensing droplets of reagent onto samples includes a probe tip to which droplets of reagent can adhere. The apparatus advances the probe tip toward a sample until a droplet of reagent touches the sample and is pulled off from the probe tip. A sensor detects that the droplet has been pulled off from the probe tip and halts the advance of the probe tip before the probe tip touches the sample. Such apparatus may be used to automatically dispense small volumes of reagent onto fragile samples.

TECHNICAL FIELD

This invention relates to the delivery of reagents to specimens. For example, the invention has application to applying reagents to arrayed samples such as microarrays.

BACKGROUND

Some medical tests involve staining individual samples. The samples may be, for example, small pieces of tissue obtained from a subject by way of a biopsy. It is tedious and time consuming to manually stain individual samples. Manually staining individual samples also introduces the possibility of errors.

Conventional staining protocols involve batch staining by incubating volumes of pre-treatment reagent and primary reagents over an array of samples arranged on a slide. The reagents may include antibodies, immunohistochemical staining materials, other markers, or the like. Relatively large volumes of reagents can be required to ensure that all of the samples on a slide are appropriately treated. Reagents can be expensive. Consequently, batch staining is costly and can also compromise the accuracy of results. In addition, batch staining generally requires all of the samples on a slide to be treated with the same reagent(s).

There exist various devices for automatically dispensing reagents. These devices are typically not capable of reliably dispensing sub-microliter quantities of reagent. U.S. Pat. No. 5,143,849 discloses a method for automatically positioning a dispensing tip at a desired distance from a surface onto which liquid is to be dispensed. The method features the formation on the tip of a meniscus of a nominal small volume and advancing the tip and meniscus until the surface is contacted. The resulting decrease in pressure in the tip is measured, to trigger the tip to stop its advance and to start dispensing.

Some existing apparatus for depositing reagents onto samples cannot effectively deliver reagents through liquid coverslip layers that can be used to prevent dessication of samples.

There is a need for efficient and cost effective methods and apparatus for applying reagents to samples in the medical testing field and in other fields.

SUMMARY

The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are given as illustrative examples and are not limiting in scope.

One aspect of the invention provides apparatus for dispensing a reagent onto a sample. The apparatus comprises: a controller; a probe tip, a sensor and an actuator. The sensor is for detecting adhesion of a droplet of reagent on the probe tip. The sensor communicates with the controller. The actuator is coupled to the probe tip and operative to advance the probe tip toward a sample and retract the probe tip from the sample under control of the controller. The controller includes a logic mechanism that causes the controller to: advance the probe tip toward the sample until the sensor detects an alteration in an adhesion of the droplet to the probe tip; and, withdraw the probe tip from the sample in response to the detection of an alteration in an adhesion of the droplet to the probe tip.

Another aspect of the invention provides a method for depositing a reagent onto a sample. The method comprises forming a droplet of the reagent on a probe tip; placing the probe tip near the sample; advancing the probe tip toward the sample and monitoring for an alteration in adhesion of the droplet to the probe tip; allowing the droplet to contact the sample and thereby altering an adhesion of the droplet to the probe tip; and, upon detecting the alteration in adhesion of the droplet to the probe tip, withdrawing the probe tip from the sample.

Another aspect of the invention provides a method for depositing a reagent onto a sample. The method comprises: forming at least one droplet of the reagent on a probe tip comprising first and second electrodes; placing the probe tip near the sample; advancing the probe tip toward the sample and monitoring electrical conductivity between the first and second electrodes; and, upon detecting an alteration in the electrical conductivity between the first and second electrodes, halting advance of the probe tip toward the sample.

Another aspect of the invention provides a probe tip for delivering sub-microliter droplets of reagent to samples, the probe tip has an end surface for holding a droplet of a reagent, a plurality of hydrophilic areas on the end surface and a hydrophobic area between the hydrophilic areas.

Further aspects of the invention and features of various embodiments of the invention are described below and/or shown in the appended drawings.

DESCRIPTION

Small quantities of reagent can be transported to and deposited onto a sample by adhering a droplet of reagent on a probe tip and placing the probe tip near to the sample. If the sample has a sufficient affinity for the reagent then some or all of the reagent will remain on the sample when the probe tip is withdrawn. It is not necessary for the probe tip to contact the sample.

FIG. 1is a conceptual schematic view of apparatus10that includes a probe11having a tip12for delivering reagents to a sample. A droplet14of a reagent is shown adhering to probe tip12.FIG. 1is not to scale. A position control system is provided to move probe tip12relative to a slide16carrying an array of samples18to be treated with one or more reagents. Slide16may be made of glass or some other suitable material. Wells19containing reagents are located near slide16.

The motion control system comprises a controller20that controls actuators22X,22Y and22Z (collectively actuators22). Actuators22may comprise linear actuators, stepper motors, servo motors or the like. Controller20includes drivers suitable for controlling the operation of actuators22. Actuators22X,22Y and22Z are respectively coupled to probe11by linkages23X,23Y and23Z to permit movement of probe tip12along X, Y and Z axes. Any suitable mechanisms may be used to control the position of probe tip12relative to slide16. A wide variety of such mechanisms are known. Controller20may receive feedback regarding the position of probe tip12from position sensors (not shown) or may operate in an open loop mode.

Controller20may comprise one or more data processors executing suitable software, suitable hardware logic circuits or both. In some embodiments, controller20comprises a CPU, such as a CPU in a programmable controller or a computer running software such as, for example, a Labview™ program.

It is convenient but not mandatory that the X and Y axes be orthogonal to one another. All that is necessary is that controller20be able to guide probe tip12to a location adjacent a desired sample18. X and Y actuators22X and22Y could be coupled to move slide16relative to probe tip12. Positioning mechanisms such as rotary tables, sliding tables, linear actuators, rotary actuators or the like could be provided to bring probe tip12and a desired sample18adjacent to one another such that probe tip12can be advanced toward the sample18under the control of controller20.

Controller20includes logic that can be configured to control actuators22to dip probe tip12into a container19to pick up a droplet14of a reagent, move probe tip12until it is adjacent to a sample18and then advance probe tip12toward the sample18until the droplet14is partly or entirely pulled off from probe tip12by its interaction with the sample18.

Probe tip12includes a detector30that provides a signal32to controller20. Signal32changes when droplet14is partly or entirely pulled off from probe tip12by its interaction with the sample18. As described below, detector30may be of a type that can detect the pulling off of a droplet from probe tip12using any of a wide variety of mechanisms. For example, detector30may measure any of:electrical conductivity;capacitance;vibration amplitude and/or amplitude of a vibrator;reflected light;other physical parameters that change when a droplet of reagent is pulled off from probe tip12; or,some combination of these.

Signal32changes when droplet14is partly or entirely pulled off from probe tip12by its interaction with the sample18. By monitoring signal32, controller20can halt the advance of probe tip12toward a sample18before probe tip12contacts the sample18but after reagent from droplet14has been transferred to the sample18.

The volume of each droplet14is determined by the dimensions, configuration and surface properties of probe tip12as well as on the surface tension and other characteristics of the reagent and the rate at which the probe tip is withdrawn from the reagent. Retracting the probe tip from the reagent quickly results in larger droplets adhering to the probe tip while retracting the probe tip from the reagent more slowly results in smaller droplets adhering to the probe tip.

It is practical to make a probe tip12capable of delivering very small quantities of reagent to individual samples. For example, a probe tip12can be designed to apply reagents in quantities of less than 1 μl. Typical designs for probe tip12deliver aqueous reagents in quantities in the range of 1 nl to 1 μl. It can be convenient to dimension probe tip12to carry droplets14of reagents that have volumes of about 10 nl.

Since a system10can deliver small quantities of reagent to each sample18, the volume of reagent needed to treat an array of samples18can be lower as compared to batch staining processes. This can lower the cost of performing assays.

FIG. 2shows an example of a typical microarray slide16. In this non-limiting example, slide16is a tissue microarray. Samples18may be, for example, approximately 2 mm apart and approximately 1 mm in diameter. Reagents can be applied to tissue samples18using a delivery device according to the invention.

The surface of probe tip12is of a material for which the reagents being used have sufficient affinity that a droplet14of reagent can be retained on probe tip12until the droplet is brought into contact with a sample18. The reagent has, overall, a weaker affinity for probe tip12than for samples18so that upon touching a droplet14of reagent to a sample18and then withdrawing the probe tip12, all or at least a significant part of droplet14is transferred from the probe tip12to the sample18.

A probe tip12having these desired characteristics may have some areas that exhibit a relatively high affinity for the reagent and other areas that exhibit a relatively lower affinity for the reagent. For example, where the reagent is water-based, probe tip12may have one or more hydrophilic areas and one or more hydrophobic areas.

FIGS. 3 to 5show a particular probe tip12A that has hydrophilic and hydrophobic areas. Probe tip12A comprises, a pair of relatively hydrophilic areas40. Hydrophilic areas40may comprise, for example, hydrophilic metal surfaces, such as areas of stainless steel. Hydrophilic areas40are surrounded by a relatively hydrophobic material42. A strip43of hydrophobic material separates hydrophilic areas40.

Examples of a hydrophobic material that can be used for material42include PTFE (e.g. Teflon™), Parylene™, epoxide, silicone or hydrophobic plastics such as polyethylene, polypropylene or polystyrene. Hydrophobic areas may also be provided by texturing selected areas of the surface of probe tip12A to provide dense regions of tiny pointed features such as sharp spikes that are hydrophobic because their geometry prevents wetting.

In the probe tip12A ofFIGS. 3 to 5, hydrophilic areas40are provided by the ends of electrodes44. In one embodiment, each electrode44extends through an electrically insulating sleeve45. Sleeves45prevent electrodes44from touching one another. Sleeves44may comprise, for example, tightly fitting glass sleeves.

In probe tip12A illustrated inFIGS. 3 to 5, electrodes44and optional sleeves45, pass through a body of solid, relatively hydrophobic material42. Individuals skilled in the art will recognize that various materials are suitable for use as electrodes44and various other materials are suitable for use as hydrophobic material42. The specific materials chosen are a matter of design convenience.

Probe tips for transferring reagents may have any of various suitable geometries. In the embodiment ofFIGS. 3 to 5, probe tip12A has a flat end surface48. Tips of electrodes44and hydrophobic material42are arranged or ground flat so that the tip surfaces40of electrodes44are flush with end surface48. In some embodiments, end surface48has a diameter in the range of about 0.01 mm to 5 mm. In some embodiments, end surface48has a diameter in the range of about ½ mm to 1 mm. In a prototype embodiment, end surface48has a diameter of ¾ mm.

FIG. 5shows probe tip12A assembled to a probe base49according to a prototype design. In base49, electrodes44connect to larger diameter conductors50. Electrical connections51A and51B connect the electrodes to a resistance measuring device. For example, electrical connection51A may be connected to ground potential while electrical connection51B is connected in series with a resistor to a source of electrical current having a potential of a few volts positive or negative relative to ground. In the illustrated embodiment, electrical connection51B is connected in series with a resistor R to a source at a potential of +5 volts. Current flowing between electrodes44may be determined by measuring a voltage across resistor R. Other suitable means for measuring current flowing between electrodes44could also be provided. Probe base49is held together by heat-shrink tubing52and joined to probe tip12A by an adhesive53, such as a suitable epoxy.

A prototype probe tip constructed substantially as shown inFIG. 3, had the dimensions shown in Table I. This prototype probe tip was used to transfer droplets having volumes of approximately 10 nl to samples.

TABLE IConstruction of Example Prototype Probe TipDiameter of end surface 48 (mm)0.75Diameter of Electrodes 44 (mm)0.25Spacing of electrodes 44 (mm)0.15Material of electrodes 44Stainless steelMaterial around electrodes 44Teflon

Probe tips for transferring reagents may have any of various suitable geometries. Instead of the flat-ended configuration ofFIGS. 3 to 5, it is also possible for a probe tip12to have other configurations. For example,FIG. 6Ashows a probe tip12B having an end surface48A that includes a concave depression55.FIG. 6Bshows a probe tip12C having an end surface48configured as a dihedral. One electrode44is exposed to provide a hydrophilic surface40on each face56of the dihedral.FIG. 6Cshows a probe tip12D having a flat end surface48having an edge57that forms an acute angle with side surfaces58of probe tip12D.FIG. 6Dshows a probe tip12E comprising a pair of spaced-apart electrodes44A having hydrophilic end faces and separated by an air gap59. In the illustrated embodiment, the end faces of electrodes44A are, flat and in the same plane. Other configurations are possible, for example,FIG. 6Eshows a probe tip12F having electrodes44B. The end surfaces of electrodes44B are angled toward one another. As also illustrated inFIG. 6Ethe end surface of each electrode may have an edge that forms an acute angle with side surfaces of the electrode. A probe tip may permit the spacing between electrodes44B to be varied and/or permit the angle of electrodes44B relative to one another to be varied. Any suitable adjustment mechanism may be provided.

FIG. 6Fis an end view of a probe tip12G having first and second coaxial electrodes. A central electrode44C is within and concentric with an annular outer electrode44D. An annular region of a hydrophobic material lies between the electrodes.

Various mechanisms may be provided to detect the adhesion of a droplet to a probe tip. Probe tip12A ofFIGS. 3 to 5uses hydrophilic areas40as electrodes to sense the presence of a droplet14of reagent. This may be done by measuring an electrical current passing between electrodes44. In the illustrated embodiment, an electrical source is connected between electrodes44and an electrical current flowing in one or both of electrodes44is measured. In the absence of a droplet14of reagent on the end of probe tip12electrodes44are electrically well insulated from one another and the electrical current is small or zero. When a droplet14is present on the end of probe tip12A, an electrical connection is created between the two electrodes44so that a measurable electric current flows in at least one of electrodes44.

Other mechanisms for detecting the presence of a droplet of reagent adhering to a probe tip12may also be provided. For example,FIG. 7Ashows a probe tip12E which includes an optical fiber60that terminates at a window62on an end64of probe tip12E. A light source66delivers optical radiation through a beam splitter67into optical fiber60. A light detector68monitors optical radiation reflected at window62. The nature of the reflected radiation will depend upon whether or not a droplet14of reagent is present on the end64of probe tip12E.

Window62may be made partly or entirely hydrophilic. In the alternative, window62may be made hydrophobic and may be located between hydrophilic areas40as shown inFIG. 7B. Hydrophilic areas40may be patterned onto the end of probe tip12E or may be exposed parts of bodies of hydrophobic material embedded in the end of probe tip12E. Hydrophilic areas40may be provided in the form of one or more annular regions surrounding window62.

FIG. 7Cshows a probe tip12F that incorporates another mechanism for detecting the presence of a droplet of reagent on the end of probe tip12F. Probe tip12F includes a vibrator70such as a small piezoelectric element. The frequency of vibration of vibrator70depends upon whether or not a droplet of reagent is adherent at the end48of probe tip12F. An electronic driving circuit72drives vibrator70and monitors its frequency of vibration. In some embodiments, the surface of probe tip12F near vibrator70is hydrophilic. In other embodiments, hydrophobic the surface of probe tip12F near vibrator70is hydrophobic and is located between two or more hydrophilic areas on probe tip12F.

The ability to detect detachment of a droplet of reagent from the end of a probe tip12permits droplets of reagent to be applied to samples without having the probe tip contact the samples.FIG. 8illustrates one method80for applying a reagent to a sample. In block82a droplet of reagent is applied to the end of a probe tip12. Block82may involve, for example, dipping the probe tip12into an appropriate reagent. A small droplet of the reagent adheres to the end of the probe tip when the probe tip is withdrawn from the reagent. In some embodiments, wells or other containers each containing a quantity of a reagent are located at positions known to a controller20(seeFIG. 1for example). Controller20operates X-axis and Y-axis actuators22X and22Y to position probe tip12over a container holding a desired reagent and then operates Z-axis actuator22Z to advance probe tip12into the reagent and then to withdraw probe tip12.

In optional block84, controller20uses a droplet detection mechanism to verify that a droplet of reagent is present on the end of probe tip12. For example, where the probe tip12is a probe tip12A as shown inFIG. 3, controller20may measure a signal indicative of the electrical resistance between electrodes44and may proceed if the electrical resistance is lower than a threshold value.

In block86probe tip12is positioned over a sample to which the reagent is to be applied. Block86typically comprises controller20using coordinates of the sample in a suitable coordinate system and operating X-axis and Y-axis actuators22X and22Y to position probe tip12over the sample.

The droplet is dispensed in block88. To dispense the droplet onto a sample, probe tip12is advanced toward the sample until the droplet14of reagent comes into contact with the sample.FIG. 9shows a probe tip12having a droplet14touching a sample18. The droplet has an affinity from the sample and is attached to and/or absorbed into the sample18. Sample18may be, for example:a tissue biopsy,a mass of cells from an animal, plant, bacteria or person, or the like,a composition including viruses,a single cell from an animal, plant bacteria or person, or the like,a deposit of biological material such as DNA or RNA, ora material comprising DNA or RNA.
The interaction between the droplet14and sample18is such that the droplet at least sticks to sample18.

InFIG. 9, samples18are shown as being covered by a layer19of a liquid coverslip material. A liquid coverslip may be applied to prevent samples18from drying out and/or to guard against any fluid crosstalk between different samples18. Layer19may comprise a hydrophobic layer (for example a layer of a hydrocarbon-based liquid coverslip). Layer19provides a fluidic boundary between samples18. Layer19may be, for example, 2-5 mm in thickness.

In some cases droplet14absorbed by the sample after it comes into contact with the sample and is consequently pulled away from the end of probe tip12. In such cases, controller20can determine that droplet14has come into contact with sample18when detector30detects that droplet14has been pulled away from the end of probe tip12. In such cases block88may comprise advancing the probe tip toward the sample and halting the advance when detector30detects that droplet14has been pulled away from the end of probe tip12.

A droplet14not in contact with a sample18projects from the end surface48of probe tip12by an amount D (seeFIG. 9A). The droplet thickness, D is determined primarily by the physical dimensions of end surface48, the nature of the interaction between the reagent, the material(s) of end surface48, and the nature of the medium surrounding droplet14. In a number of the embodiments described above, droplet14is attracted to two or more hydrophilic areas40and bridges a hydrophobic area43between the hydrophilic areas. Where droplet14bridges a hydrophobic area, the energy of the droplet14is increased. This tends to make it easier for droplet14to be pulled off of a probe tip12when the droplet comes into contact with a sample18.

In some embodiments, block88comprises serially advancing probe tip12toward sample18by increments ΔZ that are smaller than D and then checking detector30to determine whether or not droplet14has been pulled away from the end of probe tip12after each incremental advance. If droplet14has been pulled away from the end of probe tip12then controller20halts the advance. If not, controller20advances probe tip12by another increment ΔZ. Preferably ΔZ is significantly less than D, and is preferably less than ½D.

In other embodiments, probe tip12is advanced toward sample18in small discrete steps. Each step moves probe tip12closer to sample18by a distance increment ΔZ+. After each step, probe tip12is retracted by a distance increment ΔZ−and detector30is checked to determine whether or not droplet14has been pulled away from the end of probe tip12. In such embodiments, ΔZ+can be larger than D as long as ΔZ+−ΔZ−is smaller than D. Preferably ΔZ+−ΔZ−is less than ½D or ¼D. In some embodiments, ΔZ+−ΔZ−is about 10% of D or less. In some embodiments, ΔZ+−ΔZ−is on the order of about 100 μm.

FIG. 10illustrates a possible trajectory of a probe tip12. The vertical axis illustrates the distance from the end of probe tip12to a sample18. The horizontal axis indicates time. Probe tip12is initially positioned a safe distance above sample18. Then probe tip is advanced by a distance ΔZ+and retracted by the distance ΔZ−. Since ΔZ+>ΔZ−the net result of this motion is that the probe tip moves closer to the sample by a distance ΔZ+−ΔZ−. After each retraction, controller20checks to determine if electrical contact is still present between the electrodes. When the electrical connection is lost, it is known that droplet14has been pulled off from the end of probe tip12. The probe tip is then retracted. As long as the controller20detects that the drop has been pulled off from probe tip12before probe tip12touches sample18, the probe tip is prevented from ever coming into contact with the sample18.

A dispensing apparatus may incorporate a mechanism to prevent damage to a probe tip12in case the probe tip12is driven into a slide16due to some failure. Any one or more of a wide variety of such safety mechanisms may be provided. For example, a force sensor may be provided to detect forces applied to the probe tip. Controller20may be configured to monitor an output of the force sensor and to stop moving the probe tip if excessive forces are detected.

Another safety mechanism shown inFIG. 11mounts an assembly100carrying probe tip12on a sliding mount102. Sliding mount102can slide vertically. In the illustrated embodiment, sliding mount102comprises a collar comprising low-friction rollers (not shown) that engage a vertically-extending rail106. Z axis actuator22Z (not shown inFIG. 11) is connected to move rail106vertically.

Normally the weight of probe tip assembly100and sliding mount102keeps probe tip12in a lowermost position against an end stop (not shown). If Z axis actuator22Z attempts to drive the end of probe tip12into a rigid surface, such as slide16, sliding mount102moves upward on rail106, thereby preventing probe tip12from being driven into the surface. The force on the end of probe tip12is essentially limited to the weight of probe tip assembly100and sliding mount102.

A detector110detects vertical motion of sliding mount102. Operation of Z axis actuator22Z in a direction toward the slide16is inhibited in response to detector110detecting motion of sliding mount102. In a simple embodiment, motion of sliding mount102opens an electrical connection between an electrode112A mounted to sliding mount102and another electrode112B that is fixed in the Z-axis direction. When this electrical connection is broken, motion of probe tip12in a downward direction is immediately stopped.

FIGS. 12 and 13are schematic front and top views of a prototype apparatus for delivering droplets of reagent to samples. In this prototype, a probe tip12is carried on a three-axis positioner120made up of an X-axis stage120X that carries a Y-axis stage120Y. A Z-axis stage120Z is mounted to Y-axis stage120Y by supports123. The stages of positioner120include suitable actuators that can be controlled by a controller (not shown inFIG. 12or13) to move probe tip12in three orthogonal directions.

A fixture125is mounted in front of positioner120. Fixture125holds one or more slides16and supplies of reagents. In the illustrated embodiment, fixture125has a number of wells127for holding reagents.

It can be desirable to provide a mechanism for cleaning probe tip12. A cleaning procedure may be performed between spotting reagents onto samples to remove any liquid coverslip or any reagent that might still be adhering to probe tip12. A cleaning procedure may, for example, involve dipping probe tip12into a suitable solvent and then delivering a short blast of air to probe tip12. The air blast removes any solvent from the probe tip. Any suitable solvent may be used. Solvents that may be appropriate for certain applications include but are not limited to: aromatic or aliphatic hydrocarbons, xylene, ethanol, methanol and combinations thereof.

In an example embodiment of the invention, after delivering a spot of a reagent to a sample18, controller20automatically moves probe tip12to a location over a container of a suitable solvent, advances probe tip12into the solvent, retracts probe tip12, moves probe tip12to a location over an orifice, and opens a valve to cause a blast of air to be delivered through the orifice. The cleaned probe tip12is then moved to a location to collect a droplet of reagent for the next sample18. Optionally the cleaning cycle may involve sequentially dipping probe tip12into two or more solvent baths.

Fixture125may include a cleaning station comprising one or more reservoirs containing solvents or other cleaning materials and/or an air blast orifice.FIG. 13shows a fixture125having an air blast orifice128. Solvents may be provided in one or more of wells127.

For controller20to move probe tip12to a location corresponding to a particular sample, controller20must have available information defining a the location of the sample. This information may be made available to controller20in any of a number of ways. The following are some examples of such ways:Controller20may control the initial placement of samples18on slide16. For example, samples18may be placed using probe tip12or a suitable manipulation device mounted in place of probe tip12.Samples18may be placed at predefined locations on slide16. The predefined locations are defined relative to reference features on slide16, such as edges of slide16. Apparatus10may include pins or other registration members so that each slide16can be repeatably mounted with its registration features at known locations in a coordinate system used by apparatus10. In the embodiment ofFIGS. 12 and 13the registration members may be fixed to fixture125.As shown inFIGS. 12 and 13, a camera130may be located above slide16. Camera130may acquire one or more images of slide16in which samples18can be seen. The locations of samples18in a coordinate system of controller20can then be determined from the locations at which the samples18appear in the images acquired by camera130using any suitable image-processing techniques. Various algorithms suitable for locating objects in images are well known and are therefore not described herein. Such algorithms may be applied by providing computer software executing on a data processor, by providing hardware that implements the algorithms or by some combination of these. To facilitate locating samples18it is desirable to illuminate slide16obliquely. For example, light may be directed onto slide16from a homogenous light source132at an angle of, for example, 45 degrees. This light is internally reflected in slide16and liquid coverslip19. Samples18scatter light. This causes samples18to show up as bright spots against a dark background. Marks may be provided on the apparatus within a field of view of the camera for use in calibrating the camera.

The apparatus described above provides probe tips12that can hold small droplets of reagent. The reagent has a smaller overall affinity for the probe tip than it does for the sample. When the droplet of reagent is put in contact with a sample, the droplet adheres to the sample. When the droplet is adherent to the sample, he droplet will be pulled off of the probe tip if the probe tip is retracted. By approaching the sample in a manner that steps toward the sample and then steps back a sensor associated with the probe tip can detect when the droplet has adhered to the sample because the sensor can detect that the droplet has been pulled off of the probe tip when the probe is withdrawn. In some cases, the droplet may have such a strong affinity for the sample that it is drawn into or onto the sample. If this occurs strongly enough then it may not be necessary to approach the sample in a way that involves stepping back as the droplet will be pulled off from the probe tip as soon as it contacts the sample.

FIGS. 14 and 15show an alternative mode of operation. These Figures show a probe tip212that has a pair of electrodes244. Electrodes244are spaced far enough apart from one another that each electrode244retains a separate droplet214of a reagent as probe tip212is removed from a reservoir of the reagent. The volume of each small droplet of reagent is defined primarily by the diameter of each electrode244.

A controller220monitors an electrical conductivity between electrodes244. The electrical conductivity is initially very low, as indicated by line221because droplets214are not touching and electrodes244are electrically insulated from one another.

Probe tip212is then slowly lowered toward a sample18while monitoring the electrical conductivity between electrodes244. As the droplets214of reagent on the electrodes contact sample18, sample18is wetted and an electrical circuit is established between electrodes244. This causes controller220to measure an increase in conductivity, as indicated by line222inFIG. 9. The increase of conductivity indicates that the reagent has been transferred to the sample18. Upon detecting such an increase in conductivity, controller220can stop the advance of probe tip212toward the sample18.

The methods and apparatus described herein may be applied, without limitation, to deliver reagents for immunohistochemical staining (IHC) or probes for fluorescence in situ hybridization (FISH). The samples may be, for example, individual tissue biopsies, fine needle aspiration biopsies that are arrayed as tissue cores, or groups of cells.

In some embodiments, an array of samples is provided by embedding an array of tissue biopsies in a block of paraffin. A thin slice of the paraffin block is placed onto a slide. The paraffin can them be removed, for example by applying standard techniques using ethanol and xylene, to leave the array of biopsies on the slide. A liquid coverslip such as a suitable oil coating can then be applied over the array of biopsies to prevent the biopsies from drying out.

It can be appreciated that devices as described above may be made and operated to provide a number of advantages. These include:An automated system may be controlled to apply different reagents to adjacent samples in an array of samples.Fluid crosstalk between array elements is prevented.Very tiny droplets of reagents may be dispensed, thereby reducing consumption of expensive reagents in comparison to batch methods.Droplets can be dispensed onto a sample that is covered by a liquid coverslip layer or a layer of an oily liquid.The probe tip does not need to contact the samples (unlike pin printers, for example). This is especially advantageous where the samples are fragile samples that might be damaged by contact.An automated system can deliver reagents to samples that are closely spaced apart on a high-density microarray slide.An IHC system that applies methods and apparatus as described herein can allow for customized tissue core pre-treatment incubation with reagents followed by application of antibody probes to specifically designated tissue cores or cell groups on a tissue or cell microarray. For example, these methods and apparatus can facilitate a protocol for immunohistochemical staining which includes customized application of small volumes of pre-treatment reagents to break protein cross linking, followed by small volume application of antibody probes to each specific tissue biopsy within the microarray.
Except as specifically recited in the appended claims, it is not mandatory that all or any of these advantages be provided by any specific embodiment of the invention.

In some cases it may be desirable to provide mechanisms for preventing fluid crosstalk between samples18in addition to or instead of a liquid coverslip. In such cases crosstalk can be prevented by placing the array of samples18on a hydrophobic substrate. The process used to bind samples to slides should be compatible with the hydrophobic substrate. For example, where the hydrophobic substrate comprises a coating on the surface of a slide and a paraffin process will be used to bind samples to the slide, the coating should be robust enough to withstand heating and immersion in a solvent during removal of paraffin from the samples.

In the alternative, hydrophobic boundaries between samples18may be applied after an array of samples has been loaded onto a reagent delivery system as shown for example inFIG. 12and sample locations have been determined. Hydrophobic boundaries may be created, for example, by spotting a hydrophobic material onto the surface of a slide16with an inkjet-type spotter capable of dispensing hydrophobic fluid, or by using a contact/stamp method.

In cases where samples are sensitive to heat or humidity, sample preservation can be enhanced by strictly controlling both the temperature and humidity of the samples. Sample temperature can be controlled by thermally contacting the array backing to a cooler, for example, a Peltier cooler, and a temperature sensor, for example, a thermocouple. In some embodiments, the controller may be configured to operate the cooler to maintain a slide carrying a plurality of samples below a threshold temperature until the apparatus has applied reagent to a plurality of samples on the slide; and, subsequently warm the slide. A closed loop humidity control can also be provided if the microarray is enclosed so that a controlled atmosphere can be provided at the locations of the samples.

As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. For example:A delivery device as described herein can be used to both pick up, and deposit fluidic reagents to each sample.It is not necessary that the samples be held fixed while the probe tip moves. All that is required is an ability to position a probe tip12relative to a sample18. Samples18may be moved while probe tip12is held fixed or samples18may be movable in one or more dimensions while probe tip12is movable in one or more dimensions. For example, samples18and supplies of any required reagents may be supported on a two-axis adjustable stage. Probe tip12may be mounted on a one-axis stage. After the two-axis stage has been adjusted to align a sample with probe tip12, the one-axis stage can be used to advance probe tip12toward the sample.By using smaller probe tips one can deposit smaller amounts of reagent. For example, by using probe tips having diameters on the order of 1 μm in diameter one can obtain droplets of reagent having volumes on the order of 1 femtoliter. Such small volumes of reagent could be used, for example, to treat single cells. Probes of such small sizes may be made by micromachining techniques.Dispensing apparatus as described herein may be used to deposit droplets of oily liquids. In this case, the probe tip should have an affinity for the oily liquid to be dispensed that is lower than an affinity of the oily liquid for the sample such that the droplet of oily liquid will adhere to the sample such that it will be pulled off of the probe tip if the probe tip is withdrawn and/or be pulled off of the probe tip by being absorbed into or onto the sample when the droplet touches the sample. In some cases it can be advantageous to provide on the probe tip oleophilic areas separated by an oleophobic area (e.g. by replacing the relatively hydrophilic areas with relatively oleophilic areas and by replacing the relatively hydrophobic areas with relatively oleophobic areas in the embodiments described above).A probe tip does not necessarily have to have areas that are relatively hydrophilic and hydrophobic or relatively oleophilic and oleophobic. The surface of the end portion of a probe tip could be uniformly hydrophilic, hydrophobic, oleophilic and/or oleophobic as long as one or more droplets of the desired reagent can be carried on the probe tip.
Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.