Acoustic transducer refocusing for uninterrupted acoustic drop ejection

Abstract

A method of operation for an acoustic droplet ejection (ADE) system includes ejecting a series of droplets of a fluid from a well at an ejection rate. According to this method, the droplets are ejected by emission of acoustic tonebursts emitted at an ejection rate, and directed towards and focused at or near a surface of the fluid. Between ejections of successive droplets, a location of the surface of the fluid is estimated relative to the acoustic transducer. The transducer is selectively moved, without interrupting the ejection of the series of droplets of the fluid at the ejection rate, to adjust the distance between the transducer and the well based on the determined location of the surface of the fluid such that the focal point of the transducer follows the descending fluid surface across multiple ejections. This adjustment shifts the focus the transducer at an updated focal point relative to the surface of the fluid.

Description

[0001] P2024-0307-WO-D491 -Pl 5734

[0002] ACOUSTIC TRANSDUCER REFOCUSING FOR UNINTERRUPTED

[0003] ACOUSTIC DROP EJECTION

[0004] BACKGROUND

[0005] The present invention relates generally to dispensing of fluid, and more particularly to acoustic drop ejection.

[0006] Many applications demand precise transferring of dispensed fluid, and some such applications require fluid to be dispensed in extremely small increments. Acoustic droplet ejection (ADE) allows droplets as small as 1 pL - 1 pL to be moved using acoustic energy, without making mechanical contact with the fluid. In ADE, ultrasonic pulses are emitted from a transducer situated below and directed towards a sample of fluid contained with a reservoir. Each ultrasonic pulse for dispensing fluid (toneburst) is focused on a surface of the fluid sample, and tuned in power to perturb that fluid surface such that a droplet or fluid jet (producing a mist or multiple droplets) is ejected upward toward a target.

[0007] SUMMARY

[0008] In one embodiment, this disclosure presents a method of operation for an acoustic droplet ejection (ADE) system, the method comprising: ejecting a series of droplets of a fluid from a well at an ejection rate, each droplet of the series of the droplets being ejected by emission of an acoustic toneburst from an acoustic transducer directed toward the fluid within the well, and focused at or near a surface of the fluid; between ejections of successive of the series of droplets, estimating a location of the surface of the fluid relative to the acoustic transducer; and without interrupting the ejection of the series of droplets of the fluid at the ejection rate, selectively moving the transducer to adjust a distance between the transducer and the well based on the determined location of the surface of the fluid, such that the transducer is refocused at an updated focal point relative to the surface of the fluid; wherein adjustment of the distance between the transducer and the well causes the adjusted focal point to follow the fluid surface as it descends across the ejection of the series of droplets.

[0009] The method of the preceding paragraph can optionally include, additionally and / or alternatively, any one or more of the following features, configurations and / or additional components:

[0010] A further embodiment of the foregoing method, wherein estimating the location of the surface of the fluid relative to the acoustic transducer comprises: tracking a volume of the fluid ejected in the series of droplets since a last movement of the transducer; and retrieving a correlation between fluid ejection volume and height within the well; and estimating the location of the surface of the fluid by applying the correlation between fluid ejection volume and height within the well to the tracked volume of the fluid ejected.

[0011] A further embodiment of the foregoing method, wherein estimating the location of the surface of the fluid relative to the acoustic transducer comprises: emitting an acoustic ping from the transducer before each acoustic toneburst; receiving an acoustic return of each acoustic ping via the transducer; identifying at least one reflected feature from each acoustic return; and determining, by a controller communicatively coupled to the acoustic transducer, the location of the surface of the fluid within the well, relative to the transducer, based on the reflected features.

[0012] A further embodiment of the foregoing method, further comprising ingesting calibration data to the controller, the calibration data including data characterizing both the well and the fluid, wherein the determination of the location of the surface of the fluid from the acoustic return is dependent upon the calibration data.

[0013] A further embodiment of the foregoing method, wherein determining the current location of the surface of the fluid comprises evaluating a travel time between the emission of the acoustic ping and the reception of the corresponding acoustic return.

[0014] A further embodiment of the foregoing method, further comprising: ascertaining, from the current locations of the surface of the fluid across multiple of the acoustic tonebursts, a total movement of the surface of the fluid since a last movement of the transducer; and determining, by the controller, whether total movement of the surface of the fluid exceeds a threshold distance, wherein the controller commands the moving of the transducer in response to determining that the total movement of the surface of the fluid exceeds the threshold distance.

[0015] A further embodiment of the foregoing method, wherein the threshold distance is no more than 50 micrometers.

[0016] A further embodiment of the foregoing method, further comprising: emitting a plurality of acoustic pings including monitoring pings before and after each of the plurality of acoustic tonebursts; receiving an acoustic return of each monitoring ping via the transducer; identifying at least one reflected feature from each acoustic return; and determining, from the reflected features, by a controller communicatively coupled to the transducer: mound shapes reflecting perturbed shape the surface of the fluid due to each of the acoustic tonebursts; and the location of the surface of the fluid within the well, relative to the transducer. A further embodiment of the foregoing method, wherein each of the plurality of acoustic pings between successive of the plurality of acoustic tonebursts is separated from adjacent of the plurality of acoustic pings by between 10 and 200 (is.

[0017] A further embodiment of the foregoing method, further comprising identifying, from the mound shapes, a power anomaly in a respective of the acoustic tonebursts.

[0018] A further embodiment of the foregoing method, further comprising adjusting at least one of the ejection rate and a power level of the acoustic transducer in response to the identification of the power anomaly.

[0019] In another embodiment, this disclosure presents an acoustic droplet ejection (ADE) system comprising: a well configured to hold a fluid, the well extending along a well axis from a well bottom to a well opening, such that the fluid forms a fluid surface; an acoustic transducer oriented along the well axis and focused at a focal point; a motor connected to the acoustic transducer and configured to move the acoustic transducer parallel to the well axis; a controller communicatively coupled with the acoustic transducer and the motor, and configured to: command the acoustic transducer to emit tonebursts capable of ejecting droplets of the fluid from the well; track movement of the fluid surface as the fluid is depleted by ejection of the droplets; and in response to the tracked movement of the fluid surface exceeding a threshold distance, command the motor to reposition the acoustic transducer between at least some of the tonebursts, such that an updated focal point is retained at the fluid surface, wherein the threshold distance is greater than a minimum step size of the motor, but less than 50 pm.

[0020] The ADE system of the preceding paragraph can optionally include, additionally and / or alternatively, any one or more of the following features, configurations and / or additional components:

[0021] A further embodiment of the foregoing ADE system, wherein tracking movement of the fluid surface comprises: commanding the acoustic transducer to emit a first acoustic ping prior to a subset of the tonebursts; receiving a first acoustic return of each first acoustic ping from the acoustic transducer; and determining a location of the fluid surface prior to each acoustic ping based on a time of flight of the corresponding first acoustic return.

[0022] A further embodiment of the foregoing ADE system, wherein the controller is further configured to identify power anomalies by: commanding the acoustic transducer to emit a second acoustic ping following each of the subset of tonebursts, while the fluid surface remains perturbed by that toneburst; receiving a second acoustic return of each second acoustic ping from the acoustic transducer; and determining shape of the fluid surface immediately following each toneburst, based on a waveform of the corresponding second acoustic return.

[0023] A further embodiment of the foregoing ADE system, wherein the threshold distance is no more than 50 pm.

[0024] The present summary is provided only by way of example, and not limitation. Other aspects of the present disclosure will be appreciated in view of the entirety of the present disclosure, including the entire text, claims, and accompanying figures.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is schematic diagram of an acoustic droplet ejection (ADE) system.

[0027] FIG. 2 is an isolated schematic diagram of a transducer assembly of the ADE system of FIG. 1.

[0028] FIG. 3 is simplified cross-sectional diagram with schematic illustration of transducer movement during operation of the ADE system of FIG. 1.

[0029] FIG. 4 is a simplified top view of a container plate of the ADE system of FIG. 1.

[0030] FIG. 5 is schematic illustration of a transducer movement path across multiple containers within a subset of the container plate of FIG. 4.

[0031] FIG. 6 is a method flowchart illustrating open-loop control of transducer location during operation of the ADE system of FIG. 1.

[0032] FIG. 7 is a method flowchart illustrating closed-loop control of transducer location during operation of the ADE system of FIG. 1.

[0033] FIG. 8 is a combined cross-sectional and waveform diagram illustrating reflected feature identification by the transducer during operation of the ADE system of FIG. 1 according to the method of FIG. 7.

[0034] FIG. 9 is a method flowchart illustrating closed-loop control of both transducer location and power during operation of the ADE system of FIG. 1.

[0035] FIG. 10 is an illustrative plot of ping time-of-flight over successive pings for dynamic surface analysis in the method of FIG. 9.

[0036] While the above-identified figures set forth one or more embodiments of the present disclosure, other embodiments are also contemplated, as noted in the discussion. In all cases, this disclosure presents the invention by way of representation and not limitation. It should be understood that numerous other modifications and embodiments can be devised by those skilled in the art, which fall within the scope and spirit of the principles of the invention. The figures may not be drawn to scale, and applications and embodiments of the present invention may include features and components not specifically shown in the drawings. CE

[0037] DETAILED DESCRIPTION

[0038] An ADE system ejects metered portions of a sample fluid from a well or reservoir using an acoustic transducer. This transducer expels a droplet from the well by emitting an ultrasonic pulse focused at or near a meniscus surface of the sample fluid. Over time and / or over the course of operation of an ADE system, depletion of the sample fluid causes this surface to move as fluid within the well is depleted. Continued operation of the ADE system therefore requires not only initial calibration to identify and direct a focal point of the transducer at the location of the fluid surface, but also periodic adjustment of the transducer location (e.g., by vertical translation of the transducer) to adjust that focal point to account for the shifting meniscus location of the surface of depleting sample fluid.

[0039] Existing ADE systems adjust transducer distance (i.e., vertical distance between the transducer and sample fluid surface) by halting ejection when a transducer begins to go out-of-focus, e.g., when sensed or expected fluid transfer velocity crosses a predetermined limit set to avoid deterioration of droplet ejection quality or consistency. This limit can, for example, be based on fluid ejection properties, well geometry, and focal range of the transducer. After halting ejection, a new meniscus location is sensed or otherwise determined for the fluid surface. The transducer is then brought back into focus with the new meniscus location through movement to a new location selected to realign its focal point in view of the updated meniscus location. Transducer power is then adjusted and validated, and periodic ejection restarted. These “stop-and-resolve” events interrupt periodic droplet ejection.

[0040] This disclosure presents methods and ADE systems that partially or completely obviate the need to halt droplet ejection to accommodate transducer movement and recalibration. This is accomplished by maintaining alignment of transducer focal location relative to meniscus location through multiple tonebursts while adjusting transducer location nearly continuously. Rather than reacquiring and revalidating focus after allowing a meniscus of surface 103 to move to an edge of its allowed focus range (i.e., such that the transducer begins to go out-of-focus), the approach set forth herein makes micro-adjustments to transducer location / distance with a granularity limited only by minimum motor step size. These adjustments can be made via an open-loop lookup process based on known fluid properties and ejection history, or through closed-loop control wherein surface ping returns are used to identify changes in surface location between scheduled tonebursts. The approaches set forth herein allow droplets to be ejected at a constant repetition rate for a longer period of time (i.e., longer between stop-and-resolve events), which is particularly advantageous in systems (e.g., for mass spectrometry) wherein a substantially constant fluid metering rate is desirable or required. In addition, the reduction in stop-and-resolve events reduces interruption, increasing average drop ejection rate.

[0041] FIG. 1 is schematic diagram of acoustic droplet ejection (ADE) system 100, and FIG. 2 is an isolated schematic diagram of transducer assembly 110 of ADE system 100. FIG. 1 provides a schematized cross-sectional view of transducer assembly 110, container plate 120 (with multiple containers 122 holding a samples 101), and receiver plate 130 (with multiple receiver wells disposed to receive ejected liquid 102 from sample 101). Container plate 120 can, for example, be a microplate, and containers 122 can be wells in that microplate. FIG. 1 also illustrates electronics 140, X / Y / Z motor(s) 150, and coupling liquid 160. FIG. 2 depicts transducer assembly 110, which includes transducer 112 and acoustic lens 113. This disclosure describes FIGs. 1 and 2 together.

[0042] ADE system 100 can ascertain characteristics (i.e., acoustic properties) of both containers 122 and samples 101, and can cause liquid to be ejected from respective containers 122 through ultrasonic tonebursts. Each sample 101 is a dispensable liquid of interest held within a particular container 122. Although this disclosure focuses on containers that are wells of microplates, techniques described herein can be used with other containers such as tubes, flasks, and beakers, as well as any samples contained therein, as will be recognized. Containers 122 can, as illustrated in FIG. 1, be open-topped wells oriented such that samples 101 are retained in containers 122 gravitationally.

[0043] In order to cause ejected liquid 102 to be ejected from sample 101, transducer 112 generates a pulse (i.e., a toneburst) of acoustic energy (e.g., ultrasonic energy), which is focused by acoustic lens 113 into beam 170 directed towards bottom wall 123 of container 122. While beam 170 is shown as a perfect triangle, beam 170 can more generally have any shape focusing at a desired location, e.g., at, slightly forward of (above), or slightly behind (below) surface 103. In most embodiments, beam 170 is focused at a point on or within a small number of beam wavelengths of the meniscus of surface 103. Although illustrated schematically in two dimensions, it should be understood that beam 170 is three dimensional, and that beam 170 can have a three-dimensional shape tailored to produce a focal point F at a desired location (as discussed below), i.e., depending on its three-dimensional environment (e.g., the shape, location, and acoustic properties of all materials and structures between transducer 112 and focal point F). Furthermore, some embodiments of ADE system 100 can facilitate adjusting a location of focal point F by adapting the configuration of acoustic lens 113 and transducer 112 within transducer assembly 110, e.g., by adjusting the focal length of beam 170. One such transducer assembly 110 is described in U.S. Appl. No. 16 / 369,780 (U.S. Publ. 2019 / 0302063). In addition, the location of the focal point F can be adjusted by operation of X / Y / Z motor(s) 150.

[0044] X / Y / Z motor(s) 150 include a vertically-traversing linear motor, i.e., capable of translating other elements of transducer assembly 110 along a z-axis (that is, a vertical dimension between container 122 and transducer assembly 110, as shown in FIG. 1). In some examples, X / Y / Z motor(s) 150 can be or include a stepper motor or servo motor, or more generally any element capable of precisely adjusting a location of transducer assembly 112 (and in at least some embodiments also acoustic lens 113, therewith) along the z-axis in very small increments, e.g., in step sizes of less than 50 micrometers. In some embodiments, X / Y / Z motor(s) 150 can provide for movement of the transducer assembly 110 in multiple dimensions (e.g., along x, y, and z-axes, or along two dimensions). As shown, j rL motors 150 are coupled (directly or indirectly) to transducer assembly 110, but these or other motors may equivalently be coupled (directly or indirectly) to container plate 120 and / or receiver plate 130 in order control the relative movement between transducer assembly 110, container plate 120, and / or receiver plate 130. In some examples, X / Y / Z motor(s) 150 or other motors be used to adjust relative locations of transducer assembly 112 and container plate 120 by different means with respect to different dimensions, e.g., adjusting relative Z-location by moving transducer assembly 112 and X / Y-location by moving container plate 120. In other examples, transducer assembly 112 can be held stationary, with all relative positioning accomplished by movement of container plate 120. Although Y -ilL motor(s) 150 are described together, different types X / Y / Z motor(s) 150 can include multiple types and orientations of motors, e.g., to facilitate finer positional control along some axes.

[0045] This disclosure primarily discusses movement of the focal point F of acoustic energy beam 170 in terms of vertical translation of transducer 112 (e.g., together with acoustic lens 113) using X / Y / Z motor(s) 150, but the methods and systems disclosed herein are also applicable in the most general case to beam adjustments made via other control of transducer assembly 110, as introduced above.

[0046] FIG. 1 illustrates beam 170 as focused on (upper) surface 103 of sample 101, which can for example define the interface between sample 101 and the air above sample 101. During optimal operation, however, beam 170 can in some embodiments be focused at locations slightly offset from surface 103 along the z-axis, e.g., slightly above / forward of surface 103, to facilitate energetic droplet ejection. Beam 170 is emitted from transducer 112 and focused by acoustic lens 113, then passes through coupling liquid 160, a bottom wall 123 of container 122, and the depth of sample 101 to reach the surface 103 of sample 101.

[0047] Electronics 140 include processor 143, a motor controller 142, transmit signal circuitry 144, receive signal circuitry 145, and temperature sensor circuitry 141. Although shown as separate, discrete components for explanatory purposes, portions of electronics 140 can be combined, integrated, divided, or distributed in some embodiments. Furthermore, some components shown may include multiple different subcomponents not specifically shown. For example, processor 143 may include multiple processors, either located together in a single chip or distributed in different locations.

[0048] Processor 143 is or includes a logic-capable device that causes or controls transmit signal circuitry 144 to generate a control signal (e.g., an analog electrical signal such as a radio frequency signal or sequence of discrete pulses) that is communicated to transducer 112. Transducer 112 then vibrates in response to the control signal (e.g., based on amplitude and / or frequency in the case of an analog signal), such that a corresponding acoustic signal is emitted. Transducer assembly 110 may also receive acoustic signals or “pings” (e.g., acoustic signals reflected from container 120 and / or samples 101 in response to the emitted acoustic signal) and vibrate responsively. This vibration may generate a sensor signal (e.g., an analog electronic reception signal), which is then communicated to receive signal circuitry 145. Processor 143 can, in many embodiments, receive information corresponding to the received acoustic signals from the receive signal circuitry 145 in the form of an electronic reception signal. Information in the electronic reception signal is analyzed by processor 143.

[0049] Processor 143 can also communicate with motor controller 142 to control the location of transducer assembly 110. Motor controller 142 controls one or more of X / Y / Z motor(s) 150 to move transducer assembly 110 relative to container plate 120. Motor controller 142 can control one or motors 150 to position transducer assembly 110 underneath a given container 122 in container plate 120 (e.g., transducer assembly 110 is centered with respect to a center of given container 122), and then to move transducer assembly 110 underneath another given container 122 in container plate 120.

[0050] To accommodate temperature-dependent acoustic characteristics of some materials in ADE system 100, some embodiments of ADE system 100 can include temperature sensor(s) (not shown) located, e.g., in coupling liquid 160, or in a region between container plate 120 and receiver plate 130. Temperature sensor circuitry 141 receives signals (e.g., electrical or wireless) from any such temperature sensor(s), and communicates with processor 143 such that temperature(s) (e.g., of coupling liquid 160, containers 122, samples 101, air temperature) can be measured and controlled.

[0051] In some embodiments, transducer assembly 110 can have a cylindrical shape. In some examples, instead of using a single transducer 112 as a two-way transceiver to both transmit and receive acoustic signals, transducer assembly 110 can include separate transmitter and receiver hardware, for example as disclosed in U.S. Patent No. 10,787,670. According to one technique, receiving transducer hardware can substantially surround the transmitting transducer and acoustic lens.

[0052] FIG. 3 is a simplified cross-sectional diagram with a schematic illustration of transducer assembly 110 illustrating movement of transducer assembly 110 relative to container plate 120 when performing ADE on multiple samples 101 in separate containers 122 within container plate 120. As shown in FIG. 3, X / Y / Z motor(s) 150 (see FIG. 1) move transducer assembly 110 to locations of successive containers 122 along the x-axis. Although illustratively depicted in two dimensions, transducer assembly 110 can also move along the y-axis to additional containers 122 (not shown) as further described with reference to FIG. 5. For each container 122, transducer assembly 110 can be centered underneath container 122 through x- and y-axis operation of X / Y / Z motor(s) 150. Transducer assembly 110 then moves vertically (i.e., along the z-axis) to emit and receive acoustic signals at z-positions beneath container 122. Transducer assembly 110 can be positioned along the z-axis to focus beam 170 on the surface 103 of sample 101 to cause ejected liquid 102 to be ejected (ADE). The z-axis position of transducer assembly 100 relative to a container 122, and of transducer 112 and acoustic lens 113 in particular, can be finely and frequently changed via micro-adjustments as described below with reference to FIGs. 6-9 to facilitate continuous (uninterrupted) ADE operation.

[0053] FIG. 4 shows a top view of container plate 120 having a plurality of containers 122. Container plate 120 shown is a 384- well microplate (e.g., a polypropylene microplate, designated 384-PP). FIG. 5 a top view of a plurality of container wells 122 and an example traversal pattern (a serpentine pattern) for performing ADE and / or other ultrasonic techniques on each container well 122 and sample 101 therein, in series, as described with respect to FIG. 3. In this example, X / Y / Z motor(s) 150 are configured to translate the transducer assembly 110 along the x- and y-axes to position it under various container wells 122. Any other suitable pattern may be used (e.g., a raster pattern), depending on geometry of container plate 120 and distribution of relevant samples 101 within containers 122. Additionally and / or alternatively, x-y positioning of container plate 120 relative to transducer assembly 110 can be adjusted by movement of container plate 120 to achieve similar effects.

[0054] FIGs. 3 and 4 provide schematic illustrations of movement of transducer assembly 1 10 in simplified examples. More generally, X / Y / Z motor(s) 150 can be actuated to drive transducer assembly 110 to x / y locations (see FIG. 4) of suitable wells for fluid dispensing in any order or arrangement suitable for specific tasks, including in other patterns than those shown in FIG. 4, and / or based on criteria other than pattern following. Similarly, although FIG. 3 depicts generally symmetrical z-axis movement (i.e., moving transducer 110 towards and then away from each well 122), a person skilled in the art will understand that movement need not be, and will in most instances not be, symmetric. Rather, starting and ending z-axis locations transducer 110 for each well can be determined on a per- well basis based, for example, on height of surface 103 and material properties of the fluid and well.

[0055] FIGs. 6, 7, and 9 all illustrate control methods for refocusing transducer 112 (i.e., moving focal point F) coincidently with surface 113 to facilitate uninterrupted ADE, obviating the need for, or reducing the frequency of, stop-and-resolve events. As noted briefly above, the term “stop-and-resolve event” is used herein to describe interruptions to ordinary ADE (i.e., continuous operation with periodic ejection of droplets at an expected frequency defining a rate of fluid flow) to reacquire proper focus. More specifically, such events occur when transducer 112 becomes unfocused relative to surface 103, forcing droplet ejection to be halted until a new meniscus location can be determined, and transducer 1 12 brought back into focus relative to surface 103 by realigning and revalidating the location of focal point F, e.g., by translating transducer assembly 110 using X / Y / Z motor(s) 150. “Out of focus,” for the purpose of this discussion, refers to a condition of ADE system 100 wherein a distance between focal point F and a target focal point is large enough to result in poor or inconsistent droplet ejection (including uneven droplet size, form, ejection occurrence, velocity, and / or ejection direction) due to unaccounted drift of surface 103. Methods discussed below with reference to FIGs. 6, 7, and 9 facilitate micro-adjustments to transducer location between tonebursts during ordinary ADE operation, thereby improving retention of focus.

[0056] FIG. 6 is a flowchart illustrating method 600, an open-loop control method whereby micro-adjustments to focal point F are made according to system parameters known or estimated a priori. First, processor 143 ingests well and fluid data reflecting a priori understanding of level and fluid characteristics of sample 101 , geometry and material composition of container 122, and material composition of coupling fluid 160, as well as location of transducer assembly 110. (Step 602a). These parameters can variously be ascertained immediately prior to ADE operation for each sample 101, tested before operation with respect to known fluids or container plates 120, and / or retrieved from trusted records of expected parameters (e.g., as provided by a supplier of container plates 120, coupling fluid 160, or sample 101). More generally, a priori data can include any material, geometric, or operational information provided as an input to method 600, rather than obtained through method 600. Such data can, for example, include a focal length of transducer 112, geometry and acoustic characteristics of container 122 (and particularly of bottom wall 123, assuming that acoustic energy beam 170 passes substantially exclusively through bottom wall 123 and not through other portions of container 122 - calibration data can include a thickness of bottom wall 123), speed of sound through coupling fluid 160, fluid of sample 101, and material of container 122 (e.g., plastic making up bottom wall 123). A priori data can also include fluid level of sample 101, e.g., sensed prior to method 600, and echo time of flight for expected signals based at least in part on fluid level of sample 101. In some cases a priori data can also include a focal offset correction for ideal drop ejection, i.e., an ideal distance between focal point F and surface 103. Other parameters, such as fluid viscosity (including viscosity as a function of stress, for nonNewtonian fluids) can also be provided as necessary.

[0057] Based on a priori data assembled at step 602a, processor 143 determines an initial position for transducer 102 and commands X / Y / Z motor(s) 150 to reposition transducer 102 accordingly via motor controller 142. (Step 604). This determination of initial position can, for example, be an analytic computation performed by processor 143 based on a multi-layer geometric focus model or other conventional heuristic model of the a priori data set forth above. In other embodiments at least some such input data, or some quantities derived therefrom, can instead be used to retrieve corresponding preferences, e.g., via lookup table, without real-time analytic modeling. As noted above, some embodiments of transducer assembly 110 may enable focal point F to be adjusted by means other than or in addition to movement of transducer 112 by X / Y / Z motor(s) 150. In such embodiments, processor 143 more generally determines and commands an adjustment to the location of focal point F that can include movement by X / Y / Z motor(s) 150 and / or other actuation within transducer assembly 110. In still further embodiments, processor 143 may determine necessary adjustments to transducer location, or otherwise to the location of focal point F, via a trained machine learning model.

[0058] After setting an initial position of transducer 112 or otherwise adjusting an initial position of focal point F, processor 143 commands ejection of one or more droplets of fluid via transmit signal circuitry 144, directing transducer 112 to emit at least one ultrasonic toneburst. (Step 606). During sustained operation, Step 606 is repeated over multiple ejections at a precisely determined ejection rate. Each toneburst ejects a fluid droplet from sample 101 as discussed above with respect to FIGs. 1-3. During ADE operation, as the fluid level of sample 101 is depleted through ejection of one or more successive droplets, the height of surface 103 changes in a predictable manner based on known droplet size, droplet count, and geometry of container 122. Decrease in height of fluid surface 103 due to reduced volume from one or more droplet ejections can thus be ascertained from a priori data introduced at step 602a in combination with total dispensed fluid volume (e.g., based on droplet size and rate or count). As with initial transducer positioning, surface height movement due to fluid depletion by droplet ejection from sample 101 can be computed analytically or, more simply, by can be pre-cached and retrieved from a lookup table reflecting container geometry. (Step 602b). Although represented as separate step in FIG. 6, the retrieval of volume vs. height data, or more generally of a correspondence between droplet ejection count or volume and expected movement of a meniscus of surface 103, can be an extension of the ingestion of a priori data, more generally.

[0059] After one or more tonebursts, an expected meniscus location for surface 103 is determined based on a droplet count or (equivalently) total droplet volume and the projected correspondence between ejection amount and surface location. (Step 608). Where optimal focal point location F is not coincident with surface 103, as discussed above, processor 143 can generate a focal point adjustment based on the estimated change in location of surface 103. In some embodiments, however, it may be sufficient to approximate an appropriate z-axis adjustment to the location of focal point F as the estimated z-axis movement in a meniscus of surface 103.

[0060] If a z-axis travel distance of surface 103 exceeds a minimum threshold value (Step 610), or alternatively a corresponding z-axis adjustment to focal point location exceeds such a threshold value, processor 143 commands motor controller 142 to move or otherwise refocus transducer assembly 110 by a distance determined at step 608 (Step 612). So long as this z-axis travel distance remains below the minimum threshold value, however, method 600 proceeds with one or more additional tone bursts (Step 606) without adjusting focus of position of transducer 212. In such cases, travel distance evaluated at a subsequent step 610 includes all travel since an immediately preceding adjustment or initial positioning or refocusing of transducer 212.

[0061] As noted briefly above, the selection of a minimum threshold value is essential to permitting ADE operation to continue without stop-and-resolve events. More specifically, this minimum threshold must be selected to prevent acoustic energy beam 170 from losing focus relative to surface 103. Travel distance sufficient to defocus beam acoustic energy beam 170 will depend upon material and geometric properties of ADE system 100, and particularly on fluid properties of sample 101, but defocusing can in general be best avoided by reducing the step size of z-axis adjustments. An exemplary maximum permissible step size can, for example, be 50 pm. To ensure that readjustment of transducer position before total travel exceeds this maximum permissible step size, the threshold value for triggering adjustment can be less than the maximum permissible step size by more than an anticipated surface travel distance corresponding to the tonebursts of each step 606. In some embodiments, in order to move transducer 112 as continuously as possible during ADE operation (i.e. , with via increments as small as possible) the maximum permissible step size can be selected based on a minimum z-axis step size of X / Y / Z motor(s) 150, such that motor controller 142 commands adjustment of transducer location whenever estimated travel of surface 103 exceeds that motor step size. In a non-limiting example, X / Y / Z motor(s) 150 can include a z-axis stepper motor with a minimum step size of 10 pm, with motor controller 142 commanding adjustments to transducer location in increments of 10-20 pm.

[0062] Although method 600 illustratively depicts adjustment of transducer / focal position (step 612) as contingent upon evaluation of travel distance relative to a threshold value, equivalent approaches can operate for a number of droplet ejections determined in advance. Such estimates can, for example, be based estimation of surface travel (as per step 608), or calibrated to eject a fixed amount of fluid from sample 101 (e.g. 1 pL) between adjustments of transducer location (step 612). As noted above, these travel or fluid ejection volume limits are selected to reduce step size and thereby avoid out-of-focus conditions. For example, a number of droplet ejections performed at step 606 can be selected based on anticipated resulting surface travel (step 608), such that transducer position is always adjusted (step 612) after performing the expected number of tonebursts set for step 606 (i.e., predetermining the outcome of step 610). When ejecting droplets of 2.5nl, for example, a simplified control loop for ADE system 100 may repeat approximately 400 tonebursts (1 pL total fluid transfer) before reevaluating surface travel and potentially adjusting location of transducer 112 to maintain location of focal point F relative to a meniscus of surface 103. As noted above, ideal step sizes will depend on both fluid characteristics and geometry of container 122.

[0063] FIG. 7 is a flowchart illustrating method 700, which is a closed-loop operating method for ADE system 100. Where method 600 relies on a priori projections of surface location based on expected surface travel due to droplet ejections, method 700 acoustically evaluates travel distance of surface 103 relative to transducer 112 between some or all tonebursts, and adjusts transducer / focal point location when sensed total travel since a last adjustment exceeds a minimum threshold value. Steps 702, 704, and 710 generally parallel steps 602, 604, and 606, respectively, with an initial position of transducer 112 set (704) based on ingestion of a priori well and fluid data (702) before any tonebursts are emitted (710). Method 700 differs from method 600 in that acoustic sensing of surface travel (steps 712 and 714, discussed below) replaces estimation of surface travel (step 608) based on volume vs. height projections (step 602b). Steps 716 and 718 of method 700 are likewise generally equivalent to steps 610 and 612, respectively, of method 600, and cover adjustment of transducer position based on identified surface drift. As noted above, distance from transducer assembly 112 to surface 103 tends to decrease across multiple tonebursts as sample 101 is depleted by droplet ejection. In some cases, distance to surface 103 near the focal location can also be transiently increased by mound formation due to tonebursts.

[0064] Method 700 is described together with FIG. 8, which provides a combined cross-sectional and waveform diagram illustrating pings and reflected feature identification for acoustic sensing of surface location. FIG. 8 illustrates sample 101, transducer 110, fluid surface 103, container 122 (with container bottom wall 123), and coupling liquid 160 as discussed above. Container 122 is depicted alongside corresponding ping map 802 (with various reflected feature locations) corresponding to illustrative acoustic waveform plot 804 (showing various event times for ping returns).

[0065] According to method 700, processor 143 of ADE system 10 ascertains whether ejection power has been set (Step 706) after initialization of a starting transducer position (Step 704). Ejection power refers to toneburst power produced by transducer 112 to generate beam 170, and can be adjusted situationally based on multiple factors, such as material characteristics of individual samples 101, bottom wall 123, and / or coupling fluid 160, expected distance of transducer 112 from bottom wall 123 and / or surface 103, and target droplet size. Insufficient ejection power can, for example, result in erratically directed (e.g., non-vertical) ejection, non-ejection, or deviations from desired droplet sizes. Excessive ejection power can also affect droplet characteristics (e.g. size), and can generate unnecessary turbulence in surface 103. In general, higher ejection power will increase time required for surface 103 to recover from each ejection toneburst, limiting possible toneburst rate. Excessive ejection power can therefore unnecessarily slow toneburst rate. Initial ejection power is determined (step 708) based on estimated or measured values of the aforementioned factors, e.g. based on a priori data ingested at step 702, to set initial configurations of transducer 112. Once these configurations are set, an initial ejection toneburst is triggered (Step 710) generally as described above with reference to Step 606 of method 600.

[0066] Method 700 differs from method 600 following the emission of one or more ultrasonic tonebursts at step 710 (corresponding to step 606), in that processor 143 commands transmit signal circuitry 144 to trigger the generation of at least one comparatively low power acoustic “ping” pulse. (Step 712). This ping pulse is generated by transducer 112 at time tPmg(defining the zero of time-of-flight plot 804), and has substantially the same orientation as a preceding toneburst (i.e., towards focal point F). Acoustic signals received from transducer 112 via receive signal circuitry 145 are analyzed to identify ping returns and thereby changes in the meniscus location of surface 103. Signal (sonar) returns from the emitted ping pulse corresponding to features of acoustic reflections at material transitions from coupling liquid 160 to container bottom wall 123 (R- PlateBottom), from container wall bottom 123 to sample 101 (R-PlateTop), and from sample 101 to surrounding air at a meniscus of surface 103 (R-FluidSurface), are illustrated by ping map 802, as derived from reception times tR.piateBottom, tR.piateTop, tR-FiuidsUrface, respectively, of acoustic waveform 804. Processor 143 assesses the current location of a surface 103 relative to transducer 1 12 by assessing a reflected feature location (R- FluidSurface) from corresponding ping return time-of-flight tR-FiUidSurface. (Step 714). Rather than aggregating expected travel steps through successive drop ejections as described above with reference to FIG. 6, method 700 directly assesses the location of surface 103 irrespective of drop count. If this surface location has traveled more than a minimum threshold distance since an immediately previous transducer position adjustment (or initial transducer positioning) (Step 716), processor 143 commands adjustment of transducer z- axis position by X / Y / Z motor(s) 150 via motor controller 142. Unlike similar step 612 of method 600, each increment of adjustment at step 718 is selected based on sensed drift of surface 103 relative to transducer 112, per an immediately previous reflected feature evaluation (at step 714). Otherwise, threshold evaluation at step 716 and transducer position adjustment at step 718 operate substantially as described above with respect to steps 610 and 612, respectively.

[0067] Processor 143 can, in the most general case, command emission of ping pulses (step 712) any time between successive tonebursts, so long as sufficient time is left for return receipt and processing (e.g. 10-200 ps). Acoustic pulse travel time determines a minimum time between successive ping pulses. A subsequent ping pulse should be transmitted until the echo of a preceding ping pulse has been received. In an illustrative embodiment wherein transducer 212 is at focus on a surface or target of interest with a focal length of approximately one inch, this acoustic travel time can for example be 10-40 ps. Where processing occurs at least partially in parallel with pulse transmission, this return time defines a minimum delay between successive ping pulses. Longer delays (e.g., 60-200 ps) can be preferable to accommodate processing time and / or when observing slower effects, depending on sonogram function. In some illustrative embodiments, a droplet ejection toneburst may be followed at 90 ps by a ping pulse, with successive ping pulses following 65 ps thereafter. Mechanical movement of transducer assembly 112 can, for example, be performed in increments of 20-50 pm, and on time scales 10-100x slower than signal processing (e.g., 0.5-5ms), and tonebursts can be fired while transducer location is being adjusted, i.e., without waiting for mechanical movement to be completed. In some special cases, however, transducer location can be adjusted before a subsequent toneburst, e.g., where errors in droplet trajectory are identified. In some cases, however, processor 143 may be configured to preferentially trigger ping pulses shortly before (e.g., < 100 ps) a next toneburst. Delaying ping pulses until later in an ADE cycle in this way allows surface 103 to settle from a previous toneburst before the ping pulse is transmitted, thereby minimizing toneburst-related noise in acoustic waveform 804. Method 700 permits real-time sensing of surface location drift, rather than relying on projections based on expected values, and does not require generation or retrieval of volume vs. height correlations as described in method 600.

[0068] FIG. 9 is a flowchart depicting method 900, which is an extrapolation of method 700 including analysis of acoustic waveform 804 to identify and respond to power anomalies using dynamic surface analysis (DSA). Method 900 is described together with FIG. 10, which provides an illustrative plot of ping time-of-flight over successive pings of surface 103 for DSA. More detail regarding possible approaches to DSA for ADE systems in general can be found, for example, in PCT / US2023 / 077581, entitled DYNAMIC SURFACE ANALYSIS FOR ACOUSTIC DROPLET EJECTION and filed October 24, 2023.

[0069] Method 900 corresponds closely to method 700, with steps 902-918 being functionally similar to corresponding steps 702-718, respectively. Initial transducer position is set (904) based on ingested a priori well and fluid data (902). Ejection power is determined (908) for to droplet ejection if not already set (per 906). An ejection toneburst is then generated by transducer 112, ejecting a first droplet of sample 101.

[0070] Like method 700, method 900 emits surface pings to test surface 103. (Step 912). Where method 700 only describes locating surface 103 with a single surface location ping (or a small number of pings) at step 712, however, method 900 uses a large number of surface monitoring pings to track both the location and shape of surface 103. This tracking facilitates adjustment of transducer location to compensate for movement of surface 103 as described above, but also enables power anomalies to be identified as described below. FIG. 10 illustrates time-of-flight plot 1000 across multiple pings, with illumination in each row corresponding to a separate reflected feature. Transducer 112 can, for example, be commanded by processor 143 to emit low -power surface monitoring pings every 10-200 ps, identifying locations of surface 103 (in line with beam 170) by time-of- flight as described above with reference to FIGs. 7 and 8 for each ping. Together, reflected features of these surface monitoring pings provide an image of surface perturbation before, during, and after tonebursts. Time-of-flight plot 1000 illustrates pre-ejection surface location 1012 and post-ejection surface location 1014. Pre-ejection surface location 1012 represents the substantially unperturbed form of surface 103 prior to a toneburst, while post-ejection surface location 1014 represents the recovered form of surface 103 after turbulence from a toneburst has substantially died down. Mound phase 1014 is shown between pre- and post-ejection surfaces 1010 and 1012, and defines to the period of surface disturbance generated by a corresponding toneburst to produce droplet ejection event 1016. The mound shape generated by transducer 112 within mound phase 1014 of by time-of- flight plot 1000 can be characterized heuristically and / or by an appropriate machine learning model to detect low- or high-power anomalies, or otherwise inform possible changes in applied power to, for example, increase the size of possible fluid transfers and / or enable continuation of aggressive fluid transfer rates.

[0071] Surface level following a toneburst is determined by time-of-flight as described above with reference to step 714 of method 700, using reflected feature of postejection surface 1014 rather than a single surface location reflected feature. (Step 914). Processor 143 can, for example, ascertain the level to which surface 103 recedes following mound phase 1014 based on multiple successive pings. Although it is usually possible and often preferable to perform steps 712-714 while surface 103 is unperturbed, it can in some instances be necessary to perturb surface 103 in order to receiving a ping return, e.g., where at least a region of surface 103 is at an angle that does not facilitate ping returns. As discussed previously, surface drift based on this determination is evaluated to determine whether surface travel exceeds a specified minimum threshold set based, e.g., on step size of X / Y / Z motor(s) 150. (Step 916). If travel exceeds this threshold value, the z-position of transducer 112 is adjusted before any subsequent ejection toneburst. (Step 918).

[0072] Process 143 also evaluates reflected features from surface monitoring pings to assess ejection quality via DS A. (Step 920). As noted above, the shape (e.g., width, height, turbulence) of time-of-flight plot 1000 in each mound phase 1014 can provide indications of power anomalies, allowing adjustment of transducer power and / or ejection rate for subsequent ejection tonebursts without interruption of tonebursts at a scheduled (though potentially adjusted) rate.

[0073] If ejection quality as assessed with DS A is adequate, tonebursts can continue without adjustment. (Step 922). If DSA identifies power anomalies, ejection rate and / or toneburst power can be adjusted. (Step 924). In some cases power anomalies can indicate poor focus position, i.e., where focal point F is not aligned for maximum power transfer into droplet ejection, and can be improved through refocusing. In other instances power anomalies can indicate that an emitted power from transducer 112 is too high or too low, or that high power, though perhaps necessary for droplet ejection (e.g., where fluid volume of sample 101 in container 122 is high) or desirable faster fluid transfer (i.e., via larger droplet volume) may not be feasible, necessitating adjustments to power or ejection rate. If DSA indicates that droplet ejection is weak, for example, failed or off-target droplet ejections can be avoided by increasing power before a next toneburst. If surface 103 is unexpectedly highly or lingeringly perturbed, subsequent toneburst power can be reduced and / or ejection rate can be decreased to avoid impaired droplet ejection on subsequent tonebursts.

[0074] Although steps 914 and 916 are illustrated as preceding steps 920 and 922, with method 900 only reaching step 920 in the event that no transducer position adjustment (Step 918) is indicated by step 916, assessments of ejection quality and surface quality can more generally be performed independently and / or in parallel in some embodiments. Tn such cases, assessment of ejection quality may follow evaluation of surface travel distance regardless of the outcome of that threshold evaluation (i.e., with step 918 proceeding to step 920), or with assessments of ejection quality and surface travel occurring separately and in no particular order, with any adjustments at steps 918 and / or 924 occurring prior art a subsequent ejection toneburst.

[0075] Method 900 illustrates how identification and analysis of power anomalies can be incorporated into a closed loop ADE process consistent with method 700, and generally offers all of the advantages of method 700 while also facilitating correction of toneburst power and ejection rate, as necessary, without interruption of an expected series of ejection tonebursts.

[0076] Discussion of Possible Embodiments

[0077] Any relative terms or terms of degree used herein, such as “substantially”, “essentially”, “generally”, “approximately” and the like, should be interpreted in accordance with and subject to any applicable definitions or limits expressly stated herein. In all instances, any relative terms or terms of degree used herein should be interpreted to broadly encompass any relevant disclosed embodiments as well as such ranges or variations as would be understood by a person of ordinary skill in the art in view of the entirety of the present disclosure, such as to encompass ordinary manufacturing tolerance variations, incidental alignment variations, alignment or shape variations induced by thermal, rotational or vibrational operational conditions, and the like.

[0078] While the invention has been described with reference to an exemplary embodiment(s), it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment(s) disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

CLAIMS:

1. A method of operation for an acoustic droplet ejection (ADE) system, the method comprising: ejecting a series of droplets of a fluid from a well at an ejection rate, each droplet of the series of the droplets being ejected by emission of an acoustic toneburst from an acoustic transducer directed toward the fluid within the well, and focused at or near a surface of the fluid; between ejections of successive of the series of droplets, estimating a location of the surface of the fluid relative to the acoustic transducer; and without interrupting the ejection of the series of droplets of the fluid at the ejection rate, selectively moving the transducer to adjust a distance between the transducer and the well based on the determined location of the surface of the fluid, such that the transducer is refocused at an updated focal point relative to the surface of the fluid; wherein adjustment of the distance between the transducer and the well causes the adjusted focal point to follow the fluid surface as it descends across the ejection of the series of droplets.

2. The method of claim 1 , wherein estimating the location of the surface of the fluid relative to the acoustic transducer comprises: tracking a volume of the fluid ejected in the series of droplets since a last movement of the transducer; and retrieving a correlation between fluid ejection volume and height within the well; and estimating the location of the surface of the fluid by applying the correlation between fluid ejection volume and height within the well to the tracked volume of the fluid ejected.

3. The method of claim 1, wherein estimating the location of the surface of the fluid relative to the acoustic transducer comprises: emitting an acoustic ping from the transducer before each acoustic toneburst; receiving an acoustic return of each acoustic ping via the transducer; identifying at least one reflected feature from each acoustic return; anddetermining, by a controller communicatively coupled to the acoustic transducer, the location of the surface of the fluid within the well, relative to the transducer, based on the reflected features.

4. The method of claim 3, further comprising ingesting calibration data to the controller, the calibration data including data characterizing both the well and the fluid, wherein the determination of the location of the surface of the fluid from the acoustic return is dependent upon the calibration data.

5. The method of claim 1 , wherein determining the current location of the surface of the fluid comprises evaluating a travel time between the emission of the acoustic ping and the reception of the corresponding acoustic return.

6. The method of claim 1 , further comprising: ascertaining, from the current locations of the surface of the fluid across multiple of the acoustic tonebursts, a total movement of the surface of the fluid since a last movement of the transducer; and determining, by the controller, whether total movement of the surface of the fluid exceeds a threshold distance, wherein the controller commands the moving of the transducer in response to determining that the total movement of the surface of the fluid exceeds the threshold distance.

7. The method of claim 6, wherein the threshold distance is no more than 50 micrometers.

8. The method of claim 1 , further comprising: emitting a plurality of acoustic pings including monitoring pings before and after each of the plurality of acoustic tonebursts; receiving an acoustic return of each monitoring ping via the transducer; identifying at least one reflected feature from each acoustic return; and determining, from the reflected features, by a controller communicatively coupled to the transducer: mound shapes reflecting perturbed shape the surface of the fluid due to each of the acoustic tonebursts; and the location of the surface of the fluid within the well, relative to the transducer.

9. The method of claim 8, wherein each of the plurality of acoustic pings between successive of the plurality of acoustic tonebursts is separated from adjacent of the plurality of acoustic pings by between 10 and 200 microseconds.

10. The method of claim 8, further comprising identifying, from the mound shapes, a power anomaly in a respective of the acoustic tonebursts.

11. The method of claim 10, further comprising adjusting at least one of the ejection rate and a power level of the acoustic transducer in response to the identification of the power anomaly.

12. An acoustic droplet ejection (ADE) system comprising: a well configured to hold a fluid, the well extending along a well axis from a well bottom to a well opening, such that the fluid forms a fluid surface; an acoustic transducer oriented along the well axis and focused at a focal point; a motor connected to the acoustic transducer and configured to move the acoustic transducer parallel to the well axis; a controller communicatively coupled with the acoustic transducer and the motor, and configured to: command the acoustic transducer to emit tonebursts capable of ejecting droplets of the fluid from the well; track movement of the fluid surface as the fluid is depleted by ejection of the droplets; and in response to the tracked movement of the fluid surface exceeding a threshold distance, command the motor to reposition the acoustic transducer between at least some of the tonebursts, such that an updated focal point is retained at the fluid surface, wherein the threshold distance is greater than a minimum step size of the motor, but less than 50 pm.

13. The ADE system of claim 12, wherein tracking movement of the fluid surface comprises: commanding the acoustic transducer to emit a first acoustic ping prior to a subset of the tonebursts;receiving a first acoustic return of each first acoustic ping from the acoustic transducer; and determining a location of the fluid surface prior to each acoustic ping based on a time of flight of the corresponding first acoustic return.

14. The ADE system of claim 13, wherein the controller is further configured to identify power anomalies by: commanding the acoustic transducer to emit a second acoustic ping following each of the subset of tonebursts, while the fluid surface remains perturbed by that toneburst; receiving a second acoustic return of each second acoustic ping from the acoustic transducer; and determining shape of the fluid surface immediately following each toneburst, based on a waveform of the corresponding second acoustic return.

15. The acoustic droplet ejection system of claim 14, wherein the threshold distance is no more than 50 pm.

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