Measurement of dynamic systems

Abstract

A method for performing measurements includes effecting relative movement between a container and a cover over a measurement time period that includes multiple non-overlapping time periods. The distance between the container and the cover varies along a first axis over at least a portion of each time period. An apparatus for performing measurements includes a container including a support and a set of wells within the support, and a cover. The cover includes: (1) a device configured to engage the container, (2) a control subsystem configured to effect relative movement between the container and the cover, and (3) a measurement subsystem.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 725,787, filed Aug. 31, 2018, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Various techniques have been used to measure oxygen consumption by cells. Some techniques are open-air techniques that measure conditioned cells (e.g., intact monolayers of cultured cells). Some techniques allow for in vivo cell measurements. Some techniques allow for measurements of cells in multiwell plates (e.g., cells in the wells of a microtiter plate). Some techniques use a probe to make measurements between two different positions in a gradient to estimate the flux (e.g., using a self-referencing microsensor). Summary of the Invention

[0003] At least some aspects address the need to continuously (e.g., over days or weeks) measure oxygen consumption of a biological sample while maintaining a sterile environment for the biological sample, maintaining gaseous contact between the sample (e.g., the surface of the fluid containing the sample) and the environment, and minimizing the disruption of oxygen concentration gradients in the fluid. In at least some embodiments, the system includes a sterile disposable probe lid mounted beneath a durable handheld electronic array of optoelectronic sensors. The probe lid and electronic sensor array are placed on top of a standard multi-well tissue culture plate. An optical probe integrated inside and extending vertically from the disposable lid is inserted into the fluid of multiple tissue wells. The system is designed to avoid interfering with the overall delivery of oxygen to cells and minimize the disruption of oxygen concentration gradients. The lid is sterile and the system can be placed in an incubator. As such, the system is ideal for continuous use during cell culture experiments, up to several weeks. The system is ideal for measuring the response of cells to treatments (e.g., measuring changes in oxygen consumption after adding a drug).

[0004] In one aspect, generally, an apparatus is used to measure molecular levels (e.g., concentration, consumption or production rate) of a fluid in a vessel. The apparatus includes a device configured to be mounted as part of a cover over a vessel, the device comprising one or more wells each holding a biological sample immersed in a fluid. The device includes a plurality of optoelectronic sensors positioned to acquire optical signals from a probe immersed in the fluid. These optical signals represent the molecular levels of the fluid at the probe sensors. The device also includes a set of actuators configurable to adjust a relative position of the device and the vessel. A control circuit for the device is used to control the set of actuators to vary the relative position of the device and the vessel and acquire the optical signals at a plurality of relative positions.

[0005] An advantage of the device forming part of the cover on the container is that the device is portable with the container (e.g. a container covered with the device is "hand-held" and can be easily moved from one location to another, placed on an incubator shelf, etc.).

[0006] Some aspects can include one or more of the following features.

[0007] A plurality of actuators are provided which are together controllable by the control circuitry to control the orientation of a plane defined by the sensors of the probe relative to a plane defined by the set of wells of the vessel. An advantage of this feature is that each sensor can be held at the same height above a well floor and / or at the same depth in the fluid as other sensors.

[0008] The control circuitry is configured to maintain parallel relative orientations of a plane defined by the sensor of the probe and a plane defined by the set of wells of the vessel.

[0009] The change in relative position between the device and the container spans a range of motion, hi some examples, the range of motion is between 10 micrometers and 3 millimeters.

[0010] In another aspect, generally, an apparatus includes a lid, or the apparatus described above further includes a lid configured to attach to the device to form the cover, the lid including a surface configured to cover the well of the vessel (i.e., the combination of the device and the lid covers the vessel), and the lid also includes a set of probes, each probe extending substantially perpendicularly from the surface of the lid.

[0011] The lid is configured such that a position of each probe on the lid corresponds to a position of an optoelectronic sensor on the device, and when the lid is attached to the device, a light path is formed between a sensor on the probe and a corresponding optoelectronic sensor, e.g., the lid is further configured such that the position of each probe on the lid corresponds to a position within one of the set of wells of the container.

[0012] The lid is fixedly attached to the device and maintains alignment between the probe of the lid and the optoelectronic sensor of the device, i.e., the device is fixed, mounted, or positioned such that it is rigid and does not easily move relative to the lid and its probe.

[0013] The lid comprises a sterile material, for example, the lid is configured to separate a surface of the device from the well when attached to a container, thereby maintaining a sterile environment within the well. An advantage of using such a lid separate from the device is that the device does not need to maintain sterility without significant risk of contaminating the biological sample in the well.

[0014] The lid is configured to provide a gas path between the wells and the environment without providing a linear path from within or above any well to an environment external to the device, such that the environment serves as a reservoir for the molecules.

[0015] The lid includes a bent portion of the lid near a periphery of the lid, the bent portion having greater flexibility than other portions of the lid, and upon movement of the device relative to the container, at least a first portion of the lid remains in contact with the container and at least a second portion of the lid separated from the first portion by the bent portion remains in contact with the device.

[0016] The cover formed from the device and the lid is configured to be removable from the container without disturbing the container.

[0017] 10. An apparatus according to any preceding claim, wherein the device has a mass of less than 1000 grams or a mass of less than 250 grams.

[0018] A set of bent portions form a spring with force, the device has sufficient weight to compress the bent portions across the varying relative positions of the device and the container, and the spring applies sufficient force to maintain contact of the lid to the device (i.e., maintain a fixed attachment of the lid to the device) across the varying relative positions.

[0019] The device includes a guide element that engages with a corresponding element on the lid to fix the relative position of the lid and the device. For example, the guide element may include a protrusion and / or a recess in a surface of the device. As another example, the guide element may include a magnet for engaging with a magnet in the lid.

[0020] The device includes a data interface for receiving data from a corresponding element in the lid. For example, the data interface includes electrical contacts that electrically contact corresponding electrical contacts of the corresponding element in the lid. As another example, the data interface includes an image sensor for acquiring an image of a portion of a surface of the lid. As yet another example, the data interface includes a radio frequency communication device.

[0021] The data interface is coupled to the control circuitry for controlling the actuator and the acquisition of the optical signal from the sensor.

[0022] The apparatus further includes the container.

[0023] The lid is attached to the device and positioned between the device and the container to maintain a sterile barrier between the surface of the fluid and the environment (e.g., such that there is no direct gas path from the surface of the fluid to the environment outside the device), and there is at least some gas passage between each well and the environment.

[0024] The vessel includes a plurality of wells and the lid is configured to include at least one probe associated with each well, or alternatively, the vessel includes a single well and the lid is configured to include multiple probes associated with the single well.

[0025] The apparatus further comprises a data processing system configured to accept data representative of sensor values ​​obtained at multiple relative positions.

[0026] The data processing system is configured to combine the received data to calculate a rate of consumption of the molecule by the biological sample.

[0027] The data processing system includes a display for displaying quantities derived from the sensor values. The data processing system is configured to display the quantities derived from the sensor values ​​in combination with reference quantities.

[0028] The molecule comprises oxygen.

[0029] In another aspect, a lid of the type generally described above is configured for use with a device and a vessel. A separate lid has the advantage that it is a consumable part of the apparatus that can be prepared separately from the device to be reused across multiple experiments, which is cost-effective. Furthermore, different types of lids (e.g., interchangeable lids), e.g., with different sensors, can be used to measure levels of different molecules.

[0030] In another aspect, a method for measuring molecular levels of a fluid in a container generally uses any of the apparatus described above. The method includes adjusting the relative position of the device and the container, the adjusting step including moving the sensor within the fluid without substantially disturbing the gradient of the molecular level of the fluid while maintaining a gas path from the well of the container to an environment outside the container. Sensor signals are acquired at a plurality of the relative positions (e.g., at various positions in the gradient field of the molecular level). Data determined from the acquired signals is communicated from the device to a data processing system separate from the device.

[0031] In another aspect, a method for performing measurements generally includes providing relative movement between a container and a set of probes over a measurement time including multiple time periods (e.g., discrete, non-overlapping portions of the measurement time). A distance between the container and the probes varies along a first axis over at least a portion of each time period. The container includes a set of wells (e.g., a plurality of wells). At least one well (e.g., each well) supports a sample and includes a fluid, the fluid having a top surface exposed to a gas, the gas comprising a concentration of molecules, and the sample consumes or otherwise interacts with the molecules in the fluid. Each probe is configured to measure a concentration of the molecules in the fluid at a distance from a bottom surface of the well that is dependent on the distance between the container and the probe, the relative movement being along an axis substantially perpendicular to a plane of the top surface of the fluid. The method further includes obtaining measurement data from the set of probes over each of the multiple time periods. For at least a first probe configured to measure the concentration of the molecule in the fluid contained in a first well of the set of wells: the measurement data acquired from the first probe includes at least two data samples acquired at different respective distances from the bottom surface of the first well within a first period of the plurality of periods, and the maximum elapsed time between successive data samples acquired within the first period is less than a predetermined threshold based, at least in part, on, for example, the square of the difference between the respective distances at which successive data samples were acquired, i.e., a time constant associated with the square of a characteristic length (e.g., the diameter of a cross-section of the probe parallel to the fluid surface) and / or a diffusion constant associated with the flow of the molecule within the fluid.

[0032] In some embodiments, the first well is sufficiently deep such that there is at least 10 microns between the bottom surface and the top surface of the fluid within the first well.

[0033] In another aspect, generally, an apparatus for performing a measurement includes a container including a support and a set of wells (e.g., a plurality of wells) within the support (e.g., a "well plate" or "multi-well plate"), and a cover. The cover includes a lid and a device. The lid is configured to engage the container. The lid includes a set of probes, each of which extends from a surface of the lid into a corresponding well of the set of wells, the lid is substantially fixedly attached to the device to maintain the position of the probes on the lid relative to the device, and is shaped to maintain a sterile barrier between the wells and the environment (e.g., forming only a tortuous path and preventing a straight line path between any portion of the air gap above the wells and the atmosphere outside the air gap). a control subsystem configured to effect relative movement between the container and the device, wherein the distance between the container and the device changes along a first axis based on movement of a plurality of mechanical actuators, each of which is configured to move a movable element within the device, the movable element contacting the container when the device engages the container; and (3) a measurement subsystem configured to provide excitation light propagating from two or more of the plurality of probes and to collect emission light propagating into two or more of the plurality of probes.

[0034] In some embodiments, the movable element comprises a flexible diaphragm.

[0035] In some embodiments, the device has a small form factor and low power consumption. A small form factor, i.e., ideally very small, on the order of 127.8 x 85.5 mm (i.e., the footprint of some containers), with a small mass (e.g., less than about 250 grams or less than 1000 grams), allows the device to be placed on top of such plastic containers and can be a handheld device. The method described herein can be used to achieve a small form factor. Finally, the device uses less than 2.5 watts of power, ideally less than 0.5 watts on average, or more ideally less than 0.1 watts of power, and the entire device can be powered by USB and placed in an incubator (i.e., a sealed temperature and humidity controlled environment) without appreciably increasing the temperature of the surroundings.

[0036] Some aspects may have one or more of the following advantages.

[0037] The techniques and systems described herein can be used to measure the consumption and production of compounds in the fluid-holding wells of the vessel. Where oxygen is consumed by the cells, a gradient is formed between the surface of the fluid, where the oxygen concentration is in equilibrium with the environment (i.e., the gas reservoir of the compound, e.g., oxygen), and the cells, where the oxygen concentration is below saturation due to consumption. At steady state, oxygen consumption is proportional to the concentration gradient and the oxygen diffusion constant. Thus, the oxygen concentration in the well can be measured and converted to a cellular oxygen consumption rate. In some embodiments of the system, an array of small diameter oxygen probes is inserted inside the well and moved vertically (perpendicular to the plane of the well bottom) in a fixed pattern. Oxygen concentrations can be read out throughout this movement pattern. Although probe movement may disrupt the oxygen gradient and the system may not necessarily reach a perfect steady state, these concentration measurements can be used as input to an algorithm to generate a time series of values ​​corresponding to cellular oxygen consumption, which is a strong indicator of metabolic activity. The readings can also be used to infer several other system properties, including, for example, a measure of the equilibrium state of compounds within the fluid-holding wells of the vessel or the spatial distribution of cells present. This measurement technique offers significant advantages over prior art techniques in terms of accuracy, minimal disturbance to the cells, and measurement robustness.

[0038] Other features and advantages of the invention will become apparent from the following description and claims. [Brief description of the drawings]

[0039] [Figure 1] 1A-B are cross-sectional views of an exemplary measurement unit. [Diagram 2] 1 is a plot illustrating the coordinate translation of a well. [Diagram 3] 3A-D are cross-sectional views of an example of a retractable pin. [Figure 4] 4A-C are cross-sectional views of one example of an exemplary hinged lid between the device and the culture plate. [Diagram 5] 5A-B are cross-sectional views of an exemplary flex and flex cover. [Figure 6] 1 is a plot illustrating a probe envelope. [Figure 7] 1 is a block diagram of an exemplary measurement process. [Figure 8] 1 is a block diagram of an exemplary measurement algorithm. [Figure 9] 1 is a plot of exemplary measurements corresponding to different embodiments of a measurement system. [Figure 10] 10A-10B are plots of exemplary measurements corresponding to different embodiments of a measurement system. [Figure 11] 11A-11B are plots of exemplary measurements corresponding to different embodiments of the measurement system. [Figure 12] 12A-12B are plots of exemplary measurements corresponding to different embodiments of the measurement system. BEST MODE FOR CARRYING OUT THEINVENTION

[0040] 1 Overview Various embodiments of a measurement system including a measurement unit and associated computation and control circuitry for controlling and receiving data from the measurement unit (some of which may be included in and / or coupled to the measurement unit) are used to measure oxygen consumed or produced by cultured cells or to measure oxygen equilibrium with the surrounding environment. The system can provide non-invasive, continuous, real-time oxygen consumption measurements. This is useful, for example, for measuring oxygen consumption when there is little or no fluid convection. Furthermore, this is suitable for measurements where the cells are cultured in an open well format and the primary source of oxygen delivery to the fluid is via an air-liquid interface at the top of the well. Furthermore, this is useful when the cells are mammalian cells and the cells are attached to the bottom of the culture well. In various embodiments, different aspects of the system are adaptable to any of a wide variety of culture form factors.

[0041] 1A-B, an exemplary measurement system includes a measurement unit 100 with a sterile disposable (e.g., consumable) probe lid 130 mounted below a durable (e.g., non-disposable) device 110 supporting an array of optoelectronic sensors 112. FIG. 1A is a horizontal cross-sectional view of point A shown in FIG. 1B, and FIG. 1B is a cross-sectional view of point B shown in FIG. 1A. The probe lid 130 and the array of optoelectronic sensors 112 are placed on top of a culture plate 120 (also called a "receptacle"). The device 110 and lid 130 are collectively called a "cover," which is a surface that is coupled to the device and covers the culture plate 120 containing multiple wells 122. An optical probe 160 is integrated into and extends vertically from the inner surface of the disposable lid 130 and is inserted into the fluid 174 of the tissue well 122. For example, the probes may be integral and molded of the same material as the rest of the lid, although in some instances the probes include a light guide made of a different material and molded into the rest of the lid. As described below, each of the probes 160 includes a probe tip 162 that is immersed in the liquid 174 and converts a property, such as oxygen concentration, into an optical emission that is passed through the corresponding probe to an optoelectronic sensor 112 in the device 110 (e.g., in response to an optical signal emitted from the sensor 112 exciting the probe tip 162). Circuitry 118 in the device then stores the measurements and / or communicates them to an external processing system. Also, as described further below, the device includes electrically controlled actuators 114 (only one of which is shown in cross-section in FIG. 1A ) that can change the separation of the device 110 and the plate 120, for example, by extending a separator pin 116 that presses against the top surface of the plate 120 through the lid 130, thereby controlling the change in the position of the probe tip 162 within the well 122.

[0042] The system may also include a control and data storage hub that interfaces with the device 110, for example, via circuitry 118. This interface may be wireless or wired. The control hub may interface with multiple measurement units simultaneously. Additionally, the control hub may provide an interface for remote access to the data and may act as a host for visualization of the data. Such data may also be visualized in real time via a remote machine accessing the data via the hub or via another remote machine relaying the data collected by the hub.

[0043] The exemplary system is designed to avoid interfering with the overall delivery of oxygen to the cells and minimize disturbance of the oxygen concentration gradient. The lid is sterile and the measurement unit is placed inside the incubator. As such, the exemplary system is ideal for continuous use during cell culture experiments, up to several weeks.

[0044] Within the well, oxygen is supplied to the fluid primarily at the air interface, and as cells at the bottom or suspended within the well consume oxygen, a gradient of oxygen concentration is formed between the oxygen-saturated fluid at the top of the well and the fluid with reduced oxygen concentration at the bottom of the well (or at the location of the cells within the well). Alternatively, the cells in the well may produce or consume another compound that is in equilibrium with this gas in the air above the well, where it is useful to measure the production or consumption of this gas or to monitor the equilibrium of solutes contained in the fluid with their surrounding environment. The properties of this gradient reveal the localized transient state of oxygen transport occurring within the well throughout the experiment. These properties are described by Fick's law and are directly related to oxygen consumption by the cells.

[0045] While not intending to be bound by theory, Fick's first law relates diffusive flux to concentration assuming steady state conditions (an assumption that is valid at infinitesimal length scales).

[0046]

number

[0047] where J is the diffusion flux, D is the diffusion coefficient, φ is the concentration, and x is the spatial position. Fick's second law predicts how diffusion will change the concentration with respect to time t.

[0048]

number

[0049] In operation, the probe moves up and down within the well (on a length scale of about one millimeter) and the system records the oxygen concentration within the gradient. These concentration readings, processed and combined with the known movement of the system, give the user a readout of oxygen consumption.

[0050] In some embodiments, the system moves the probe precisely relative to a previous probe position, but does not provide an absolute position relative to the location of the fluid surface or the location of the cells. Thus, the terms height (relative to the bottom of the well) and depth (relative to the fluid surface) are not intended to imply absolute height or depth certainty relative to the exact location of the physical fluid surface (which may vary during an experiment) or the bottom of the well (which may vary due to manufacturing variations), but rather an idealized or approximate height or depth relative to a reference point in space that corresponds to an approximate (nominal) location.

[0051] The computational subsystem of the system can be configured to run estimation algorithms based on measurements of relative position, so that all that is needed is to ensure that the probe is within the gradient of the measurement target. In some cases, this is a useful improvement over fixed probe approaches, for example, since the system can tolerate absolute position inaccuracies while still providing an accurate readout. Such absolute position inaccuracies can arise from a number of sources, such as: Variation in well plate dimensions (e.g., depth, volume, rim height, etc.). Fluid volume variations (e.g. due to evaporation, improperly calibrated or inaccurate pipettes, or plate volume variations). · Variations in manufacturing various physical components of the system itself, including the working parts and the lid.

[0052] There are many commercial benefits that result from the system's tolerance to absolute position inaccuracies and that can be exploited in some embodiments. These include: Compatible with a variety of plates from a variety of different vendors. Low cost manufacturability due to the ability to produce with low tolerance manufacturing processes, for example low cost injection moulding rather than precision milling to produce the lid. · Customer ease of use. Effects of evaporation and fluid volume inaccuracies are minimized. No recalibration is required to maintain absolute position accuracy. Longer system life. The system can tolerate wear on various components that can affect position accuracy.

[0053] 2 Exemplary Measurement Units Referring again to FIG. 1, the measurement unit 100 includes a cell culture plate 120 with an array of open wells 122 containing cells 172 submerged in a liquid 174. A sterile disposable lid 120 is placed over the plate 120 with an array of probes 160 extending vertically downward therefrom into the cell culture wells. An oxygen sensitive material is attached to the tips 162 of these probes contained within the culture fluid 174 of each well 122. An array of optoelectronic sensors 112 in the durable device 110 is coupled to the probe lid 130 (e.g., optically by maintaining contact with the lid 130 of the device 110), and the sensors 112 read the oxygen concentration at the probe tips 162 by processing optical emissions from the probe tips 162 that reach the sensors 112 through the probes 160. The probes 160 are moved up and down within the wells to measure the oxygen concentration at multiple locations. An actuator 114 vertically moves the device 110 relative to the plate 120, thereby resulting in relative movement of the probe tips 162 relative to the wells 122. The unit 100 connects to a data and power hub that processes, stores, provides access to, or displays data collected by the sensors. Various embodiments of different features of an exemplary measurement unit are now described.

[0054] 2.1 Durable devices supporting sensors and actuators 2.1.1 Direct coupling of optical sensors to probes In general, in various embodiments, the probe tip 162 is configured to move up and down relative to the cell culture plate 120. In some embodiments, this relative movement can be achieved by fixing the probe lid, such as in an enclosure, and vibrating the well plate vertically. In other embodiments, the probe lid can be vibrated vertically while the well plate remains stationary. This can be done, for example, on an incubator shelf where the plate rests on the shelf and the probe lid vibrates above the well plate. This latter type of embodiment is described below.

[0055] The probe tip can be configured to be coupled to optoelectronics. Optical sensors typically use a flexible optical cable to separate the sensing material on the probe from the optoelectronics. A flexible cable coupling the sensing electronics to the probe lid allows the lid to vibrate freely. Alternatively, in some optical sensing environments, the optical cable is omitted and the optical signal is read directly from the material, for example when used with a microscope. This allows the sensor electronics to move freely relative to the probe material, for example with a plate reader. In the embodiments described below, optical fibers are substantially rigidly attached to optoelectronic sensors of the sensor array in the device such that vertical vibration of the optoelectronics directly corresponds to vertical vibration of the probe tip in the well of the plate.

[0056] In some embodiments, direct coupling is advantageous because it allows a minimum length of fiber or light pipe to be employed, reducing system complexity and size, reducing the number of interfaces where light can be lost, and increasing signal strength.

[0057] 2.1.2 Reversible binding The durable device 110, including the array of optoelectronic sensors 112, is fixedly attached to the disposable lid 130. This fixed attachment is reversible so that the disposable lid 130 can be removed and replaced. In some embodiments, magnets (e.g., 135 and 136 shown in FIGS. 1A-B) are used to hold the two parts together, and alignment features on both parts ensure that they are repeatably connectable (e.g., to align the sensor 112 with the corresponding probe 160). Optionally, the magnetic holding feature can be turned on or off using an opposing permanent magnet that can be centered or misaligned using an electromagnet, or by temporarily turning on an electromagnet to counteract the action of the permanent magnet. Alternatively, a sliding lock or other mechanical mechanism can be used to hold and align the lid to the device.

[0058] 2.1.3 Electronic Devices and Data Storage The durability device 110 includes electronics 110 that assist in processing the oxygen concentration readings, monitoring additional sensor inputs, executing decision-making algorithms based on these inputs, providing sampling and actuation instructions, and communicating with the data hub. These additional electronics include microcontrollers for general processing and specialized digital processing devices such as field programmable gate arrays (FPGAs) for high speed control and calculations. The electronics also include non-volatile memory for storing device specific information including device calibration variables. These calibration variables can potentially account for differences in the electronics including oscillators, amplifiers, gain resistors, capacitors, etc.

[0059] Each sensor in the array has certain optical components. These components can include at least some of the following: lenses, dichroic mirrors, first surface mirrors, emission filters, and excitation filters. The light emitting elements (light emitting diodes LEDs) and light sensors (photodiodes) often have an integrated lens.

[0060] The device may also include wireless communication capabilities to provide data to a data hub. It may further include built-in power storage (e.g., a battery) to allow continued operation in the event of a disturbance in the external power supply. Backup power sources also allow for more extensive shutdown procedures. For example, the operational features may be retracted to a safe location within the ruggedized device.

[0061] 2.1.4 Additional Sensors Optional additional sensors include air pressure, temperature, and humidity sensors that can be used to calculate local oxygen saturation values. An accelerometer may also be included to monitor the orientation of the device and to ensure that it is being transported or is stable on the incubator shelf. This information is useful for automatic initiation and termination of movement patterns. A temperature sensor can be used to monitor the stability of the incubator environment, such as when the door is opened. An ambient light sensor can also be used for this purpose, which can be useful in minimizing power consumption. The oxygen, temperature, and acceleration sensors can also infer when media is exchanged in the wells. Relative humidity measurements are also useful for general incubator monitoring, as low humidity levels increase the rate of media evaporation.

[0062] Patterns and thresholds derived from sensor readings can automatically trigger the initialization or termination of measurements or continuous operations. In this way, the device can intelligently predict user intent and eliminate the need for specific user interface mechanisms such as buttons. The accelerometer can also be configured to detect motion through actuation functions. In this configuration, the accelerometer can be used to monitor system operation and take corrective action if the system is not operating as expected.

[0063] 2.1.5 Three-point actuation system In some embodiments, the vertical (i.e., z-axis) motion of the device 110 and lid 130 relative to the plate 120 is accomplished by a single actuator and linear guide, where the actuator maintains the parallel alignment of the device and plate, so that the plane through the probe tip remains parallel to the plate. In a preferred alternative embodiment, the vertical position of the probe in the well can be controlled by setting the height at three positions in the xy plane of the device 110 and the fixedly attached lid 130. This distributed positioning system is more complex than an arrangement using multiple linear guides and a single actuator, but the advantage of three-point actuation is that it allows the system to be relatively large in the plane perpendicular to the direction of travel and small along the dimension parallel to the direction of travel. These three actuation points can be actuated in synchrony with each other to keep the plane of the probe tip parallel to the plate. Alternatively, the actuators can be operated to achieve different separations between the device and the plate at different actuator points, so that the tilt of the corresponding planes of the lid and the probe tip can be controlled. For example, in such an alternative, some probes move a greater distance than other probes in the system.

[0064] The actuation points can be set within the footprint of a standard multi-well plate, where they can press against a flat surface near the top of the well. Alternatively, cells may be removed from three wells, and the device can press against the well bottom to move away. Actuation points can also be located outside the footprint of a standard plate, for example against an incubator shelf, or against a specially designed platform that also holds the well plate. Features of the well plate or the specially designed platform can receive the actuation points. These features can be simple dimples, or a combination of dimples / slots / flats can be used as a kinematic coupling to achieve precise positioning.

[0065] To determine the vertical position of the probe given the positions of the three actuation points, the x-y coordinate system can be translated so that one of the actuation points is located at the origin and the second point is located on the x-axis.

[0066] The center of each well is then translated to the new set of axes. Next, the slope is found between the first operating point at the origin and the second operating point on the new x-axis. Next, the slope is found between the new x-axis and the value of the third operating point along the new y-axis. At this point, the z position of each well center can be found using the slope and the coordinates of each well center along the new set of axes.

[0067] An image of the translated coordinate system is shown in FIG.

[0068] 2.1.6 Encoders or steppers, limit switches The actuator is designed to provide a near absolute position of the probe in the well and a precise positional relationship to its previous position. In one configuration, the actuation points are rods 116 extending from the durable device 110. Among many possible designs, these rods can be coupled to the durable device via threaded bushings in the actuator 114. As the rods are rotated about their cylindrical axes, they extend and retract relative to the device. In another configuration, a tapered disk abuts the top of the pins, allowing the pins to extend from the device at the thickest part of the disk and retract into the device when the thinnest part of the disk is aligned with the pins. Yet another configuration utilizes a flexure to convert horizontal motion to vertical motion. In a further configuration, the actuator is held at a fixed height and three lead screws are used to raise and lower the durable portion of the device.

[0069] There are many options for actuators. They can be actuated by a piezoelectric device combined with a position encoder. Alternatively, a DC motor can be used in combination with an encoder, or a servo motor can be employed. The actuation provided by a stepper motor is advantageous because each cycle rotates the stepper motor a fixed angle. Thus, steppers are effective for translating precise relative distances.

[0070] Stepper motors can be integrated with limit switches that can be used during device initialization. The limit switches provide an absolute position reference for the actuator. Additionally, acceleration sensors can be used to help determine absolute position, e.g. the orientation of the gravity vector, or to monitor missteps at the translation limits by an increase or absence of an acceleration signal pattern.

[0071] In some embodiments, a horizontally oriented stepper motor is coupled with a gear head to increase torque and decrease step size. The gear head then drives a bevel gear that converts the axis of rotation from horizontal to vertical. The actuation pin is finely threaded and coupled to a matching bushing housed in a durable device. Each step of the stepper motor translates the extension of the pin by approximately 1 μm (target translation per step is 0.01-10 μm). A larger step size is ideal for power consumption since fewer steps are required over the entire range of travel, but a smaller step size allows for smoother operation and improved precision.

[0072] When the direction of travel is reversed, backlash occurs. This backlash can be measured and accounted for during operation by adding an additional step each time the direction is reversed. Furthermore, the effect of rotational backlash can be minimized by choosing a lead screw with a fine thread pitch. Thus, the nominal angular backlash is translated into a smaller vertical backlash. By balancing the mechanical advantage provided by the gearbox attached to the motor, the tooth ratio of the pinion and bevel gear, and the thread pitch of the lead screw, the overall mechanical advantage of the system for converting stepper steps into vertical position changes can be maintained. For a given angular backlash, concentrating the mechanical advantage generated by the lead screw is advantageous in minimizing backlash.

[0073] 2.1.7 Internal telescopic pin If the pin is directly coupled to the stepper motor through a bevel gear, the position of the bevel gear should remain constant as the pin retracts into the device. There are several ways to make the pin slide relative to the bevel gear. If a keyway (or a non-circular shape) is machined into the shaft of the pin, the bevel gear will slide axially along the pin shaft but cannot rotate. In this configuration, the device must be large enough to accommodate the pin when it is fully retracted, as the top surface of the pin will rise as it retracts into the device. In some embodiments, the bevel gear is rigidly fixed to a shaft that contains a keyway (or another non-circular shape such as a hexagon). This shaft is inserted into another cylindrical shaft with alignment features. The outside of this second shaft is threaded and connects with a threaded bushing of the electronics. This format is vertically compact as the bevel shaft telescopically retracts inside the actuation pin. Figures 3A-D show an exemplary embodiment of a telescoping pin 300.

[0074] 2.2 Disposable Probe Lids 2.2.1 Inserting the probe into the well The lid 130 includes an array of probes 160, e.g., the probes are formed as integral rods extending from the inner surface of the lid. When the lid is secured to the device 110, each probe is coupled to a sensor 112 in the sensor array of the device. The lid (or the combination of the lid and device) constrains all the probes to move together in a plane. The probes 160 are configured to extend along a vector perpendicular to the plane of the lid (i.e., a horizontal plane parallel to the top of the plate 120). The probes are configured to be aligned with the array of wells in the well plate 120 in the xy plane. The probes can be configured to be aligned to the center of the well or other specific positions within the well (e.g., offset from the center, at a fixed distance from the sidewall, etc.), and multiple probes can be configured to cover a pattern within a single well. Alignment of the lid probes with the well plate center can be achieved by a ridge on the inner surface of the vertical portion of the lid that connects with the tapered outer wall of the well plate. To allow flexibility with tapered walled connecting plates and plates with various external dimensions, these ridges can be configured as bends. The probe is configured to penetrate the top surface of the fluid in the well and is further configured such that a nominal clearance between the cell at the bottom of the well (or suspended within the well) and the probe tip is always maintained. This can be achieved by a positive stop on the surface of the lid that interfaces with a feature on the top surface of the plate such that the probe cannot move within some nominal distance from the well bottom (typically 50-500 μm, more preferably 100-400 μm). The cross-sectional area of ​​the well perpendicular to the direction of travel of the probe is substantially constant. The constant cross-sectional area minimizes the importance of the absolute position of the probe within the well.

[0075] 2.2.2 The lid realizes vertical translation The lid translates along the axis of the probe (slight tilt of the lid is ignored). If the cells are uniformly distributed across the cross section of the well, a linear oxygen gradient may form within the well. Both the uniform distribution of cells and a substantially constant well cross-sectional area are important aspects of the design of some embodiments, as they minimize the importance of the absolute position of the probe.

[0076] The lid is placed on the culture plate to facilitate the effect of a sterile barrier over the top of the culture wells. Thus, the non-sterile actuation pins should not directly contact the surface of the culture plate, but instead contact the lid, which in turn contacts the culture plate (if the actuation pins are contained within the footprint of the culture plate). Thus, the lid can be configured to include features that allow translation of actuation initiated by circuitry in the durable device to change the relative position of the probe with respect to the well plate. More specifically, the lid can be configured to allow an array of optoelectronic sensors to move the probe vertically within the well.

[0077] Several features of the lid are used to achieve this functionality. The lid can be configured to include a rigid portion that constrains the probe and connects the durable device with its sensor array. One aspect of the connection between the lid and the sensor array device is that the connection can be configured to constrain the lid so that as the device moves, the lid moves with it. This constraining can be achieved, for example, when there is a magnetic attraction force that couples the lid and the device (see section 2.1.2). Alternatively, this can be achieved by allowing the lid to naturally force itself to extend as far away from the well plate as possible, and the device providing sufficient weight against the strength of the opposing force the lid exerts on the well plate such that the lid collapses completely flat against the well plate. The lid can also be configured to provide flexible or sliding regions that allow translation of motion in the plane of the lid without breaking the sterility barrier.

[0078] Also, in configurations where a magnet reversibly holds the lid against the durable sensor array, the magnet may be positioned and moved between positions such that the holding force can be turned on and off, or an electromagnet can be turned on and off or turned on to oppose a permanent magnet.

[0079] In an alternative configuration, where the lid feature can slide vertically up and down within the lid, the actuation pins can be positioned around the lid's sterility barrier while remaining within the footprint of the plate. When pressed by the actuation pins, these passive pins can similarly press against the horizontal surfaces of the culture plate.

[0080] There are several configurations that allow translation of motion through the plane of the lid while simultaneously maintaining a sterile barrier at the plane of the lid. In one configuration, a flexible diaphragm is utilized. The diaphragm can be folded or loosely held in place to minimize resistance to translation of the actuation pin. Potential drawbacks of this approach are that a thick membrane may not maintain a consistent thickness when pressed by the lid, and a relatively thin membrane relative to the thickness of the lid creates a dead space where the actuation pin may be configured to move before the probe moves relative to the well.

[0081] In a second configuration, a pin is provided that is vertically slidable within the lid. To maintain the vertical orientation of the pin, it is useful to maximize the vertical distance between the planar constraint of the bottom of the lid on the pin and the planar constraint of the top of the lid on the pin. The planar constraint may be, for example, a cylindrical perforation. In some embodiments, it may be desirable to minimize the thickness of the lid, for example to facilitate injection molding. If the thickness of the lid is minimal, the vertically sliding pin may be located on a thin vertical wall of the lid. The vertical travel limit of the pin may be constrained to maintain the coupling of the pin and the lid. This vertical constraint feature may consist of two vertical constraint features on the pin and one vertical constraint feature on the lid located between these two constraint features on the pin. Alternatively, the vertical constraint feature may consist of two constraint features on the lid and one vertical constraint feature on the pin located between these two vertical constraint features on the lid.

[0082] In a third configuration, the flexures allow translational motion from the durable device and the sensor array supported by the device through the disposable lid plane to the surface of the well plate. Advantages of this configuration include that the translation feature is constrained to the lid in the same plane of the lid (as opposed to along its axis of translation) and the thickness of the flexure along the axis of translation is a fixed value that can be selected by design. In particular, the setting of the thickness of the flexure along the axis of translation can minimize dead space. Finally, the flexures better utilize the available area of ​​the lid plane. In some embodiments, the flexures are located near the periphery of the lid (e.g., as illustrated in FIG. 1B).

[0083] 2.2.3 Bend Load and Neutral Position If a flexure is used to translate motion through the plate, the flexure can be configured such that its relaxed, neutral position is located anywhere inside or outside the limits of travel. In particular, the neutral position of the flexure can be selected near or beyond the maximum range of the actuation pin so that the spring action of the flexure does not tend to separate the lid from the durable sensor array.

[0084] In this configuration, the stiffness of the flexure is set so that the weight of the handheld sensor array (approximately 250 g) fully deforms the flexure and lowers the probe to its lowest position in the well. Figures 1A-B show the lid flexure with a relatively flexible section 132 bonded to pad 133. Another view is shown in Figures 4A-C, which show three use states where the flexures 132, 133 support the weight of the disposable lid when the durable sensor array is not placed on the lid (Figure 4A) and collapse completely flat under the weight of the durable sensor array (Figure 4B). When the actuation pin 116 is pressed against the top surface of the flexure pad 133, the weight of the sensor array is lifted off the disposable lid and the flexure relaxes as the lid is raised (Figure 4C). Magnets (e.g., 135, 136 in Figures 1A-B) or other coupling features can be used to maintain a tight coupling between the lid and the lid reader as the lid is raised. Additionally, the lifting force from the lid flexure reduces the force from the actuation pins required to lift the sensor array and lid.

[0085] Generally, the bent portion 132 has greater flexibility than other portions of the lid 130 (e.g., due to the material composition or due to the geometric shape of the cutout in the lid that forms the bent portion) such that as the device moves relative to the container, at least a first portion of the lid remains in contact with the container and at least a second portion of the lid separated from the first portion by the bent portion (e.g., bent pad 133) remains in contact with the device.

[0086] 2.2.4 Bends built into lids with stand-alone covers Features of the lid 130 that allow translation may require gaps in the plane of the lid's surface, which may reduce the sterility barrier formed by the lid. Figure 5A shows an exemplary gap 510. A separate cover 520, shown in Figure 5B, can be inserted over the bend so that a tortuous path 512 is formed and the sterility barrier is maintained. The use of a separate cover facilitates fabrication of the lid. In an alternative configuration, the tortuous path feature is built directly into the lid, and the bend is a separate piece that is attached to the lid during assembly.

[0087] The cover 520 may be permanently attached to the lid 130 via adhesive, solvent bonding, press fit, ultrasonic welding, or any other technique. In one embodiment, the cover is a membrane that maintains an integral sterility barrier.

[0088] 2.2.5 Lid Translucency In some embodiments, the lid is colorless and translucent so that light can pass through the lid for imaging (e.g., to illuminate the sample through the lid for viewing through the transparent bottom of the container). For example, the lid is formed from translucent or transparent acrylic or polystyrene. Although the electronics contained in the device are opaque, the device and contained electronics can be easily separated from the lid without disturbing the sterility barrier formed by the lid.

[0089] 2.2.6 Light focusing characteristics of the lid In some embodiments, the lid includes light focusing features that capture light from a large area and focus the light onto a small probe material. These focusing features also collimate or focus the light emitted from the sensing material onto a light collection device (e.g., a photodiode). The light focusing and moving features can include optical fibers, which may be coated with a hard material such as hard plastic or stainless steel tubing. The ends of the light focusing features present within the cell culture well typically have a minimum diameter between 250-800 μm in diameter cross section. Figure 6 shows the amount of disturbance in oxygen concentration levels that depends on the diameter of a probe held in place at a constant height within the well. This figure assumes that oxygen consumption by the cells is followed by a zero-order oxygen reaction (i.e., not substrate limited).

[0090] The cross section is minimized so as not to disrupt oxygen delivery to the cells in the well and to minimize disturbance of the fluid as the probe moves through the well. The light focusing features may be cylindrical or conical in shape, with a larger area for focusing light on the top side of the plate, and may have a diameter of up to 4 mm, or even up to 6 mm. Optionally, the probe may be manufactured as a stand-alone component and integrated into the lid, for example using the techniques described in section 2.2.4.

[0091] 2.2.7 Integral Fluid Dispensing Ports The lid can include a reservoir for holding a small amount of fluid, such as a drug to be added to the cells in the well. After the reservoir and cover are equilibrated, for example in an incubator, the fluid can be added to the sample and the change in oxygen consumption can be measured to evaluate the effect of the fluid on the sample. The array of reservoirs can be aligned with the wells of the plate. There are various methods for dispensing the fluid, including pneumatic or mechanical methods, for example, U.S. Pat. No. 8,318,479. Additionally, the design of the reservoir can include multiple bends that are closed in the natural state. The number of bends can be as many as 4 to 8 or 16, such that when they are separated, there is a minimum surface area to hold a droplet of fluid. This small amount of fluid can be, for example, up to 20 microliters or less than 100 microliters. The maximum surface area of ​​any bend can be set such that the force of gravity on the droplet of fluid is greater than the force from the surface tension between the bend surface and the droplet. The surface tension between the droplet and the multiple bends in the closed position can be greater than the force of gravity on the droplet. The flexure may also include a tab at the top of the receptacle such that when the pin is inserted into the top of the vessel, the flexure opens and allows the fluid contained within the vessel to drop into the well below. In some embodiments, the tab is positioned such that the pin never touches the fluid held within the vessel. The lower end of the receptacle flexure may be designed with a point to minimize the surface tension available to hold the drooping fluid.

[0092] Another option for incorporating a fluid dispensing port is to provide a tilted well with a shallow slope that steepens depending on the actuator position and allows the fluid to fall to mix with the fluid in the container below. Generally, a method of dispensing fluid can be used that utilizes the operation of an existing system.

[0093] 2.2.8 Calibration Features The disposable lid can include a unique identification code (e.g., embedded in read-only memory 139 as illustrated in FIGS. 1A-B) that can be read by durable electronic device 110 electronically (e.g., by reader 138 making electrical contact with memory 139 as illustrated in FIGS. 1A-B), optically, or otherwise. This identification code or process can be used to verify the authenticity of the lid. The lid can also include electronics for storing calibration and usage information as well as verifying the authenticity of the lid. This calibration information can potentially include the Stern-Volmer constant, which indicates the relationship between light attenuation rate and oxygen concentration, and the calibration can include information for performing temperature compensation, information for factoring in the useful life of the sensor, and calibration information related to the response time of the probe and / or associated diffusion constants.

[0094] 2.2.9 Built-in Lid Electronics The lid can include electronics (e.g., EEPROM) for storing data such as probe calibration constants, identification codes, manufacturing dates, authentication signatures, etc. This data can be read by the optoelectronic sensor array via a communications interface. The lid electronics can also include functionality for detecting removal and attachment of the lid.

[0095] 3 Exemplary Measurement Process Referring to FIG. 7, an example of a measurement process 700 that can be used in operating the measurement system includes three different types of procedures, some aspects of which can be performed simultaneously: controlling the probe movement pattern (702), taking sensor readings (704), and running an algorithm to estimate a parameter of the well environment, i.e., cellular oxygen consumption (706).

[0096] 3.1 Continuous movement pattern In some embodiments, the measurement system moves a set of probes vertically according to a movement pattern. The described pattern differs from a pattern configured to perform a series of measurements at various fixed heights where the measurements are processed to have a one-to-one correspondence with the heights at which they were measured (step pattern). In a step pattern, the motion is configured to pause so that measurements at a certain height are not correlated with previous heights of the probe (or a more general pattern). In the described system, the movement pattern can be seen as an input waveform that is transformed by the measurement environment to generate an output signal. Considered in this way, the techniques used for signal analysis and processing can be used to characterize and estimate the measurement environment in order to optimize the measurement process.

[0097] One type of movement is continuous movement (e.g., substantially continuous on the time scale of mass movement in the fluid) without significant pauses during the movement (e.g., pauses greater than those inherent in the use of stepper motors to control the actuators). Another type is stepped movement, in which the probe is moved relatively quickly between two positions followed by a pause for a significant amount of time. In some cases, a pause is made that is long enough for the gradient to be established. This type of stepped pattern using a "clear stop" where the gradient is at least partially established is not optimal for some embodiments of the measurement process 700 because a clear stop (e.g., longer than about 50 time constants) reduces the responsiveness of the measurement system. An important aspect of some embodiments of the measurement process 700 performed using the described measurement system is that they can operate using a movement pattern without a clear stop that some other systems may require for the gradient to be re-established and / or the sensor readings to stabilize. Furthermore, the movement speed is variable, but is typically slow enough not to exceed a speed limit set by a dimensionless number threshold determined by the Stokes flow, and possibly a speed limit set by the diffusion constant.

[0098] In some embodiments, the movement pattern includes a "continuous movement pattern." As used herein, "continuous movement" is intended to be understood as either strictly continuous movement (i.e., no pauses) or stepwise movement in which the duration of the pauses in the stepwise motion is relatively short compared to the time constant of the system (e.g., less than about 20 time constants, or less than about 10 time constants). In some examples, the system time constant can be based on the diameter of the probe (e.g., when it comes to the speed of movement), in other examples, the system time constant can be based on the distance traveled by the probe (e.g., when waiting for stabilization for the probe to disturb the gradient), in other examples, the time constant can be based on the distance between the probe and the sample, and in other examples, the time constant can be based on the distance between the sample and the air-liquid interface. Such continuous movement can provide continuous and accurate disturbance of the concentration gradient in a manner that can be modeled without requiring a delay for the disturbance to dissipate.

[0099] 3.1.1 Fixed movement patterns The system can operate with various kinds of continuous movement patterns. These movement patterns include sinusoidal, multiple sinusoidal at different frequencies, sawtooth, sink, chirp, or stochastic / irregular patterns (e.g., random walk). In general, patterns that cover a larger range give better resolution measurements for a given probe sensor resolution (i.e., more sensitive to different gradient levels, especially lower / flatter gradient levels corresponding to low oxygen consumption). On the other hand, patterns that repeat more frequently (i.e., fast / short period patterns) can achieve higher time resolution and detect changes in consumption sooner. For example, a sinusoidal pattern with a relatively high frequency and low amplitude (i.e., small excursion) will respond more quickly to higher gradients that change more rapidly, while a slower frequency and larger amplitude pattern will give better accuracy for low (i.e., shallower) gradients at the expense of response time. Furthermore, chirp movement patterns can be indicative of the response time of a given probe. Irregular patterns can ensure that measurement errors are not introduced by external processes that would interfere by correlation with regular patterns.

[0100] Known patterns can also be used to filter out noise in sensor readings, i.e., an effective gain in sensor sensitivity. For example, if the movement pattern is sinusoidal and the environment is modeled under Fick's law, the signal can be filtered with a low-pass or band-pass filter that attenuates the noise while allowing the signal resulting from the underlying concentration gradient to pass unaffected. As a waveform, the step pattern occupies a wide band of frequencies, and as a result, so does the signal of interest contained in the sensor reading. Thus, when using step patterns, it is difficult to filter sensor noise without attenuating the signal of interest.

[0101] Since the selection and configuration of migration patterns results in trade-offs in system performance, the system can be configured to operate in different modes depending on the type of experiment and the properties the user is interested in. In one configuration, the user may select a deeper migration pattern (closer to the cells) to measure higher frequency changes in cell behavior. In another configuration, the user may select a pattern that covers a wider area to obtain better resolution, as described above. In another configuration, the user may select a migration pattern that operates higher in the well to minimize the effect of the probe on oxygen delivery to the cells. In another configuration, the user can select a migration pattern that tailors oxygen delivery to the cells with a specific profile, using the system simultaneously for both measurement and environmental control.

[0102] The system can be configured to prompt the user to select the desired performance from a subset of options or via a short questionnaire, and the actual travel pattern is determined based on the input from the user. Additionally, the ideal travel pattern can be determined automatically, as described below.

[0103] 3.1.2 Dynamic Movement Patterns In addition to the modes and patterns that can be selected based on user interest, the system can automatically update the movement based on actual external events. These different pattern modes can be automatically triggered based on environmental conditions ascertained by the system's sensors or external sensors. For example, if a low oxygen gradient is detected in most or all wells, the optimal movement pattern may be deeper, closer to the cells. If a steep oxygen gradient is detected in most or all wells, a movement pattern optimized for high resolution at steeper gradients can also be used. The movement pattern may also be automatically determined based at least in part on determining which direction most requires additional information (e.g., by using the uncertainty state of a Kalman filter for the wells). Additionally, the movement pattern can be set independently for each operating point so that all probes do not move along the same vertical pattern. Dynamic movement patterns can be achieved in some embodiments of the measurement system using any number of the following features: (1) The lid contains either an electrical, active or passive, or non-electrical (e.g., optical, magnetic) feature that allows electronic equipment to detect when the lid is attached. (2) The electronic device has built-in sensors, possibly including acceleration, temperature, relative humidity, oxygen partial pressure, ambient light, oxygen, carbon dioxide or other gas concentration, or other sensors. (3) The start or end of a sequence of movements can be triggered by the installation (removal) of a lid or the reading of other sensors. (4) The travel pattern includes a zeroing step utilizing limit switches and is triggered by an accelerometer / lid attachment. (5) The state of the motion pattern (determination of the next action) is conditioned not only by the current state of the system (dam, pre-programmed pattern) but also by the state of the environment (system under test) measured by one or more built-in sensors. For example, the sensor readings can include any number of the following: a. Accelerometer readings indicate that the plate has been lifted or lowered or is not level on the shelf. b. An overall low dissipation rate may cause the probe to move lower or to a greater extent within the well, whereas a high dissipation rate may trigger the opposite. c. If the gradient is stable, the probe can be shifted to a lower position in the well, the actuation rate can be slowed, or the amplitude can be increased. d. Temperature readings below atmospheric (or other environmental factors such as humidity) may indicate recent changes in the medium and it may be desirable to keep the probe in a single location.

[0104] 3.2 Sensor Readings Sensor readings to obtain measurements of dissolved oxygen concentration can be obtained in a variety of ways. A material can be used whose optical properties are sensitive to the surrounding oxygen environment. The material can be excited with light from an LED and return to its ground state by emission of a photon or energy transfer to molecular oxygen (quenching). The amount of light emitted and the rate of decay of the light emission are inversely related to the oxygen partial pressure via the Stern-Volmer relationship. The intensity of the excitation light source can be modulated between two stages (typically on and off) in an irregular pattern. This irregular pattern can be generated via a pseudorandom code, which can be low-pass filtered and then discretized into two levels. This code can be output from a logic circuit such as an ASIC or FPGA. The response can be read using an analog-to-digital converter and the digitized response can be processed on the logic circuit. Each reading of the ADC light emission signal can be processed in the FPGA, for example using a recursive method, and the final system response after some predefined reading time can be further analyzed and converted into a decay rate. Examples of such techniques are described in more detail, for example, in U.S. Pat. No. 9,075,011, which is incorporated herein by reference. (The terminology used in the incorporated documents may differ slightly from the terminology used in this specification, but in the event of a conflict in terminology, the terminology used in this specification shall control.)

[0105] Because the process is recursive, the sensor read time can be set arbitrarily. Short sensor read times (1-100 ms) may result in less accurate readings for each sensor compared to longer read times (100-600 ms). That is, to obtain greater sensitivity and / or resolution in the measurement, sensor reads of longer duration may be performed, or shorter sensor read times may sacrifice measurement sensitivity and / or resolution. For short reads, the previous reading of a given sensor may be used to initialize the recursive pattern such that the algorithm startup error is minimized. Multiple short readings may be processed together to produce a single high-precision reading for downstream processing.

[0106] The properties of this sensor methodology can be used to maximize the overall performance of the system. Sensor read times can be altered to match movement patterns, measurement modes, and experiment types. For example, movement patterns tailored for better time resolution can be matched with the use of shorter sensor read times to produce even better time resolution. Alternatively, if maximum sensitivity of oxygen consumption / gradient measurements is desired, longer sensor read times can be used in combination with movement patterns tailored for higher resolution. To satisfy other system constraints such as cost, size, or power, alternative configurations may use longer sensor read times in combination with movement patterns of smaller range. As with dynamically selected movement patterns, this trade-off selection can be performed dynamically based on detected environmental conditions.

[0107] In general, the configurable read time vs. resolution tradeoff offered by this sensor read method allows great flexibility in system design and operation.

[0108] 3.3 Estimation Algorithm 3.3.1 Algorithm Overview In some embodiments, an example of the steps of an algorithm for estimating parameters of a well environment is as follows: (1) A sensor reading is taken at time t along the motion cycle. (2) The reading is converted to an oxygen concentration measurement, c(t), using the calibration and temperature data. (3) An estimated measurement pattern height is calculated based on the sensor response transfer function and the cycle pattern to give a concentration measurement as a function of depth c(z, t). (4) Using the new measurement and a set of previous measurements, a concentration versus height regression is performed to interpolate concentrations at one or more discrete pattern heights. (5) Kalman observations are generated based on the interpolated concentrations. (6) New observations are applied to the Kalman filter to update the estimate according to a state transition model based on Fick's law.

[0109] Referring to FIG. 8, an example of an estimation process that can be used in operating the measurement system includes the steps of: receiving a concentration measurement at time c(t) and applying (802) an inversion of the sensor transfer function to give a concentration measurement at height c(z, t); performing (804) concentration interpolation at discrete heights to give a Kalman observation; and applying (806) a Kalman filter to give a processed measurement.

[0110] 3.3.2 Considering sensor response The sensor probe has a response delay that can be accounted for to provide accurate measurement data. One way to account for the response delay is to stop at a given depth and discard the readings until the sensor has had enough time to stabilize. This approach may not be preferred in some embodiments because the cell consumption rate may be variable and a long fixed pause will reduce the transient response. In a continuous migration pattern, the sensor readings do not necessarily stabilize at a value that corresponds to the concentration at the depth where the probe is currently located. One approach is to use a migration pattern that is slow enough relative to the sensor delay so that the readings at a given point in the migration pattern correspond to the actual concentration at the given depth within a certain threshold.

[0111] Another approach considers the sensor response delay as a system transfer function whose input is the waveform of the motion pattern. This sensor transfer function can be measured or estimated and then compensated to obtain sensor readings as a function of height. This correction may require time shifting and scaling depending on the motion pattern. Assuming a roughly steady-state slope for a short time window, for a sinusoidal pattern, the sensor transfer function can be modeled as a low-pass filter that attenuates and phase shifts the sensor readings. This filtering can then be compensated by deconvolution (i.e., phase shifting and magnitude scaling). This transfer function can be dynamically calculated by assuming a steady-state linear slope or a non-steady-state slope (e.g., modeled using an erf function) and then running a constrained optimization algorithm (e.g., brute force error minimization) to estimate the phase shift (sensor lag).

[0112] 3.3.3 Observation model As shown in FIG. 11B, after the sensor response is compensated, the concentration readings at the steady-state gradient are a linear function of height. This is consistent with the expectation that a diffusion gradient will form with a steady-state flux (i.e., cellular oxygen consumption) at the bottom of the well and stable atmospheric conditions at the top surface. At this point, these values ​​can be used to evaluate concentration estimates at a set of uniform, discrete heights, providing Kalman observations for input to the Kalman filter. An example of linear interpolation using a set of small heights is shown. This interpolation step allows the use of a smaller set of Kalman states of concentration values ​​while still using sensor readings at any point in the migration pattern.

[0113] 3.3.4 Kalman filter states The Kalman filter state is a set of uniformly spaced relative heights c0...c N-1 A set of concentrations at , the bottom flux into / out of the cell, q c , upper flux q tThe state updates can be calculated according to Fick's law using finite differences.

[0114] For example, a state x with N height cardinality is:

[0115]

number

[0116] 3.3.5 Kalman State Transition Model The Kalman state transition updates, in some embodiments, may be calculated based at least in part on Fick's law for the modeled well environment.

[0117] 3.3.6 Output measurements The primary measurement parameter output of this system is flux q c The Kalman estimate of is because it is directly related to cell activity and can be further processed into specific measurements of cell activity such as oxygen consumption rate. In addition to this, all other state variables of the Kalman filter can be used as output measurement estimates. From these measurements, higher level measurements and events can also be inferred. If the concentration estimate as a function of height matches a particular shape, then a non-uniform cell seeding density is inferred. Furthermore, if a low but increasing gradient is detected in many or all wells, then a medium change event can also be inferred. These events can automatically trigger a change in migration pattern as described elsewhere herein.

[0118] In general, if oxygen consumption by the cells is uniform, then at steady state the gradient within the well will reduce to a uniform one-dimensional linear gradient. This assumes that the cell density is also uniform. If consumption is greater directly below the path of the probe, the gradient will be higher towards the bottom of the well and lower towards the top of the well, creating an upwardly curving gradient along the path of the probe. If consumption is greater away from the probe than directly below the probe, the opposite will be true, resulting in a downwardly curving gradient along the path of the probe. Using this information, the system can inform the user of non-uniform distribution of cells within the well, or inform the user of expected bulk consumption measurements or the range of measurements at the well surface.

[0119] In other embodiments, a nonlinear space-time model (e.g., a nonlinear autoregressive moving average model with exogenous inputs, or NARMAX model) is employed (see, e.g., Stephen A. Billings. Nonlinear System Identification: The NARMAX Method in the Time-Frequency-Space-Time Domain. Wiley, September 2013. ISBN 978-1-119-94359-4). In this scenario, both the current probe position and the concentration reading are entered as inputs into a nonlinear model, which is used to estimate parameters of the underlying system involving the transport of the molecule of interest through the region of interest.

[0120] 4 Handling of Data 4.1 Software The measurement system includes a computational subsystem that can be configured to execute a number of software components capable of performing a variety of useful operations. These components include embedded software that operates on circuitry within the device and, in some embodiments, is coupled to the optoelectronic sensor array (see "Disposable Probe Lid" section), data processing and storage software, and user interface software for data visualization, device control, and general laboratory functions.

[0121] 4.1.1 Lid integration software In some embodiments, software installed on an embedded element (e.g., a processor or other computing circuit) within the device and coupled to the optoelectronic sensor array includes functionality relating to any of the following: Light sensor readout Auxiliary sensor readouts (temperature, humidity, accelerometer, air pressure, ambient light, etc.) Actuation control to execute movement patterns Lid installation detection -Read / write lid data Lid data verification and authentication ·Communication interfaces with data processing and storage systems

[0122] 4.1.2 Data processing and storage The data generated by the embedded elements may be additionally processed and stored by software running on a coupled secondary system. This software may run, for example, on a desktop, a server, or on an embedded OS. This software may be distributed among the hardware components and may be local to the measurement environment or remote (i.e., via communication with "cloud" software). This software may store the data measurements in a database for later analysis. This software may forward this data to user interface software for real-time visualization and monitoring. This software may also communicate with the device in a variety of ways and may rely on a variety of protocols, such as USB, bluetooth, TCP / IP, WiFi, etc. Additionally, this software may perform data backups for redundant storage and data recovery.

[0123] 4.1.3 User Interface The system may be monitored and controlled through a user interface managed by software configured to render the user interface on a display and may be configured to receive input through one or more input devices. The software may reside on the same system as the data processing and storage software or may reside on a separate hardware device such as a touch screen tablet or workstation. The software may be a traditional desktop application, a web application, a mobile application, or a combination thereof. The software may provide visualization of system measurements provided to the user. The software may allow for operational control of the device and configuration of various system parameters.

[0124] The software may also include general features useful for conducting experiments, such as logging of experiment start times, end times, summaries, notes, events, conditions, etc.

[0125] The software can also display the results of an experiment, or display aggregated results from past experiments for comparison with any experiment as the experiment runs in real time. These results from previous experiments can be from experiments run on the same device, on other devices, or from other laboratories. These previous results can be displayed as is, or can be aggregated and adjusted using various techniques.

[0126] 4.1.4 Experimental Verification The system software can also provide an experiment authentication feature for auditing and verification of laboratory experiments. Fabrication, falsification, and theft of scientific data are serious problems. Using digital signature algorithms (e.g., ECDSA) and optionally on-board tamper-proof components, the system helps to ensure that for a given experiment: Data integrity: Data has not been altered (secure on-board digital signature). · Attributes: Data is recorded by a specific device (secure on-board digital signature). Data is recorded at a specific date and time (on-board tamper-proof clock). Data is recorded at specific locations (on-board tamper-proof GPS). · Compliance with Good Laboratory Practices (GLP).

[0127] 5 Example measurement data Figure 9 shows an example of just over an hour of average centered measurements (uncalibrated) from four sensors using an exponential chirp movement pattern. Because the sensors are operating at a steady-state linear slope and the amplitude of the chirp movement pattern is constant, the change in the amplitude of the readings indicates a frequency dependent attenuation of the readings that can be modeled as an LTI filter or other system transfer function acting on the movement pattern signal. Note: the y-axis is time in experimental time.

[0128] 10A and 10B show examples of concentration measurements from four sensors using a sinusoidal motion pattern (FIG. 10A) and these concentration values ​​as a function of probe depth at the time of reading without compensating for the sensor response of the readings to this cycle pattern (sensor lag) (FIG. 10B). Because the concentration versus height profile does not match the profile expected based on the law of diffusion, the data is further processed.

[0129] 11A and 11B show examples of concentration measurements from four sensors using a sinusoidal motion pattern (FIG. 11A) and these concentration values ​​as a function of probe height after compensating for an approximately 80 second lag in readings for this cycle pattern (FIG. 11B). After compensation, the concentration versus height profile appears linear following a steady-state linear slope.

[0130] Figures 12A and 12B show an example of the conversion from a set of lag-compensated concentration values ​​for four sensors to Kalman observations for an N = 3 height model. The bubbles represent the clusters of values ​​used for regression and interpolation at each of the three heights. The dashed lines represent the linear regression of each cluster. The concentration estimates (c1, c2, c3) at each height are then interpolated to update the Kalman state. The slopes of these lines (dC / dz) can also be used to estimate the degree of nonlinearity across the profile. This could indicate either a rapidly changing flux or a non-uniform consumption distribution (i.e., non-uniform cell density). NOTE: The x-axis represents the height of the probe above the well bottom in micrometers, and the y-axis represents the oxygen concentration in micromolar units.

[0131] 6 Alternatives and implementation examples Several alternatives to the described embodiments can be used, for example the probe is moved relative to the well, e.g., to sense oxygen gradients at various depths, but in some alternatives it is the vessel that remains stationary and the probe moves relative to a fixed reference (e.g., a shelf in an incubator), rather than moving with, e.g., the device or some other cover.

[0132] In many embodiments, standard culture trays are used having an array of 6, 12, 96, etc. wells. Alternatively, single well vessels such as cell culture flasks may be used, with one or more, typically multiple, probes placed in a single well.

[0133] The techniques described above can be implemented using a program that includes instructions for execution on a device or module (e.g., a control module and / or an analysis module), which includes one or more processors or other circuits for executing the instructions. For example, the instructions execute software or firmware procedures that run on one or more programmed or programmable computing devices or modules, which include at least one processor and at least one data storage system (e.g., including volatile and non-volatile memory and / or storage media). The program may be provided on any non-transitory medium, including a computer-readable storage medium such as a CD-ROM, and may be readable by a general-purpose or special-purpose programmable computer, or may be distributed over a communication medium, such as a network, to a computer on which it is executed. Each such program may be stored in a storage medium (e.g., a solid-state memory or medium, or a magnetic or optical medium) readable by the device, or downloaded to the device, to configure and operate the computing device when the storage medium is read by the device to execute the program procedures.

[0134] The above description is intended to be illustrative, and not limiting, of the scope of the invention, which scope includes the appended claims. Other embodiments are within the scope of the following claims.

Claims

1. 1. An apparatus comprising a device (110) for measuring molecular levels of a fluid in a container and configured to be mounted as part of a cover (110, 130) over a container (120) having a set of wells (122) for holding a biological sample (172) immersed in a fluid (174), 4. The device comprising: a plurality of optoelectronic sensors arranged to acquire optical signals from a probe (160) immersed in the fluid, the optical signals being indicative of a molecular level of the fluid at a sensor (162) of the probe; a set of actuators configurable to adjust the relative position of the device and the container; a control circuit for controlling the set of actuators to vary the relative position between the device and the container and to acquire the optical signals at a plurality of relative positions of the device with respect to the container while varying the relative position; The device further includes a lid (120) fixedly attached to the device (110) to form the cover, the lid comprising: a surface configured to cover the well (122) of the container (120); a set of probes, each probe (160) extending substantially perpendicularly from the surface of the lid; the lid is configured such that a position of each probe on the lid corresponds to a position of an optoelectronic sensor on the device, and when the lid is attached to the device, an optical path is formed between the sensor (162) on the probe and the corresponding optoelectronic sensor (112); The apparatus, wherein the lid is further configured such that the location of each probe on the lid corresponds to a location within one of the set of wells of the container.

2. The apparatus of claim 1, wherein the device (110) includes a set of actuators that are jointly controllable by the control circuit to control an orientation of a plane defined by the sensor of the probe relative to a plane defined by the set of wells of the container.

3. 3. The apparatus of claim 1, wherein the control circuit is configured to maintain a plane defined by the sensor of the probe parallel to a plane defined by the set of wells of the container.

4. 4. The apparatus of claim 1, wherein the relative position of the device and the container is variable between 10 micrometers and 3 millimeters.

5. The apparatus of any one of claims 1 to 4, wherein the lid (130) comprises a sterile material.

6. 6. The apparatus of claim 1, wherein the lid (130), when attached to a container, is configured to maintain a sterile environment within the well by isolating a surface of the device from the well.

7. 7. The device of claim 1 , wherein the lid is configured to be formed over the well, maintain a gas path between the well and an environment outside the device, and maintain a sterile barrier between the well and the environment.

8. 8. The device of claim 7, wherein the lid is configured to form a serpentine path between the wells and the environment without forming a linear path in or above any of the wells and the environment.

9. 9. The apparatus of claim 1, wherein the lid includes a pair of flexures, each flexure having greater flexibility than other portions of the lid, such that as the device moves through the changing relative position of the device to the container, at least a first portion of the lid maintains contact with the container and at least a second portion of the lid separated from the first portion by the flexures maintains contact with the device.

10. 10. The apparatus of claim 1, wherein the cover formed from the device and the lid is configured to be removable from the container without moving the container.

11. 11. Apparatus according to any preceding claim, wherein the lid includes guide elements which engage with corresponding elements on the device to fix the relative positions of the lid and the device.

12. The apparatus of claim 11 , wherein the guide element comprises at least one of a protrusion and a recess in a surface of the device.

13. 13. The device of any of claims 1-12, wherein the lid is translucent.

14. 14. The device of any of claims 1-13, wherein the lid is sterile.

15. 15. The device of claim 1, wherein the lid includes a set of fluid dispensing ports, each of the ports configured to dispense a fluid stored in the lid into a corresponding well.

16. 16. The apparatus of any preceding claim, wherein the device has a mass of less than 1000 grams.

17. The apparatus of claim 16 , wherein the device has a mass of less than 250 grams.

18. 10. The apparatus of claim 9, wherein the set of flexures forms a spring with force, the device having sufficient weight to compress the flexures across the varying relative positions of the device and the container, and the spring applying sufficient force to maintain contact of the lid to the device across the varying relative positions.

19. 2. The apparatus of claim 1, wherein the lid (130) is fixedly attached to the device (110) and maintains alignment of the probe of the lid and the optoelectronic sensor of the device over a range of variation of the relative positions.

20. 20. Apparatus according to any of claims 1-19, wherein the device includes a data interface for receiving data from a corresponding element in the lid.

21. 21. The apparatus of claim 20, wherein the data interface includes electrical contacts that electrically connect to corresponding electrical contacts of the corresponding element of the lid.

22. The apparatus of claim 20 , wherein the data interface includes an image sensor for acquiring an image of a portion of a surface of the lid.

23. The apparatus of claim 20 , wherein the data interface comprises a radio frequency communication device.

24. 24. Apparatus according to any of claims 20-23, wherein the data interface is coupled to the control circuitry for controlling the actuator and the acquisition of the optical signal from the sensor.

25. 25. The apparatus of any of claims 1-24, further comprising the container (120), wherein the lid is attached to the device and positioned between the device and the container to maintain a sterile barrier between the surface of the fluid and an environment outside the device, and wherein at least some gas passage exists between each well and the environment outside the device.

26. 26. The apparatus of claim 25, wherein the container includes a plurality of wells, and the lid is configured to include at least one probe corresponding to each well.

27. 27. The device of claim 25 or 26, wherein the lid is configured to carry multiple probes corresponding to the single well.

28. 28. Apparatus according to any preceding claim, further comprising a data processing system configured to accept data representing sensor values ​​obtained at multiple relative positions.

29. 30. The apparatus of claim 28, wherein the data processing system is configured to combine the received data to calculate a rate of consumption of the molecule by the biological sample.

30. 30. Apparatus according to any of claims 28 and 29, wherein the data processing system includes a display for displaying quantities derived from the sensor values.

31. 31. The apparatus of claim 30, wherein the data processing system is configured to display the quantities derived from the sensor values ​​in combination with reference quantities.

32. 32. The device of any of claims 1-31, wherein the molecule comprises oxygen.

33. A method for measuring molecular levels of a fluid in a container using a device according to any one of claims 1-32, comprising the steps of: adjusting the relative position of the device and the vessel, the adjusting step comprising moving the sensor within the fluid without substantially disturbing the gradient of the molecular levels in the fluid while maintaining a gas path from the well of the vessel to an environment outside the vessel; acquiring sensor signals at a plurality of said relative positions while varying said relative positions; and communicating data determined from the acquired signals from the device to a data processing system separate from the device.

34. 1. A method for performing a measurement, comprising: Providing relative movement between the container (120) and the array of probes (160) over a measurement time period including a number of periods, the distance between the container and the array of probes (160) varying along a first axis over at least a portion of each period; The vessel includes a set of wells, at least one of which supports a sample (172) and contains a fluid (174), the fluid having an upper surface exposed to a gas (180), the gas having a concentration of molecules, the sample interacting with the molecules in the fluid, each probe configured to measure a concentration of the molecule in the fluid at a distance from a bottom of a well that is dependent on the distance between the vessel and the probe; providing a relative movement, the relative movement being along an axis substantially perpendicular to a plane of the upper surface of the fluid; acquiring measurement data from two or more of the plurality of probes at varying distances from a bottom surface of the well over each of a plurality of said time periods, at least a first probe configured to measure a concentration of the molecule in the fluid contained in a first well of the set of wells; acquiring, wherein the measurement data acquired from the first probe includes at least two data samples acquired at different respective distances from the bottom surface of the first well within a first period of the plurality of periods.

35. 35. The method of claim 34, wherein the step of causing relative movement causes substantially continuous movement.

36. The step of obtaining the measurement data comprises:

36. The method of claim 35, comprising acquiring successive data samples within the first time period, wherein a maximum time lapse between successive data samples is less than a time required for stabilization of the environment of the fluid following a shift in relative position.

37. 37. The method of claim 36, wherein the maximum elapsed time is less than a predetermined threshold based at least in part on a time constant associated with the smaller of a square of the difference between the distances over which the successive data samples were acquired and a cross-sectional area of ​​the probe.

38. 35. The method of claim 34, wherein the first well is deep enough such that there is at least 10 microns between the bottom surface and the top surface of the fluid within the first well.

39. 1. An apparatus for performing measurements comprising: a vessel (120) including a support and a set of wells (122) within said support; and a cover, the cover comprising: A device (110), comprising: a set of actuators configurable to adjust the relative position of the device and the container; a device including: a control circuit for controlling the set of actuators to vary the relative position between the device and the container and to perform the measurements at a plurality of relative positions of the device with respect to the container while varying the relative position; a lid (130) including a set of probes (160), the set of probes extending from a surface of the lid, each probe extending into a well of the vessel; the device and the lid are configured to engage the vessel, and when the device engages the vessel, the shape of the lid prevents the formation of a straight line path between any portion of the air gap above the well and the atmosphere outside the air gap; the cover further includes a control subsystem configured to effect relative movement between the container and the cover, where the distance between the container and the cover varies along a first axis based on movement of a plurality of mechanical actuators, each of the mechanical actuators configured to move a movable element within the device, the movable element contacting the container when the device engages the container; a measurement subsystem configured to provide excitation light propagating from the probe and configured to collect emission light propagating within the probe.

40. 40. The apparatus of claim 39, wherein the movable element comprises a flexible diaphragm.

41. 2. The apparatus of claim 1, wherein the control circuitry configured to control the set of actuators is configured to continuously vary a depth of a probe within fluid within the vessel and acquire a plurality of optical signals while continuously moving the probe.

42. 42. The apparatus of claim 41, wherein the control circuitry is configured to move the probe within the vessel according to at least one pattern from a set of patterns consisting of a sine wave, multiple sine waves at different frequencies, a sawtooth, a sincline, a chirp, or a regular pattern.

43. 1. An apparatus for measuring molecular levels of a fluid in a vessel, the apparatus comprising: a device configured to be mounted as part of a cover including a plurality of probes over a vessel having a set of wells for holding biological samples immersed in the fluid, wherein changes in the relative position of the device and the vessel vary the depth of the probes within the fluid in the wells, the device comprising: a plurality of optoelectronic sensors positioned to acquire optical signals from respective probes immersed in the fluid, each optical signal representing a molecular level in a respective probe; a set of actuators configurable to adjust the relative position of the device and the container; and a control circuit for controlling the set of actuators to vary the relative position between the device and the container and to acquire the optical signal within the container at a plurality of respective relative positions of the device with respect to the container while varying the relative position, wherein when the device is mounted as part of a cover over the container, the control circuit by its operation: and causing relative movement between the container and the plurality of probes over a measurement time period comprising a number of time periods, such that a distance between the container and the array of probes increases or decreases along a first axis over at least a portion of each time period; each probe configured to measure the level of molecules in the fluid at a distance from a bottom of a well that is dependent on the distance between the vessel and the probe; the relative movement is along the first axis substantially perpendicular to a plane of the upper surface of the fluid; The control circuitry is configured to operate to obtain measurement data from two or more of the plurality of probes over each of a plurality of said time periods, with at least a first probe configured to measure a concentration of the molecule within the fluid contained in a first well of the set of wells; The measurement data acquired from the first probe includes at least two data samples acquired at different respective locations within the first well during a first period of the plurality of periods.

44. 44. The apparatus of claim 43, wherein the control circuitry is configured to vary a rate of the movement within each period.

45. 44. The apparatus of claim 43, wherein the control circuitry is configured to vary the movement pattern between periods.

46. 10. The apparatus of claim 1, wherein each probe of the set of probes includes an elongated light guide for passing light acquired from a tip of the probe immersed in the fluid to an optoelectronic sensor of a device attached to the lid.

47. The apparatus of claim 6 , wherein the lid is configured to fully support the device placed on the lid.

48. The apparatus of claim 1 , wherein the lid is configured to induce relative movement between the lid and the container without the device being directly connected to the container.

49. The apparatus of claim 1 , wherein the position of each probe on the lid is aligned with the position of a corresponding optoelectronic sensor on the device.

50. 24. Apparatus according to any of claims 1-23, wherein the device includes circuitry for passing a signal from the photoelectron sensor of the device forming the cover to an external processing system.

51. The apparatus of claim 1 , wherein the device is configured to be handheld.

Images