Method and apparatus for single particle deposition

Abstract

A method and droplet dispenser for depositing single particles onto a target. For example, a single particle depositing method with improved rate of dispensing single particles and/or increased recovery of rare particles and/or with an extended applicability with different types of particles and/or operation conditions. The depositing method may be capable of increasing the rate of dispensing single cells without decreasing the recovery rate. Testing a single particle condition is combined with testing a zero particle condition and/or the particle type condition. The ejection and sedimentation regions are tested with regard to the presence of a single particle in the ejection, and further particle arrangements allowing a single particle deposition are identified and tested and/or the particle type detection is added to the dispenser control. Accordingly, the speed and recovery rate of dispensing single particles of interest can be improved.

Claims

1. A method of depositing single particles via a droplet dispenser onto a target, wherein the droplet dispenser has a suspension reservoir and a nozzle having an ejection region and a sedimentation region upstream the ejection region, the droplet dispenser being capable of dispensing operation wherein a particle suspension having the particles flows from the suspension reservoir through the nozzle towards a terminal opening of the nozzle at the ejection region and droplets of the particle suspension are dispensed through the terminal opening to the target, nozzle camera is arranged for a particle detection including detecting particles in the particle suspension within the nozzle, a controller is arranged in electronic communication with the nozzle camera and with a testing unit including an image data storage for storing image data of the ejection region and the sedimentation region; the method comprising: loading the particle suspension having the particles to the droplet dispenser having the suspension reservoir and the nozzle, wherein the droplet dispenser is capable of dispensing droplets having the particle suspension at a first droplet volume; detecting a presence of the particles in the particle suspension in the nozzle via the nozzle camera; controlling the droplet via the controller dispenser based on a test procedure, which includes (i) testing a single particle condition of the droplet dispenser via the testing unit to determine the single particle condition is fulfilled when an ejection region of the nozzle adjacent the terminal opening includes one single particle of the particles and the sedimentation region adjacent to and upstream the ejection region is free of the particles, wherein in the sedimentation region the particles are displaced to the ejection region by sedimentation between the step of detecting the presence of the particles and the step of controlling the droplet dispenser, and (ii) testing at least one of a zero particle condition of the droplet dispenser via the testing unit to determine the zero particle condition is fulfilled when a volume equal to or larger than a 2-fold volume of the ejection region beginning at a tip of the nozzle to the sedimentation region is free of particles to be dispensed, and a particle type condition via the testing unit to determine the particle type condition is fulfilled when the detected particles have a predetermined particle type identified by at least one of size, shape, colour emission and absorption; and operating the droplet dispenser via the controller, wherein, in accordance with the testing determinations of (i) the single particle condition, and (ii) at least one of the zero particle condition and the particle type condition, one of the droplets is dispensed from the droplet dispenser onto the target or at least one of the droplets is discarded from the droplet dispenser into a collection reservoir.

2. The method according to claim 1, wherein the step of testing the zero particle condition is conducted when the single particle condition is not fulfilled, and includes determining the zero particle condition is fulfiled when the volume of the droplet dispenser is free of the particles, wherein the step of operating the droplet dispenser includes discarding at least two droplets from the droplet dispenser when the zero particle condition is fulfilled, and discarding one droplet from the droplet dispenser when the zero particle condition is not fulfilled.

3. The method according to claim 2, wherein when one of the particles is detected within the volume of the droplet dispenser, the step of operating the droplet dispenser includes discarding a number of the droplets from the droplet dispenser required for moving the detected particle from the position within the ejection region.

4. The method according to claim 3, wherein the controller determines the number of droplets to be discarded during the controlling the droplet dispenser step by tracking particles in the nozzle during dispensing droplets, determining a particle displacement per dispensing step and determining the number of droplets to be discarded by dividing the volume that is free of particles by the particle displacement per dispensing step.

5. The method according to claim 3, wherein a number of droplets to be discarded is determined via the controller by partitioning the nozzle into droplet regions being numbered according to the number of droplets required for moving one of the detected particles from the respective droplet region to the ejection region, and determining the number of the droplet region of the one detected particle.

6. The method according to claim 2, wherein the predetermined volume has an axial length along the nozzle equal to a field of view of the nozzle camera used for testing the zero particle condition.

7. The method according to claim 1, wherein, when the single particle condition within the ejection region is not fulfilled, testing the single particle condition further includes reducing the ejection region in size to an ejection region subsection and determining whether the ejection region subsection includes one single particle and the sedimentation region adjacent to the ejection region subsection is free of particles, wherein the step of controlling the droplet dispenser includes dispensing a fractional droplet having a fraction of the first droplet volume onto the target when the single particle condition is fulfilled with the ejection region subsection.

8. The method according to claim 7, wherein, when the single particle condition within the ejection region subsection is not fulfilled, testing the single particle condition further includes reducing the ejection region subsection in size and testing the single particle condition with the reduced ejection region subsection.

9. The method according to claim 7, wherein the step of testing a single particle condition of the droplet dispenser ejection subjection includes testing a plurality of ejection region subsections, each having a different size.

10. The method according to claim 7, wherein the fraction of the first droplet volume is determined by the controller according to the position of the single particle in the ejection region subsection.

11. The method according to claim 1, wherein the step of testing the particle type condition via the testing unit includes defining types of particles in the droplet dispenser by at least one of size, shape, colour emission and absorption, and testing whether the particle in the ejection region is the predetermined particle type, wherein the step of controlling the droplet dispenser via the controller includes dispensing a droplet including the particle having the predetermined particle type onto the target or discarding the droplet into the collection reservoir.

12. The method according to claim 11, wherein the particles include at least two particle types of interest, the step of testing the particle type condition includes determining the particle type of a particle in the ejection region or ejection region subsection, and the step of operating the droplet dispenser includes dispensing a droplet at a target position selected based on the predetermined particle type particle in the ejection region or an ejection region subsection.

13. The method according to claim 12, wherein the step of operating the droplet dispenser includes dispensing droplets from the nozzle on different targets based on different particle types.

14. The method according to claim 11, wherein the particles include one particle type of interest, the step of testing the particle type condition includes determining the particle type of a particle in the ejection region or an ejection region subsection, and if the determined particle type of the particle in the ejection region or the ejection region subsection is the particle type of interest or not, the step of controlling the droplet dispenser correspondingly includes dispensing a droplet at a target position or discarding a droplet.

15. A method of depositing single particles via a droplet dispenser onto a target, wherein the droplet dispenser has a suspension reservoir and a nozzle having an ejection region and a sedimentation region upstream the ejection region, the droplet dispenser being capable of a dispensing operation wherein a particle suspension having the particles flows from the suspension reservoir through the nozzle towards a terminal opening of the nozzle at the ejection region and droplets of the particle suspension are dispensed through the terminal opening to the target, a nozzle camera is arranged for a particle detection including detecting particles in the particle suspension within the nozzle, a controller is arranged in electronic communication with the nozzle camera and with a testing unit including an image data storage for storing image data of an ejection region and a sedimentation region the method comprising: loading the particle suspension having the particles to the droplet dispenser having the suspension reservoir and the nozzle, wherein the droplet dispenser is capable of dispensing droplets having the particle suspension at a first droplet volume; detecting a presence of the particles in the particle suspension the nozzle via a nozzle camera; controlling the droplet dispenser via the controller based on a test procedure, which is capable of testing a single particle condition of the droplet dispenser via a testing unite to determine the single particle condition is fulfilled when an ejection region of the nozzle includes one single particle of the particles and eth sedimentation region adjacent to and upstream the ejection region is free the particles, wherein in the sedimentation region the particles are displaced to the ejection region by sedimentation between the step of detecting the presence of the particles and a step of controlling the droplet dispenser; and operating the droplet dispenser via the controller, wherein, in dependency on the single particle condition fulfilment, one of the droplets is dispensed from the droplet dispenser onto the target or at least one of the droplets is discarded from the droplet dispenser into a collection reservoir, wherein when the step of testing the single particle condition with the ejection region is not fulfilled, testing the single particle condition further includes reducing the ejection region according to an ejection region subsection and determining whether a current ejection region subsection includes one single particle and the sedimentation region adjacent to the current ejection region subsection is free of particles, and dispensing a fractional droplet having a fraction of the first droplet volume onto the target if the single particle condition is fulfilled with the current ejection region subsection.

16. The method according to claim 15, wherein, if the step of testing the single particle condition with the ejection region subsection is not fulfilled, testing the single particle condition further includes further reducing the ejection region and testing the single particle condition with the further reduced ejection region subsection.

17. The method according to claim 15, wherein the fraction of the first droplet volume is obtained by determining the position of the single particle in the ejection region subsection and estimating the fractional droplet volume based on the particle position in a mapping step.

18. A dispenser apparatus, being adapted for dispensing droplets including single particles onto a target, comprising: at least one droplet dispenser having a suspension reservoir and a nozzle configured to include a particle suspension, an ejection region and a sedimentation region upstream of the ejection region; a dispenser head arranged for carrying the at least one droplet dispenser; a nozzle camera arranged for detecting particles in the nozzle; a control device arranged electrical communication with the nozzle camera and for controlling the at least one droplet dispenser; and a testing unit coupled with the nozzle camera and with the control device and being adapted for (i) testing a single particle condition of the droplet dispenser, wherein during testing it is determined whether the ejection region of the nozzle includes one single particle and the sedimentation region adjacent to the ejection region is free of particles, wherein in the sedimentation region the particles are displaced to the ejection region by sedimentation during an operation delay interval between a step of detecting the presence of particles and a step of operating the droplet dispenser, and (ii) testing at least one of a zero particle condition of the droplet dispenser, wherein it is determined whether a predetermined volume equal to or larger than a 2-fold volume of the ejection region beginning at a tip of the nozzle of the droplet dispenser to the sedimentation region is free of particles to be dispensed, and a particle type condition, wherein it is determined whether detected particles have a predetermined particle type identified by at least one of size, shape, colour emission and absorption, wherein the control device is adapted for controlling the droplet dispenser based on the single particle condition, at least one of the zero particle condition and the particle type condition, a droplet is dispensed onto the target or discarded into a collection reservoir.

19. The dispenser apparatus according to claim 18, wherein the testing unit includes an image data storage being adapted for storing image data of the ejection region and the sedimentation region.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Further details and advantages of the invention are described in the following with reference to the attached drawings, which show in:

(2) FIG. 1: a flow chart illustrating features of preferred embodiments of the inventive single particle dispensing method;

(3) FIG. 2: a schematic view of features of a dispenser apparatus according to preferred embodiments of the invention;

(4) FIG. 3: a schematic cross-sectional view of a nozzle section with an illustration of an ejection region, a sedimentation region and a travel region;

(5) FIG. 4: a flow chart illustrating further details of testing the zero particle condition according to a preferred embodiment of the inventive single particle dispensing method;

(6) FIG. 5: a schematic cross-sectional illustration of partitioning the nozzle section into numbered droplet regions;

(7) FIG. 6: a schematic cross-sectional illustration of defining ejection region subsections;

(8) FIG. 7: a flow chart illustrating further details of extended testing the single particle condition according to a preferred embodiment of the inventive single particle dispensing method;

(9) FIGS. 8 and 9: examples of particle distributions detected with the extended testing the single particle condition according to FIG. 7; and

(10) FIG. 10: a flow chart illustrating further details of testing the particle type condition according to a preferred embodiment of the inventive single particle dispensing method.

DETAILED DESCRIPTION OF THE INVENTION

(11) Features of preferred embodiments of the invention are described in the following with exemplary reference to a dispenser apparatus including at least one PDC, like e.g. the apparatus sciFLEXARRAYER (manufacturer: Scienion AG, Germany). It is emphasized that the application of the invention is not restricted to this particular model or PDC-based systems, but rather correspondingly possible with other types of non-contact droplet dispensers, like e.g. solenoid valve controlled dispensers. Furthermore, particular reference is made in the following to the application of the inventive single particle depositing method and the adaptation of the dispenser apparatus for implementing the method. Details of a dispenser apparatus, which are known from conventional systems, are not described. Furthermore, exemplary reference is made to dispensing droplets including single biological cells. The application of the invention is not restricted to dispensing single cells, but correspondingly possible with other biological or non-biological particles.

(12) FIG. 1 illustrates a flow chart representing features of preferred embodiments of the inventive single particle deposition method. The method is illustrated with three tests, including testing the single particle condition (step S6), testing the zero particle condition (step S9) and testing the particle type condition (step S7). The implementation of these tests can be provided in dependency on the requirements of a practical dispensing application. With preferred embodiments, testing the single particle condition (step S6) can be combined with testing the zero particle condition (step S9) only (see FIG. 4), e. g. if there is only one particle type in the suspension liquid. Alternatively, testing the single particle condition (step S6) can be combined with testing the particle type condition (step S7) only, e. g. if the suspension liquid includes a relative high density of particles, so that testing the zero particle condition (step S9) is expected to provide always a negative result. As a further alternative, all tests can be combined as shown in FIG. 1, or only the single particle condition is tested (step S6) in extended manner as shown in FIG. 7. Details of the procedure of FIG. 1 can be modified, e. g. as shown in FIGS. 4, 7 and 10.

(13) According to FIG. 1, after the start of the procedure, a nozzle setup and alignment (step S1) and a step of collecting base line images (step S2) are conducted. With step S1, the droplet dispenser 10 is aligned relative to the collection reservoir 5 and the nozzle camera 30 at the fixed position on the platform 23 (see FIG. 2, described below). Collecting base line images with step S2 includes the collection of images of the nozzle section 12, in particular the ejection region 13 and the downstream end of the travel region 15 thereof (see FIG. 3, described below).

(14) Subsequently, mapping of the nozzle section 12 of the droplet dispenser 10 and calculating the sedimentation region 14 are conducted with step S3. To this end, a portion of the sample to be dispensed or test particles or test beads having similar properties like the particles to be deposited are loaded to the dispenser. The mapping step includes an identification of the ejection and travel regions 13, 15 for the particular sample and dispensing parameters to be used. Multiple dispensing operations are conducted with the particles or test beads loaded to the dispenser. The number of training dispensing operations is dependent on particle concentration and is typically between 100 to 10000 droplets. The sedimentation region 14 is calculated on the basis of one of the above static or dynamic calculation methods.

(15) The ejection and travel regions 13, 15 (see FIG. 3, described below) are identified by tracking and characterization of a number of particles as they pass through the nozzle section 12 during the dispensing operation. As droplets are produced by the droplet dispenser 10, particles within the nozzle section will exhibit one of the following two behaviours: travel or ejection. The mapping step identifies these behaviours and correspondingly localizes the ejection and travel regions 13, 15 within the nozzle section.

(16) The inclusion of the sedimentation region 14 upstream of the ejection region 13 prevents multiple cells from falsely being dispensed as they sediment into the ejection region 13 while the droplet dispenser 10 is in between dispensing routines. The sedimentation region 14 can be a dynamic region which is calculated based on the sedimentation velocity which is a function of the apparent particle size along with the time required for image processing and to move the PDC into position for printing.

(17) Step S3 is performed for every new droplet dispenser 10, dispensing parameters or samples (particle suspension) utilized. Thus, a unique map including the ejection region 13 and at least one sedimentation region 14 (each for one particle type) is created for each new dispensing operation, which is used in the subsequent optical feedback test for single particle encapsulation.

(18) Once trained, the particle dispensing operation can commence with the particles, e. g. cells to be dispensed. The droplet dispenser 10 is positioned in front of the optical system where each individual particle within the droplet dispenser 10 is identified and tracked in real time. For single particle encapsulation, the optical feedback system will selectively identify single particles for printing while accounting for the potential effects which particles upstream of the nozzle may have on the encapsulation outcome.

(19) The practical operation of the dispenser apparatus 100 for depositing single particles on the target 2 starts with step S4 (FIG. 1), wherein the particle suspension is loaded into the droplet dispenser 10. The droplet dispenser 10 is aligned relative to the collection reservoir 5 and the nozzle camera 30 (see FIG. 2). The droplet dispenser 10 is positioned in front of the nozzle camera 30, which acquires an image of the nozzle section with step S5. The acquired image is aligned with the base line image collected with step S2, and the presence of particles within the nozzle section 12 is detected. Each individual particle within the nozzle section 12 is identified and tracked in real time.

(20) With step S6, the single particle condition of the droplet dispenser 10 is tested as described in European patent application No. 16000699.5 or in extended manner as described below with reference to FIG. 7. For instance, according to European patent application No. 16000699.5, the acquired image of step S5 is examined for testing whether no particles are located in the sedimentation region 14 and one single particle is located in the ejection region 13 (see FIG. 3).

(21) Advantageously, the inclusion of the sedimentation region 14 upstream of the ejection region 13 prevents multiple particles from falsely being dispensed as they sediment into the ejection region 13 while the droplet dispenser 10 is in between dispensing routines. During the dispensing operation, the optical feedback system can dynamically update the map, in particular the identification of the ejection region 13 if necessary to compensate for any shifts in the dispensing parameters, e.g. shifts in droplet volume.

(22) The sedimentation region 14 (see FIG. 3) can be modelled as static or dynamic region as mentioned above. The dynamic sedimentation region can be calculated based on the expected sedimentation distance of each identified particle to account for the inherent polydisperse particle size range. This may be important as the sedimentation velocity has an exponential relationship with the particle diameter D. Alternatively, the static sedimentation region 14 would be utilized if a preset approximation of the maximum sedimentation distance expected within the droplet dispenser 10 would be sufficient for the particular deposition task.

(23) If testing the single particle condition is negative, e. g. if no or multiple particles are present within the ejection region 13, the step of testing the multiple particle condition (step S9) is conducted (see FIG. 4), resulting in discarding at least two droplets (step S10) or one droplet (step S11). Alternatively, step S9 can be omitted, and the current droplet considered is directly discarded with step S11 by operating the droplet dispenser at the collection reservoir 5. After steps S10 or S11, the next image is acquired with step S5, followed by another test with step S6.

(24) If testing the single particle condition (step S6) is positive, the particle type condition can be tested with step S7, as described below with reference to FIG. 10. Subsequently, if a particle having a type of interest is detected, the droplet dispenser 10 is moved to the target 2, and a droplet including the single particle is deposited on the target 2 with step S8. Otherwise, a droplet is discarded (step S11).

(25) After step S8, another test can be conducted with step S12, wherein it is tested whether all fields on the target 2 are spotted, e.g. by analysing a camera image of the target or using recorded position data of droplets dispensed during previous operation. If yes, the procedure stops. If not, the droplet dispenser 10 is moved again to the collection reservoir 5 and the nozzle camera 30 (step S13) for acquiring the next image with step S5.

(26) Advantageously, the optical feedback system of the invention enhanced by the mapping pre-process and the consideration of the upstream particle's motions in the sedimentation region 14 allows for a reliable single particle dispensing platform with minimized false positive droplets. A test printing was performed using heterogeneous primary lung cancer cells suspended at 100.000 cells/ml. Imaging of the grid post-dispensing in experimental tests shows that each droplet at one of 100 dispensing positions on the target encapsulated one single cell.

(27) FIG. 2 schematically illustrates a preferred embodiment of a dispenser apparatus 100, which is adapted for dispensing droplets including single particles, like single biological cells, onto a target 2. The dispenser apparatus 100 comprises at least one droplet dispenser 10, which is attached to a dispenser head 20. With practical examples, one single droplet dispenser 10 or a line or matrix array of droplet dispensers 10 can be provided.

(28) The at least one droplet dispenser 10 is a PDC, including a suspension reservoir 11 and a nozzle section 12 (see FIG. 3) as well as a piezo-electrically activated drive unit (not shown). With more details, the droplet dispenser 10 is composed of a glass capillary connected to a syringe pump (not shown) for sample loading and washing between different samples. The drive unit includes a piezo-ceramic element positioned around the glass capillary. By applying a control voltage to the piezo-ceramic element, a dispensing operation of the droplet dispenser is triggered.

(29) The dispenser head 20 onto which the droplet dispenser 10 is mounted is moveable with a translation stage 22. The translation stage 22 is adapted for translations of the dispenser head 20 in all three spatial directions (x-, y- and z-directions). The target 2 and a collection reservoir 5 are arranged in the operation range of the translation stage 22, e.g. on the common platform 23. The position of the dispenser head 20, i.e. the position where droplets are dispensed, is controlled via the movement of the dispenser head 20 using the translation stage 22 over the platform 23, in particular to the target 2 or the collection reservoir 5. Alternatively, the at least one dispenser 10 can be arranged with a fixed position, while the target 2 is mounted on a translation stage for adjusting the target position relative to the droplet dispenser position. According to another alternative, both of the at least one dispenser 10 and the target 2 can be moveable with translation stages being adapted for translations along the three or less spatial directions.

(30) A head camera 21 is mounted on the dispenser head 20. It can be coupled to a range of different light sources (not shown). The head camera 21 is adapted for both of the alignments of the target 2 and the droplet dispenser 10 relative to each other and for quality control of the deposition result. To this end, the head camera 21 is coupled with the control device 40 running an image analysis software, like “Online Array QC”. After the droplet deposition on the target 2, the head camera 21 can be used for visualizing the printed droplets 4 including the single particles 1. If necessary, the head camera 21 is used for identifying the number of particles present at the different deposition positions. Optionally, a fluorescence exciting light source can be used for illuminating the target, so that particles carrying a marker substance, e.g. fluorescently labelled cells, can be visualized and detected.

(31) The dispenser apparatus 100 is adapted for conducting the detecting and testing steps with the at least one droplet dispenser 10 according to FIG. 1, while it is operated at the collection reservoir 5. A nozzle camera 30 is placed over the platform 23 adjacent to the droplet dispenser 10 under consideration such that the nozzle section of the at least one droplet dispenser 10 can be imaged and particles in the nozzle section can be detected with the nozzle camera 30. Optionally, a light source (not shown) can be arranged for illuminating the droplet dispenser 10. As an example, a UV light source can be used for illuminating the nozzle section 12, so that the detection of e.g. the fluorescently labelled cells can be improved.

(32) The nozzle camera 30, like e.g. the camera IDS UI3240CP, is provided with a CCD-based device and a camera optic, and it is configured for imaging the nozzle section 12 and further for a visualization and monitoring of the drop formation prior to dispensing. As an example, the nozzle camera 30 is adapted for detecting particles in the nozzle section of the droplet dispenser 10 over an axial length of e.g. 700 μm to 800 μm. The nozzle camera 30 is connected with the control device 40 including the image processing unit 52. Using the location information on the particles and the a priori knowledge of the particle behaviour within the nozzle section 12, the single particle condition of the droplet dispenser can be analysed as outlined below.

(33) If multiple droplet dispensers are provided, the nozzle camera 30 is arranged for collecting images of all droplet dispensers. Alternatively, multiple nozzle cameras can be provided for collecting images of single droplet dispensers or groups of droplet dispensers, or one nozzle camera can be adjusted relative to one of the droplet dispensers.

(34) Furthermore, the dispenser apparatus 100 includes a control device 40, which is arranged for controlling the operation of the at least one droplet dispenser 10. To this end, the control device 40 comprises a computer circuitry which is connected with the drive unit of the at least one droplet dispenser 10 and the translation stage 22 as well as the nozzle and head cameras 30, 21. The control device 40 includes a testing unit 50, which is adapted for testing the single particle condition of the at least one droplet dispenser 10. To this end, the testing unit 50 is coupled with the nozzle camera 30 and provided with an image data storage 51 storing image data of the at least one droplet dispenser 10 and with an image processing unit 52, which identifies the presence and location of any particle within the nozzle section of the at least one droplet dispenser 10.

(35) FIG. 3 schematically illustrates the downstream end of a droplet dispenser 10, including the suspension reservoir 11 (partially shown) and the nozzle section 12. The droplet dispenser 10 comprises e.g. a glass capillary with an inner diameter of about 40 μm to 90 μm. The nozzle section 12 includes the ejection region 13 and the travel region 15, which are identified in preparing steps by the particle behaviour during the dispensing operation. The regular ejection region 13 is identified as the volume, which covers all positions of particles, which are ejected during a single dispensing operation with a regular droplet volume. If a fractional droplet with a reduced volume is to be dispensed (see FIG. 7), the ejection region 13 is identified as the volume, which covers all positions of particles, which are ejected during a single dispensing operation with the fractional droplet volume. The travel region 15 is identified as covering all positions of particles, which are displaced during a single dispensing operation, while being kept within the droplet dispenser 10.

(36) The particles are displaced during the dispensing operation due to the movement of the liquid within the nozzle section 12. Additionally, particles are displaced due to sedimentation everywhere inside the dispenser, in particular from the travel region 15 to the ejection region 13. Accordingly, a sedimentation region 14 is defined, which is the downstream section of the travel region 15. The sedimentation region 14 covers all positions of particles, which can be displaced by the effect of gravity during the time interval between the particle detection and the dispensing operation, including a duration of the movement of the droplet dispenser 10 from the collection reservoir 5 to the target 2. The sedimentation region 14 does not depend on the droplet volume to be ejected, i.e. it is equal for the regular or reduced ejection volumes.

(37) The volume of the sedimentation region 14, in particular the height in axial direction of the droplet dispenser 10 is calculated on the basis of the following considerations. The sedimentation velocity (v) of a particle is driven by gravity, and it can be calculated by:

(38) $v=\frac{\left({\rho }_p-{\rho }_f\right){\mathrm{gD}}^2}{18\mu }$

(39) wherein ρ.sub.p is the mass density of the particle, ρ.sub.f is the mass density of the fluid, g is the gravitational acceleration, D is the dimension, e.g. diameter of the particle and μ is the dynamic viscosity of the fluid.

(40) The displacement distance is calculated by the product of the sedimentation velocity and the time required for the dispenser apparatus 100 to test the single particle condition and to move the droplet dispenser 10 to the target 2. In practice, the time for testing the single particle condition is negligible compared with the movement time, which is provided by the dispenser apparatus 10, in particular the speed of the translation stage 22.

(41) Typically, the particle mass density is larger than the liquid mass density, so that all particles within the nozzle section 12 will sediment over time. Only particles near the upstream boundary of the ejection region 13 within the sedimentation region 14 are critical for the consideration of the single particle condition. Accordingly, the sedimentation region 14 is always adjacent to the ejection region/travel region boundary.

(42) The size of the sedimentation region 14 can be calculated in a static or in a dynamic way as follows.

(43) For the static calculation, the variables in the above equation are generalized by taking an average particle diameter and the maximum time required for the dispenser apparatus 100 to reach a desired target 2. Accordingly, one fixed sedimentation region 14 is determined.

(44) With a practical example of a cell suspension, the values are as follows: ρ.sub.p=1068 kg/m.sup.3, ρ.sub.f=1000 kg/m.sup.3, g=9.8 m/s.sup.2, and μ=1 mPa s. An average cell diameter of D=18 μm and a duration for moving the droplet dispenser 10 to the target 2 of 5 s would yield a thickness of the sedimentation region of about 60 μm. With another example, if the duration of moving the droplet dispenser 10 to the target 2 is typically less than 2 s, the thickness of the sedimentation region typically is less than 25 μm. The above calculation has been done on the assumption that the particles do not contact the inner wall of the PDC channel. This approach is fulfilled in practice.

(45) For the dynamic calculation, the values of ρ.sub.p, ρ.sub.f, g and μ are assumed to be constant. However, the diameter D and the time to target 2 are variables. The time to target 2 can be provided by the dispenser apparatus 100 as the desired target locations are predefined before the dispensing operation. The diameters D can be estimated by the apparent diameter of the particles as identified with the nozzle camera 30. Accordingly, different dimensions of the sedimentation region 14 are obtained for different diameters D and time to target 2 values. Accordingly, for each particle outside the ejection region 13 and within the field of view of the nozzle camera 30, a unique sedimentation region 14 can be determined. Advantageously, this results in a more robust consideration of the single particle condition, especially for particle samples, which have wide particle size distributions. As there is a D.sup.2 relation to the sedimentation velocity, a particle of e. g. 12 μm will only have a sedimentation velocity of 5.3 μm/s, while a particle of e. g. 18 μm will have a corresponding sedimentation velocity of 11.9 μm/s.

(46) FIG. 3 illustrates an example of a distribution of two particles 1, 1A in the nozzle section 12, providing a positive test of the single particle condition (step S6). The ejection region 13 includes one single particle only, while the sedimentation region 14 is free of particles. The second particle 1A is detected in the travel region 15, i.e. at a position where it cannot sediment into the ejection region 13 during moving the droplet dispenser 10 to the target 2.

(47) FIG. 4 shows a flow chart illustrating further details of testing the zero particle condition. Steps S1 to S6, S8, S12 and S13 are conducted as described above with reference to FIG. 1. Step S11 is not shown in FIG. 4, but can be implemented like in FIG. 1. Additionally, a multiple droplet calibration (step S3A) is conducted, which is used for implementing the zero particle condition test of step S9.

(48) The multiple droplet calibration step 3A may include at least one of the following examinations. Firstly, it can be counted how many droplets (number m) having the regular droplet volume can be generated to displace a particle from one extremity of the field of view, e. g. in an upper zone of the travel region 15 (see FIG. 3) to the other, e. g. in the ejection region 13, preferably just to the tip of the nozzle. Secondly, it can be estimated, how many droplets (number n) are to be dispensed to move a particle from any position, in particular z-position, in the nozzle section 12 to the ejection region 13. Number m and n are stored in the testing unit 50 (see FIG. 2).

(49) Step S9 of testing the zero particle condition includes testing whether an axial length of the droplet dispenser 10 beginning at the tip of the nozzle section 12 is free of particles. If the zero particle condition is not fulfilled, e. g. when no particle is located in the ejection region 13, but one particle is located in the sedimentation region 14 and multiple particles are located in the travel region 15, one droplet is discarded (step S11 in FIG. 1, not shown in FIG. 4). If the zero particle condition is fulfilled, e. g. there are no particles in the ejection and sedimentation regions, 13, 14, but one particle is located in the travel region 15, the number of droplets to be discarded is determined (step S9A) by at least one of the following methods. Firstly, it can be determined, if a particle is detected in dispenser 10, how far from tip of dispenser 10 is it arranged. Based on the multiple droplet calibration step 3A, it is deduced how many droplets (n, number of shifting steps) can be dispensed to move it to the ejection region. Secondly, it can be determined, if no particle is present in dispenser 10, it is deduced how many droplets (m, number of emptying steps) can be dispensed to discharge the current dispenser contents from the field of view. Accordingly, (m−1) droplets could be dispensed, in order to discard the current dispenser content. Subsequently, n or (m−1) droplets are discarded to the collection reservoir (step S10).

(50) FIG. 5 shows a variant of determining (step S9A) the number m of emptying steps. The schematic cross-sectional view of the nozzle section 12 shows a partitioning into numbered droplet regions 16A, 16B, 16C and 16D above the ejection region 13. The droplet regions are defined as follows. First droplet region 16A, including the sedimentation region 14, is defined as including all particles being one droplet away from the ejection region 13. Further droplet regions 16B, 16C, 16D are defined as including all particles being two, three or four droplets away from the ejection region 13. In other words, by dispensing two (m=2) droplets, the contents of the ejection region 13 and the first droplet region 16A can be emptied to a collection reservoir or one particle can be shifted from the second droplet region 16B to the ejection region 13. Alternatively, the number of droplets to be discarded in step S10 can be determined by tracking particles in the nozzle section 12 during dispensing droplets in the mapping step S3, determining the particle displacement per dispensing step and determining the number of droplets to be discarded by dividing the axial length of the nozzle section that is free of particles by the particle displacement per dispensing step.

(51) If the single particle condition (test in step S6) with the first ejection region is not fulfilled, testing the single particle condition can be modified as described in the following with reference to FIGS. 6 and 7. A considered size of the ejection region 13 is reduced to an ejection region subsection 13A or 13B, and it is determined whether the current ejection region subsection 13A or 13B includes one single particle 1 and the sedimentation region 14 adjacent to the current ejection region subsection 13A or 13B is free of particles. FIG. 6A shows (like FIG. 3) the first ejection region 13 with the particle 1 and the sedimentation region 14 with another particle 1A. In this case, the single particle condition would not be fulfilled. However, by reducing the size of the considered ejection region, the particle 1 can be dispensed as a single particle with a fractional droplet. FIGS. 6B and 6C show corresponding ejection region subsections 13A and 13B with reduced sizes, each with the sedimentation region 14. Due to the above definition of the sedimentation region 14, it has the same thickness in all three cases. Subsequently, the step of operating the droplet dispenser 10 includes setting a fractional droplet volume and dispensing a fractional droplet having a fraction of the first droplet volume onto the target 2, if the single particle condition is fulfilled with the current ejection region subsection (e. g. FIG. 6B).

(52) Details of this extended testing of the single particle condition are shown in the flow chart of FIG. 7. Steps S1, S2, S4, S5, S6, S8, S12 and S13 are conducted as described above with reference to FIG. 1. Testing the zero particle condition and the particle type condition are not shown in FIG. 7, but can be implemented like in FIG. 1. Additionally, the PDC mapping step S3 is modified and further steps of testing the single particle condition with reduced ejection regions are provided as described in the following.

(53) With the PDC mapping step S3, three different ejection zones (ejection zone and ejection region subsections) are considered like with step S3 in FIG. 1, wherein the first ejection region is the ejection region 13 tested with the regular droplet size and the second and third ejection regions correspondingly are ejection region subsections 13A, 13B (see FIG. 6) with reduced volumes (or axial lengths along dispenser extension). Each of the first, second and third ejection regions corresponds to a predetermined droplet size. In particular, the first ejection region 13 corresponds to the first droplet volume, and the second and third ejection regions correspond to fractional droplets having reduced volumes. With a practical example, the first droplet volume is 600 picoliter (pi), while the fractional droplets have reduced volumes of about 300 pl and about 150 pl. Furthermore, the PDC mapping step S3 includes calculating the sedimentation region 14. As a result of the mapping step, the extensions of the first, second and third ejection regions and the sedimentation regions as well as the volumes of the first, second and third droplets are stored in the testing unit 50 (see FIG. 2).

(54) After acquiring the dispenser image with step S5, the single particle condition is tested like in FIG. 1 with the first ejection region 13 (step S6). If the single particle condition is fulfilled, the process continues with steps S8, S12 and S13. If the single particle condition is not fulfilled, a first particle distribution test (step S6.1) is conducted. If a single particle is detected in the first ejection region 13 and at least one particle is detected in sedimentation region 14 or if multiple particles are detected in the first ejection region 13, the first ejection region subsection 13A is applied (step S6.2). Otherwise, the droplet with the first droplet volume is discarded (step 11.1).

(55) After step S6.2, the single particle condition is tested with the first ejection region subsection 13A like in FIG. 1 (step S6.3). If the single particle condition is fulfilled, i.e. there is no particle in sedimentation region 14 and one single particle in the first ejection region subsection 13A (see FIG. 6B), the process continues with steps S8.1, S12 and S13. Otherwise, if the single particle condition is not fulfilled, a second particle distribution test (step S6.4) is conducted.

(56) The second particle distribution test (step S6.4) includes testing whether a single particle is detected in the first ejection region subsection 13A and at least one particle is detected in the sedimentation region 14 or whether multiple particle are detected in the first ejection region subsection 13A. If yes, the second ejection region subsection 13B is applied (step S6.5). Otherwise, the droplet with the volume corresponding to ejection region 13A is discarded (step 11.2).

(57) After step S6.5, the single particle condition is tested again with the second ejection region subsection 13B like in FIG. 1 (step S6.6). If the single particle condition is fulfilled, i.e. there is no particle in sedimentation region and one single particle in the second ejection region subsection 13B, the process continues with steps S8.2, S12 and S13. Otherwise, the droplet with the volume corresponding to ejection region 13B is discarded (step 11.3).

(58) FIGS. 8 and 9 show examples of particle distributions detected with the extended testing of the single particle condition according to FIG. 7, wherein particles are detected in the sedimentation region 14 (FIG. 8) or multiple particles are detected in the ejection region or ejection region subsection 13, 13A or 13B (FIG. 9). An advantage of the invention can be seen in particular in FIG. 8 (top): In the event where a single particle was present in the ejection region, but another particle was present in the sedimentation region, applying an ejection region subsection allow displacement of the sedimentation region which in turn now allow fulfilment of the single cell condition using this ejection region subsection. This insures that most of the cells present in a sample are successfully isolated as single particles (and not anymore discarded). This is particularly important when working with limited samples containing few particles in order to maximise single particle isolation yields

(59) Details of testing the particle type condition (step S7 in FIG. 1) are shown in the flow chart of FIG. 10. Steps S1 to S4, S5, S6, S8, S12 and S13 are conducted as described above with reference to FIG. 1. Testing the zero particle condition is not shown in FIG. 10, but can be implemented like in FIG. 1. For testing the particle type condition, further steps of defining particle types and targets or target positions are added, e. g. after loading the particles into the PDC, and further test steps are provided as described in the following.

(60) After loading the particles into the PDC (step S4), particle types are defined in step S4.1. The particle types are indicated e. g. with “a”, “b”, “c” etc., and they are specified e. g. by the size, shape, marker substance or function of a biological cell. In practical examples, the following particle types can be defined. Particle type a can have a diameter from 4 to 9 μm, and while particle type b can have a diameter from 9 to 35 μm, and/or particle types a and b can correspondingly comprise fluorescent and non-fluorescent particles. The particle types can be differentiated e. g. in a nozzle camera image. At least one of the particle types is a particle type of interest, which e. g. is to be enriched or sorted on a target. Furthermore, specific targets or target positions are defined for each of the particle types in step S4.2. It is noted that the steps S4.1 and S4.2 alternatively can be implemented at another phase of the process. For examples, the particle types and associated targets or target positions can be prestored in the testing unit 50.

(61) After acquiring an image of the nozzle section (step S5), step S6 includes the test of the single particle condition as described above with reference to FIG. 1 or 7. If no particle is detected in the sedimentation region 14 and one single particle is detected in the ejection region 13, the process goes forward to step S7.1. Otherwise, one droplet is discarded.

(62) Step S7.1 includes determining the particle type of the detected particle. Subsequently, it is tested whether the detected particle in the ejection region 13 has the at least one particle type of interest (step S7.2). If the particle type condition is not fulfilled, i.e. there is no particle of interest, the droplet including the particle, which is not of interest, is discarded (step S11). Otherwise, if the particle type condition is fulfilled, i.e. the particle is to be dispensed, the process goes forward to step S8.

(63) With step S8, the dispenser 10 is moved to the target or target position, where the particle with the detected particle type is to be deposited. With preferred examples, particles with different types of interest can be sorted to different targets or target positions.

(64) Testing the particle type condition according to FIG. 10 can be modified, e. g. by conducting the test of step S7.2 before testing the single particle condition (step S6), or both test can be combined.

(65) Advantageously, the invention has a broad range of applications in particle handling. Examples of applications are summarized in the following.

(66) In particular, particle type selection using particle type condition (see e. g. step S7 in FIG. 1) can be used for processing a sample containing a mixture of two cell populations with different diameters, for example population 1 has diameters from 2 to 5 μm and population 2 has diameters from 5 to 12 μm. With the invention, different particles can be isolated at different positions onto the target in order to facilitate different analyses to be carried out on the two different populations. Furthermore, if only one of the population is of interest, all particles corresponding to the other population can be discarded in order to only isolate single particle of the population of interest. As a further alternative, enriching one population can be provided. If all particles from the population of interest are sorted at the same target position and all other particles are discarded, then a single pure/enriched population is obtained. Furthermore, sorting two particle populations can be obtained. If only two targets are used, i.e. one for each population then such an approach can be used for sorting two populations in order to obtain two pure populations.

(67) As a further example, using multiple discarding of droplets (see e. g. FIG. 4) for moving single particles inside the dispenser ejection zone allows a drastic increase of the speed of particle processing, and this has particular advantages when working with highly diluted samples containing few particles.

(68) Setting fractional droplet volumes (see e. g. FIG. 7) for having multiple ejection region subsections has particular advantages when working with samples containing few cells of high interest. With more details, this approach allows an increase in the proportion of single particle successfully isolated in order to ensure no particle are lost. For example, practical tests have shown over 97% of cells present in a cerebrospinal fluid sample being successfully isolated as single cells.

(69) The features of the invention disclosed in the above description, the drawings and the claims can be of significance both individually as well as in combination or sub-combination for the realization of the invention in its various embodiments.