Multimodal sensing of biological samples

Abstract

A method for sensing biological samples in a multi-well plate, for example on an ongoing basis over an extended period of time, provides an image acquisition function. For instance, such image acquisition may use multiple different illumination conditions for each sample. In some examples, a result of optical scanning is combined with sensing of dissolved gas characteristics (e.g., gradient of oxygen concentration; oxygen consumption, etc.), for example, to normalize the gas characteristics according to number of cells or volume of the biological sample. An apparatus (100) for measurement of a plurality of biological samples (124) in a multi-well plate (120), comprises: an illumination source with a plurality of controllable light sources (144) for illuminating wells of the multi-well plate; an imager (152) for acquiring images of samples in said multi-well plate; and a controller configured to control the light sources to provide multiple illumination patterns for each well, and to cause repeated acquisition using the imager of sets of multiple images in respective illumination patterns.

Description

MULTIMODAL SENSING OF BIOLOGICAL SAMPLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 671,843, titled “Multimodal Sensing of Biological Samples,” filed on July 16, 2024, which is incorporated herein by reference.BACKGROUND OF THE INVENTION

[0002] This invention relates to multimodal sensing of biological samples.

[0003] A number of embodiments for sensing gas concentration gradients as a means of inferring metabolic activity in biological samples, for example, in multi-well plates, are described in U.S. Pat. 9,075,011, titled “Irregular excitation of optical sensors” and filed 2012-05-18, U.S. Pat. 11,988,583, titled “Measurement of a dynamic system” and filed 2019- 08-30, and International Application Pub. 2022 / 265970, titled “Lid with variable interlayer gap” and filed 2022-06-13. These applications are incorporated herein by reference.

[0004] One approach to imaging biological samples is described in US Pat. Pub.2020 / 0116600 in which a colorless and translucent lid covers the wells. Illumination can pass through the lid for imaging the samples in the wells by viewing the samples through a transparent bottom of the multi-well plate. US Pat. Pub. 2023 / 0235266 describes another example of imaging in which illumination is passed through a sample and a transparent window below the samples. Another approach to imaging is described in US Pat. Pub. 2017 / 0261732 in which a light-source unit emits illumination light upward from below a sample and an image-capturing optical system that captures, below the sample, transmitted light, which is the illumination light emitted from the light-source unit that has passed through the sample by being reflected above the sample.

[0005] There is a need for improved imaging of biological samples, for instance, in conjunction with other measurements of such samples.SUMMARY OF THE INVENTION

[0006] In a general aspect, a method for sensing biological samples, for example, in a multi-well plate on an ongoing basis over an extended period of time, provides an imageacquisition function. For instance, such image acquisition may use multiple different illumination conditions for each sample. In some examples, a result of optical scanning is combined with sensing of dissolved gas characteristics (e.g., gradient of oxygen concentration; oxygen consumption, etc.), for example, to normalize the gas characteristics according to the number of cells or volume of the biological sample.

[0007] In some implementations, an apparatus includes components for image acquisition for wells in a multi-well plate. These components include an imaging device, including a fixed or movable imaging sensor and associated optics that in operation are below the plate, a controllable illumination device, and a controller to provide multiple different illumination conditions for image acquisition of each well of the plate.

[0008] In some implementations, the components for image acquisition are combined with components for sensing gas concentration and / or gas consumption (e.g., oxygen concentration gradient in a vertical direction) in the wells. For example, components as described in the incorporated patent publications are combined with the components for image acquisition. Such a combination provides a way to concurrently (or within a short delay) image a biological sample and determine its gas emission or consumption. For example, a computational procedure determines a normalized rate of gas emission or consumption per reference number of cells, a reference volume, or a reference mass.

[0009] In examples in which image acquisition is combined with the gas concentration sensing, for example as described in the prior patent publications referenced above, light emitters may be positioned on the bottom-size of the reusable device that includes the control electronics, and the light may pass through the disposable lid.

[0010] The multiple illumination conditions under which a sample in a particular well are imaged may result from control of a number of individual light emitters (e.g., LEDs). For example, light emitters in a number of different positions relative to a particular well provide oblique illumination of a well, thereby providing image data that may be useful in determining a size of a biological sample notwithstanding the sample being close to transparent. For example, the images from different illumination directions may have different patterns due to differences in the index of refraction of a sample and the solution in which the sample is placed.

[0011] In some examples, the combination of the images with different illumination are combined using a machine-learning technique (e.g., a convolutional neural network) that is trained on acquired images and reference size or mass of the samples (“ground truth”) forexample determined using fluorescent imaging techniques that are not required in normal operation after the machine-learning techniques are trained.

[0012] In some examples, the device is configured to sense wells in a sequential manner, for example, illuminating each well in turn. In this way, there may be less interference between the illumination between wells. In other examples, illumination patterns are selected to illuminate multiple (e.g., adjacent) wells at the same time, and images of multiple wells may be acquired concurrently. In association with sequential imaging of the samples, gas concentration gradients may be measured in a similar sequential manner.

[0013] In an alternative or addition to illumination from above the wells, a light emitter or a light transmission element (e.g., an optical fiber) coupled to a light emitter, may be submerged into the solution of the well, and the multiple illumination conditions for a well may be associated with different positions (depths) of the light emitter. The different positions of the lights from neighboring wells (or inter-well illumination sources) alter the illumination angle of the sample.

[0014] In another aspect, in general, an apparatus measurement of a plurality of biological samples in a multi-well plate has an illumination source with a set of controllable light sources for illuminating wells of the multi-well plate. An imager of the apparatus is used for acquiring images of samples in said plate. A controller is configured to control the light sources to provide multiple illumination patterns for each well, and to cause repeated acquisition using the imager of sets of multiple images in respective illumination patterns.

[0015] A number of optional features are used for measurement of metabolic activity and / or gas concentration measurements, thereby providing the ability to concurrently (or in close time proximity) match sequences of images with metabolic or gas measurements. Such concurrent measurements may be used to normalize the metabolic or gas measurements by the size of the biological samples. Such multiple modes can provide multiple inputs to determine characteristics of the biological samples, for example, to classify the samples according to their overall activity, for example, as a result of different environments in different wells. These features can include one or more of the following.

[0016] The apparatus includes a set of optical measurement devices for measuring fluid properties in respective wells of the multi-well plate. In some examples, these optical measurement element each has an excitation source for exciting a probe in the fluid as well as an optical detector for acquiring an emission from the probe.

[0017] The controller of the apparatus is further configured to cause repeated acquisition of measurements from the wells using the set of optical measurement devices. In some examples, the controller is further configured to vary a depth of probes in the wells with the repeated acquisition of the measurements including acquiring those measurements at differing depths in the wells.

[0018] The controller is configured to interleave over time acquisition of images and measurement of the fluid properties. For instance, the controller is configured to acquire interleaves sequences of images and metabolic / gas measurements.

[0019] A number of optional features relate to the manner in which images of the biological samples are acquired, and in particular, the lighting conditions under which the images are acquired. These features can address the technical difficulties of imaging biological samples immersed or suspended in fluid, where the samples may be almost transparent and invisible in at least some lighting and imaging configurations. These features can include one or more of the following.

[0020] The set of controllable light sources has light sources arranged around each well such that each well may be imaged while illuminated by a multiple different subset of the light sources such that each subset of light sources corresponding to a different light pattern for illuminating the well.

[0021] The subsets of light sources can form a repeated placement pattern such that each well has a substantially same placement pattern of light sources around said well.

[0022] A number of features relate to alternative placements and / or type of the light sources. The choice of placement of the light sources may affect the nature of the illumination, and may affect overall apparatus features such as overall size, ease of operation, and cost of consumable and reusable components. These alternatives can include the following.

[0023] The set of controllable light sources are arranged to provide illuminations from above the wells of the plate.

[0024] The apparatus has a device for repeated use with different multi-well places, and that reusable device houses the set of controllable light sources. Optionally, this device is configured to be positioned above in close proximity to and registered to positions of the wells of the multi- well plate.

[0025] The light sources are Light Emitting Diode (LED) light sources, and are optionally powered via an electrical (or magnetically coupled) power connection to the reusable device.

[0026] The light sources comprise passive optical elements. For instance, each of such passive optical elements include a lens and / or a waveguide, which may be integrated in the reusable device.

[0027] A number of features relate to alternative placement, type or control of imagers.

[0028] The imager is configured to acquire images from below the wells of the plate.

[0029] The apparatus has a base for repeated use with different multi-well places and configured to be positioned below the multi-well plate, and this base houses the imager. This base may also be configured to support the multi- well plate.

[0030] The imager is movable to acquire images of different subsets of wells at different imager positions, for example, being movable to acquire images of different wells at different imager positions. In such an example, the controller may be further configured to move the imager and to control the light sources to acquire images in multiple illumination patterns for each well.

[0031] Other features and advantages of the invention are apparent from the following description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a schematic side view of a sample monitoring system;

[0033] FIG. 2 is a schematic top view of the system;

[0034] FIG. 3 is a bottom view of a lid showing light sources and optoelectronic sensors;

[0035] FIGS. 4-6 are schematic side views of alternative arrangements of the system;

[0036] FIGS. 7-11 are top views of various illumination configurations.DETAILED DESCRIPTION

[0037] Referring to FIG. I, a schematic side view of a sample monitoring system 100 shows a multi-well plate 120 that is monitored by the system. The multi- well plate hasmultiple wells 122 in which biological samples 124 are immersed in solution 126. For the sake of discussion, the monitoring system 100 is considered to be separate from the multiwell plate 120 and its samples and solution that are monitored using the system. A lid 130 is held over the plate 120, providing a substantially sterile cover to the wells. One function of the system is to monitor characteristics of the solution 126 in each well. The set of elongated probes 132 are used to monitor the solution, for example, as described in US Pats. 9,075,011 and 11,988,583, and PCT Pat. Pub. WO 2022 / 265970, which are incorporated herein by reference. In such examples, the probes provide transmission of optical excitation and return sensing optical signals between optoelectronic devices 142 and sensing material at the tips of the probes, which are immersed in the solutions. The devices 142 are housed in a device 140, which sits above the lid 130. The device includes control and communication electronics and mechanical actuation devices as described in the incorporated publications. The system is configured such that the probes repeatedly move up and down under control of the device during the monitoring providing a way to measure vertical gradients of properties of the solution. In a particular use of such monitoring, vertical gradients of oxygen concentration are measured in each well, and these gradients are used to compute oxygen consumption of each of the samples.

[0038] In FIG. I, the device 140 further includes controllable imaging light sources 144, for example light emitting diodes, and the system also includes a base 150 that is used positioned below the multi-well plate 120. Light from the light sources 144 passes through the lid toward the wells and samples. The lid may have optical elements 134 that affect the transmission of the light through the lid. Examples of such elements are lenses, diffusers, collimators, fiber light guides, and the like. In effect, the combination of the light sources (light emitters) and the optional optical elements in the lid provide controllable illumination patterns for the samples in the wells. In some examples not illustrated in FIG. 1, light sources may protrude into the wells or into regions surrounding the wells, and optical elements (e.g., lens, reflectors, opaque regions, fluorescent materials) may be positioned in the regions surrounding the wells, again serving to form the illumination patterns of the samples. The base 150 includes at least one image sensor (which might include one or an array of light sensors) 152, and an associated optional optical element, such as a lens that, can acquire an image of an illuminated sample. In this illustration, the camera and associated optical element are movable to allow imaging of the samples in succession one at a time. In other examples there may be multiple cameras to acquire images of multiple samples at once, or there may be one or more cameras that have a sufficient input field to image multiple samples at once.

[0039] In operation, imaging may be performed in cycles in which each sample is imaged in turn. For each sample in one cycle, multiple images may be acquired using different patterns of illumination. The multiple images are then processed to determine characteristics of the sample, such as the physical size (e.g., volume or cell count) of the sample.

[0040] Referring to FIG. 2, a schematic top view of the system shown in FIG. 1 shows the positions of the wells 124 and of the light sources 144A and aligned optical elements 134. The wells 122 are arranged in a grid pattern, and the light sources 144 are arranged in a grip pattern in the regions between the wells. The optoelectronic sensors 142 are concentric with the wells. The light sources and optoelectronic sensors are illustrated as being round in the schematic, but other cross-sectional shapes may be used in practice, and the schematic figure is intended to illustrate the relative arrangement in the horizontal plane of the elements.

[0041] As introduced above, a particular well sample in a well may be illuminated in a variety of ways. For example, each of the four light sources 144 surrounding a well may be turned on one at a time, and a separate image may be acquired for each lighting condition. Light sources closer to or more distant from the well being imaged may also be used, for example, to provide more diffuse illumination. Light sources may also be included in the device 140. In any case, the use of various illumination patterns for different images of a sample provides more information that may be used to estimate characteristics, such as size of the biological samples. This is particularly the case when the biological sample is largely transparent, and the imaging process effectively captures refraction at the solution / sample boundaries from which the extent of the sample may be inferred, for example, by image processing and / or machine learning.

[0042] Imaging is introduced above in the context of illumination passing from the light sources to the imager through the samples. In some examples, fluorescence imaging may be used in which the light sources are used to excite fluorescent material in the samples, and the imager captures the resultant fluorescent light from the samples.

[0043] FIG. 3 shows a bottom view of a device (drawn to scale) showing an arrangement of the optoelectronic sensors 144 and the light sources 142. The optoelectronic sensors provide the optimal excitation signals that are passed via the probes 132.

[0044] An aspect of a system in which gradient measurements of the solution are acquired along with images is that the device may move up and down to cause movement of the probes 132. However, such movement also moves the light sources 144 up and down. In some examples, this movement is compensated for by using optical elements 134 that acceptvertically-directed light patterns and disperses that light toward the wells, thereby making the illumination patterns largely insensitive to the vertical position of the device 140 relative to the wells. In some examples, different images are acquired at different heights of the device, thereby providing yet more lighting patterns of the samples from which the sample characteristics may be inferred.

[0045] FIG. 4 shows a side view in which a lid 130 has a dispersive lens 134 for altering the path of illumination light. FIG. 5 shows a side view of an alternative lid in which the lid has opaque regions so that most of the light that illuminates the well bottom arrives via the fixed aperture in the base of the lid rather than the moving illumination LED. Without the arrangement of FIG. 5, the illumination path may vary as the device moves up and down. FIG. 5 shows a side view of another alternative arrangement for “side-fluorescence excitation”. Here the excitation light path is split out by a waveguide to the neighboring wells. The light path is shallow relative to the imager objective so that while it is able to fluoresce material in the well (e.g. indicator dyes), it is not in the acceptance angle of the imager objective. A benefit here is exciting measuring fluorescence in the basal media (e.g. for analyte concentration) while measuring OCR gradients in the typical well media. Imaging can also be performed to analyze the concentration of a constituent of the overall media in combination with measurements of the gradient.

[0046] Sample size, for instance cell count, may be inferred in various ways. One way is to use image processing and / or machine learning techniques that infer the boundary locations (e.g., in three dimensions) of the sample in each well based on projection techniques by using relationships between boundary locations found in the images with illumination from different directions. Another way to do this is using a machine-learning technique in which “training” image sets of samples of various sizes are used to learn (i.e., estimate settable numerical parameters of) a transformation of such image sets directly into an estimate of the sample size. For instance, a multiple layer convolutional neural network that accepts a “stack” of the multiple images (e.g., in multiple different illumination conditions and / or optimal sensing configuration, such as aperture, focal length, etc.) for a sample may be trained to provide an estimate of the sample size, or to provide an estimate of sample occupancy over an arrangement of spatial voxels. In some examples, sample size for training purposes may be determined using fluorescence imaging techniques to provide a “gold standard” for size. In runtime use, such fluorescence techniques are not required, and the sample size may be obtained by imaging without fluorescence (e.g., transmitted light in multiple lighting patterns).

[0047] Referring to FIGS. 7-11, a number of alternative arrangements of illumination elements may be used. For example, the LEDs may be arranged in concentric rings. The LEDs illumination pattern can be selectively configured for brightfield, darkfield, phase contrast, and differential phase contrast based on the distance from the sample and numerical aperture of the objective. For brightfield illumination, the LEDs positioned closest to the center of the objective are enabled. For darkfield and phase contrast, only LEDs beyond a certain radius from the center of the objective are enabled.

[0048] The LED array can be incorporated into the bottom of the device 140. It can alternatively be incorporated into a moving layer of the lid 130. There can also be a “trilayer” lid configuration consisting of an additional illumination layer, separate from the moving layer 130 controlling the probe positions.

[0049] Depending on the field of view of the objective, multiple LED rings (or sub arrays) are used per- well as shown in FIG. 11

[0050] A number of features may be incorporated into embodiments described above. For instance, the lid may have a top and a bottom layer forming a bilayer lid, for example, as described in International Application Pub. 2022 / 265970, titled “Lid with variable interlayer gap”. The optical elements 134 may be in the fixed / lower piece of the bilayer lid design that alter the light path (e.g. diverging lens) such that the illumination angle is fixed despite the illumination source moving as needed for gradient measurement. In another alternative in which there is a bilayer lid, the optical elements are the moving / upper piece of the bilayer lid design that enable / enhance the effect of the moving light source (e.g. diverging lens or potentially a diffusive element) of the optical path such that the illumination angle is controllable providing a greater variety of lighting conditions under which the samples are imaged. In some examples, optical elements are in the fixed / lower piece of the bilayer lid design that enable / enhance the effect of the moving light source (e.g. apertures in the fixed base) the optical path such that the illumination angle is controllable. In yet other examples, some or all of the illumination may be provided from below the wells, for example, with LEDs being positioned below the inter-well spaces. The plate 120 may incorporate optical elements, for example, diffusion or reflecting elements in the inter-well spaces to direct light toward the wells.

[0051] As introduced above, in a number of embodiments, the imaging is performed concurrently or in short time separation from the monitoring of oxygen consumption. The images are used to infer the sample sizes, and then the oxygen consumption and the samplesizes are combined to determine oxygen consumption per unit size (e.g., per a standard number of cells, a standard volume, etc.).

[0052] A number of optional and / or alternative features of the system, some of which are introduced above, are as follows.

[0053] The light sources for imaging the biological samples may be located in different parts of the overall apparatus, for example, in different “layers” of the stack of elements including one or more of a base, the well plates, a reusable lid dedicated to illumination, in a reusable lid that includes sensor probes (e.g., elongated probes that extend into the sample fluid), or in the device housing control and sensing electronics, potentially with lens, waveguides, or diffusers and the like in other of the layers listed above to guide illumination toward the sample. A number of embodiments are described with illumination with an LED or other light source. It should be recognized that an “LED” may be formed of a diode that emits at a first frequence or frequency range (e.g., in an infra-red range) and a fluorescent layer that is excited by that first frequency and emits light at a second frequency (e.g., visible light). In some optional or alternative embodiments, an emitter at such a first frequency may be is one layer (e.g., in the device that houses the control and sensing electronics) and fluorescent material may be embedding is another layer (e.g., in the lid), optionally optically coupled to the emitter with a waveguide. While LEDs may be controllable electrically by gating electric current through them, embodiments may make use of controlled optical “shutters” (e.g., liquid crystal devices, LCDs, or potentially mechanical devices such as piezo-electric controlled shutters or mirrors) that cause controlled changes in the illumination pattern. In such embodiments, the light source (e.g., LED) may optionally be located in a different layer than the shutter. The light sources may also be located in the base below the well plates. For example, light directed upward may be reflected or diffused back down through the sample from above the plate, for instance with a passive optical element or fluorescent material in in a layer above the plate. While embodiments are described with active light sources (e.g., LEDs) being in a layer above the well plate, some embodiments may have the sources remote from these elements with the source(s) being coupled to the device via an optical guide, such as a flexible optical fiber. The position and orientation of the light sources can be configured to provide for different illumination modes to support brightfield, darkfield, differential phase contrast, brightfield, and fourier ptycography. The height of the light sources may be dynamically adjusted to allow for a range of illumination angles and Fourier domain coverage to improve computational imaging performance (e.g. for phase retrieval). There are a variety of optional or alternative ways that light passes from a light source to the biological samples in the well plate. For example, the light passes throughlenses, diffusers, waveguides, reflectors, openings, or controlled shutters in the layers above the well plate or potentially integrated in the well plate itself. The light may be emitted from above the well plate and imaged below the plate or may pass downward through the sample and imaged from above the sample, for example, reflecting off a surface below the well plate on a base on which the well plate rests. Another type of element through which the light may pass is through a (e.g., rod-shaped) waveguide immersed in the fluid. In some such approaches, a fluorescent material may be incorporated into the waveguide (e.g., to convert IR to visible light). In some examples, such a waveguide may be combined with a sensor probe for sensing gas properties in the solution in the wells.

[0054] Reference is made to control and sensing electronics in the device that is above the well plate. In alternative embodiments, such electronics may also be in a base “layer” that is below the well plate in operation, and may be distributed among multiple layers. The device and the base may be coupled electrically, or each may be powered separately (e.g., by an electrical or magnetic connection, or with batteries) and control communication passes between them and / or a remote control device (e.g., a “hub” that resides outside of the environment where the biological samples are maintained) using a wired (e.g., USB) or wireless (e.g., Bluetooth) connections.

[0055] In embodiments in which the imaging device(s) are in a base below the well plate, the base may be coupled and / or registered to the well plate so that images can be accurately positioned below the samples in the wells. In some embodiments, the base does not require such registration or accurate coupling. Rather, a pattern of calibration illumination patterns are emitted and acquired, and using those calibration patterns, the spatial relationship between the base and the well plate is determined and used during acquisition of the images of the samples, for example, by controlling positioning of the imaging devices and / or selecting regions of acquired images.

[0056] By including a controller to control the timing of illumination for imaging relative to the timing of other sensing mechanisms (that use light) the two can optionally be interleaved. Having a system where a base plate includes an image capture element (i.e., camera, image sensor) that can move around relative to a plate, in combination with an illumination plate that moves with the well plate is advantageous because the well plate can be positioned anywhere on a platform where the camera can move around. Having the light source sit directly on top of the plate (rather than also moving around freely) has the advantage that various alternative configurations may be used, for example, by incorporating additional sensors or using waveguides. Furthermore, the apparatus has practical advantages,such as saving power by not needing to physically move an array of optics (e.g., only the camera may need to be moved). Also, the light sources (e.g., LED illuminators) may be significantly less expensive than complex arrays of lenses and image sensors.

[0057] In some examples, the imaging device includes a dynamically controllable turret of multiple objectives with different numerical apertures / magnification levels. The system can then switch objectives without changing the illumination pattern, for example, to alter the image from brightfield to darkfield. It could also be useful to dynamically alter the illumination pattern when switching objectives.

[0058] In some examples, computational imaging methods are applied to multiple acquired images of the sample. These computational imaging methods can be used to calculate quantitative phase information which in turn can be used to infer biologically relevant parameters (e.g. cell size, dry mass, etc.). Deep learning algorithms can be trained on images acquired under various illumination modes including epi-fluorescent stained images (e.g. nuclear stains, actin stains, mitochondrial stains, etc) so that images acquired in one or more other illumination modes (e.g. brightfield, darkfield, annular, etc.) can be used for label-free prediction of stained images.

[0059] A number of embodiments for sensing gas concentration gradients as a means of inferring metabolic activity in biological samples, for example, in multi-well plates, are described in U.S. Pat. 9,075,011, titled “Irregular excitation of optical sensors” and filed 2012-05-18, U.S. Pat. 11,988,583, titled “Measurement of a dynamic system” and filed 2019- 08-30, and International Application Pub. 2022 / 265970, titled “Lid with variable interlayer gap” and filed 2022-06-13. The approaches to imaging the biological samples described above can be combined with features of the means for inferring metabolic activity that are described in these referenced publications.

[0060] A number of embodiments of the invention have been described. Nevertheless, it is to be understood that the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the following claims. Accordingly, other embodiments are also within the scope of the following claims. For example, various modifications may be made without departing from the scope of the invention. Additionally, some of the steps described above may be order independent, and thus can be performed in an order different from that described.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for measurement of a plurality of biological samples in a multi- well plate, comprising: an illumination source with a plurality of controllable light sources for illuminating wells of the multi- well plate; an imager for acquiring images of samples in said plate; and a controller configured to control the light sources to provide multiple illumination patterns for each well, and to cause repeated acquisition using the imager of sets of multiple images in respective illumination patterns.

2. The apparatus of claim 1, further comprising a plurality of optical measurement devices for measuring fluid properties in respective wells of the multi- well plate, optical measurement element having an excitation source for exciting a probe in the fluid and an optical detector for acquiring an emission from the probe.

3. The apparatus of claim 2, wherein the controller is further configured to cause repeated acquisition of measurements from the wells using the plurality of optical measurement devices.

4. The apparatus of claim 3, wherein the controller is further configured to vary a depth of probes in the wells, and wherein the repeated acquisition of the measurements includes acquiring said measurements at differing depths in the wells.

5. The apparatus of any one of claims 2 through 4, wherein the controller is configured to interleave over time acquisition of images and measurement of the fluid properties.

6. The apparatus of any one of the preceding claims, wherein the controller is configured to image wells in a sequence.

7. The apparatus of any one of the preceding claims, wherein the plurality of controllable light sources comprises light sources arranged around each well such that each well may be imaged while illuminated by a plurality of sets of light sources, each set of light sources corresponding to a light pattern for said well.

8. The apparatus of claim 7, wherein the light sources form a repeated placement pattern such that each well has a substantially same placement pattern of light sources around said well.

9. The apparatus of any one of the preceding claims, wherein the plurality of controllable light sources are arranged to provide illuminations from above the wells of the plate.

10. The apparatus of any one of the preceding claims, where the imager is configured to acquire images from below the wells of the plate.

11. The apparatus of any one of the preceding claims, where the imager is movable to acquire images of different subsets of wells at different imager positions.

12. The apparatus of claim 11, wherein the imager is movable to acquire images of different wells at different imager positions.

13. The apparatus of any one of claims 11 through 12, wherein the controller is further configured to move the imager and to control the light sources to acquire images in multiple illumination patterns for each well.

14. The apparatus of any one of the preceding claims, wherein the apparatus comprises a device for repeated use with different multi- well places, said device housing the plurality of controllable light sources.

15. The apparatus of claim 14, wherein the light sources comprise Light Emitting Diode light sources.

16. The apparatus of claim 14 or claim 15, wherein the light sources comprise passive optical elements.

17. The apparatus of claim 16, wherein the passive optical elements include at least one of a lens and a waveguide.

18. The apparatus of any one of claims 14 through 17, wherein the device is configured to be positioned above in close proximity to and registered to positions of the wells of the multi- well plate.

19. The apparatus of any one of the preceding claims, wherein the apparatus comprises a base for repeated use with different multi-well places and configured to be positioned below the multi-well plate, said based housing the imager.

20. The apparatus of claim 18, wherein the base is configured to support the multi- well plate.

21. A method for measuring biological samples in a multi- well place, the method comprising imaging said samples, the imaging including repeating multiple times for the biological samples in the multi- well plate: using an illumination source coupled to the multi-well plate to provide multiple illumination patterns for each well of the multi- well plate, acquiring using an imager multiple images for each of the wells in respective illumination patterns, processing the multiple images to determine at least one physical property of the sample in each of the wells.

22. The method of claim 21, wherein the physical property of the sample in each well comprises a characterization of a size of said sample.

23. The method of claim 22, wherein the size of the sample includes at least one of a spatial extent of the sample and a volume of the sample.

24. The method of one of claims 21 through 23, wherein using the illumination source comprises illuminating the samples from above the wells, and where acquiring the images comprises capturing images from below the wells using the imager.

25. The method of any one of claims 21 through 24, wherein the method further includes measuring at least one gas property of fluid in the wells.

26. The method of claim 25, wherein measurements of the gas property and acquiring images of each well are interleaved during measuring of the biological sample.

27. The method of claim 25, wherein the gas property comprises a vertical gradient of a gas concentration in the fluid,28. The method of claim 27, wherein the method further comprises computing a relationship between the physical property of the sample in each well and the gas property in the well.

29. The method of claim 28, wherein computing the relationship comprises computing a rate of gas emission or consumption normalized by a size of the sample in each well.

30. The method of claim 29, wherein computing the rate of gas emission or consumption normalized by size comprises computing a metabolic rate of the biological sample in each well.

31. The method of any one of claims 21 through 30 using the apparatus of any one of claims 1 through 20.

32. An apparatus comprising a controller configured to cause the apparatus to perform all the steps of any one of claims 21 through 30.

3. A non-transitory machine-readable medium comprising instructions stored thereon, execution of said instructions by a controller causing said controller to cause performing of all the steps of any one of claims 21 through 30.

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