WO2021068060A1 - Système d'imagerie d'incubateur - Google Patents

Système d'imagerie d'incubateur Download PDF

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Publication number
WO2021068060A1
WO2021068060A1 PCT/CA2020/051336 CA2020051336W WO2021068060A1 WO 2021068060 A1 WO2021068060 A1 WO 2021068060A1 CA 2020051336 W CA2020051336 W CA 2020051336W WO 2021068060 A1 WO2021068060 A1 WO 2021068060A1
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WO
WIPO (PCT)
Prior art keywords
illumination
incubator
image
images
matrix
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PCT/CA2020/051336
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English (en)
Inventor
Sebastian HADJIANTONIOU
David SEAN
Original Assignee
Incuvers Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Incuvers Inc. filed Critical Incuvers Inc.
Publication of WO2021068060A1 publication Critical patent/WO2021068060A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/14—Incubators; Climatic chambers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/30—Base structure with heating device
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/362—Mechanical details, e.g. mountings for the camera or image sensor, housings

Definitions

  • the present invention pertains to an incubator imaging system for imaging live cells.
  • the present invention also pertains to a microscopy system for real-time live cell imaging under variable environmental conditions.
  • Laboratory cell incubators are used to grow and maintain cell cultures and are available with a variety of features and in a variety of sizes and types.
  • Various incubators are available which can vary the internal environmental conditions such as gas composition, temperature, and humidity, all of which affect cell growth.
  • the incubator market is divided into two main categories: gassed incubators also referred to as carbon dioxide (CO2) incubators, and non-gassed or microbiological incubators.
  • CO2 incubators are typically heated to 37°C and maintain 95% relative humidity and a CO2 level of 5 percent.
  • microbiological incubators are essentially temperature-controlled ovens that work within the biological range of 5°C to 70°C, and are often used for growing and storing bacterial cultures.
  • Most incubator units are water-jacketed, air-jacketed or use direct heat to maintain the temperature around the culture chamber.
  • Incubators can be used in a wide variety of applications including cell culture, biochemical studies, hematological studies, and pharmaceutical and food processing, and can range in size from about 1 cubic foot (table-top size) to 40 cubic feet (freezer size) or larger.
  • Various features can be found in incubators, for example shaking incubators are often used for cell aeration and solubility studies, and refrigerated biochemical oxygen demand incubators with a temperature variability range of, for example, 20°C degrees to 45°C are commonly used for applications such as insect and plant studies, fermentation studies, and bacterial culturing.
  • Various devices are available for imaging live cells during incubation.
  • United States patent US8,859,263 to Greenberger et al. describes an apparatus for incubating cells in a dynamically controlled environment in which cells can be examined with a camera while the environment is dynamically controlled and maintained in the desired condition.
  • An object of the present invention is to provide an incubator imaging system for imaging live cells under environmental control conditions.
  • an incubator imaging system comprising: an incubation chamber for maintaining an internal environment; an environmental controller for controlling the internal environment in the incubation chamber; an environmental sensor for detecting an environmental condition in the incubation chamber; and a microscopy device comprising: a stage for supporting a sample; an illumination matrix comprising a plurality of light sources; a lens for focusing light; and an optical sensor for receiving light image data.
  • the system further comprises a data storage device for storing light image data obtained by the optical sensor.
  • the illumination matrix is above the stage and the optical sensor is below the stage.
  • system further comprises an optical filter between the stage and the optical sensor.
  • the optical sensor is a camera.
  • the illumination matrix is an LED matrix.
  • the plurality of light sources in the illumination matrix are arrayed in a square or circle.
  • the environmental controller controls one or more of temperature, humidity, pH, nitrogen concentration, oxygen concentration, and carbon dioxide concentration.
  • the illumination matrix is capable of creating brightfield, darkfield, and asymmetric light patterns.
  • system further comprises a secondary illumination device.
  • a method of live cell imaging comprising: incubating a cell culture under environmental control conditions; during incubation, obtaining a first image of the cell culture under a first illumination condition; during incubation, obtaining a second image of the cell culture under a second illumination condition different than the first illumination condition; and combining the first image and the second image to generate a composite live cell image.
  • the first illumination condition is an asymmetric illumination pattern.
  • the second illumination condition is a fluorescence illumination.
  • the method further comprises: obtaining more than two images, each of the more than two images taken under different illumination conditions; and combining the more than two images.
  • the method further comprises changing the environmental control conditions based on the live cell image.
  • the method further comprises obtaining a plurality of sequential composite live cell images to provide a time lapse of the cell culture.
  • a method of live cell imaging comprising: incubating a cell culture under environmental control conditions; obtaining a plurality of images of the cell culture, each of the plurality of images obtained under a different illumination condition; and combining the plurality of images to generate a composite live cell image.
  • At least one plurality of images is obtained in an asymmetric illumination condition.
  • combining the plurality of images comprises averaging the plurality of images.
  • the illumination condition of at least one of the plurality of images is non-illuminated.
  • Figure 1 is a front perspective view of an incubator imaging system shown with the incubator door open;
  • FIG. 2 is a front view of an incubator imaging system shown with the incubator door closed;
  • FIG. 3 is a front view of an incubator imaging system shown with the incubator door open;
  • Figure 4 is a front view of an imaging system for the incubator imaging system
  • Figure 5 is a top perspective view of an imaging system for the incubator imaging system
  • Figure 6 is a cross-sectional view of an imaging system for the incubator imaging system
  • Figure 7 is a graph of an example protocol that can be used with the incubator imaging system
  • Figure 8 illustrates a set of screen shots from data obtained by the incubator imaging system for display on a smartphone
  • Figure 9a is an image taken of a sample under brightfield illumination
  • Figure 9b is an image taken of a sample under darkfield illumination
  • Figure 9c is an image taken of a sample under fluorescence illumination
  • Figure 10 is a composite image of the single images shown in Figures 9a-9c;
  • Figure 11 a is an example darkfield (left) and brightfield (right) illumination pattern
  • Figure 11 b is an example set of four asymmetric illumination patterns.
  • Figure 12 is an example method of use of the incubator imaging system.
  • cell refers to the subject of study of the present incubator imaging system, and is understood to mean all cells including those from unicellular and multicellular organisms. Examples of cells which can be observed with the present incubator imaging system include but are not limited to prokaryotic cells such as bacteria and archaea, and eukaryotic cells such as those derived from plants, animals, fungi, slime moulds, protozoa, and algae.
  • prokaryotic cells such as bacteria and archaea
  • eukaryotic cells such as those derived from plants, animals, fungi, slime moulds, protozoa, and algae.
  • the term “cell” also encompasses multi-cell structures, such as, for example, tissue samples, microorganisms, and macrostructures formed from a plurality of cells.
  • an incubator imaging system having a microscopy apparatus inside an incubator.
  • the incubator imaging system can capture images of live cells in growth conditions while having the capability of controlling, monitoring, and varying the environmental or growth conditions inside the incubator.
  • the real-time imaging capability provided by the present incubator imaging system provides researchers with an accurate picture of cell growth and behaviour under variable growth conditions, as well as high quality still and time-lapse imaging with minimal disturbance of the growing cells.
  • the system can also enable analytics of cell growth, as well as control and monitoring data of the incubator interior environment for correlative analytics to the obtained cell growth images.
  • the combination of high quality image capture and control and monitoring of incubator cell conditions provides an improved overall understanding of cell growth which is beneficial to researchers.
  • the present incubator imaging system is also capable of non-invasively quantifying and visualizing biological cells and/or tissues during cell culture under controlled conditions.
  • the environment inside the incubator can be controlled without disturbing the cells, providing aseptic and/or sterile conditions that permit monitoring and analysis of cells in a sterile environment by microscopy. Live monitoring, time lapse imaging, and real time incubator tracking is thereby enabled by a connected and controllable incubator imaging system.
  • Live cell imaging including fluorescence illumination can also be captured using real-time, still, and time lapse videos of experiments without removing cells from the incubator, enhancing the images obtained.
  • an illumination matrix to illuminate the cells under study, the imaging system can be made small enough to fit inside even small benchtop sized incubators and eliminates expensive components.
  • the illumination matrix also enables finer illumination control for illumination of the sample during microscopy, including variable brightness and illumination direction during imaging. Collection of multiple images of a cell culture under different illumination conditions in a brief period of time and combining the images provides a single high quality composite image of the live cell culture.
  • the present incubator imaging system provides an affordable combination biological incubator and imaging system that can be used effectively for cell culture research, providing an accessible tool to researchers with limited funding.
  • Small to mid-sized cellular research labs and small undergraduate university and college cell culture teaching labs can now have access to an accurately controlled and monitored as well as robust incubator imaging system for wider access to cell biology research.
  • FIG 1 is a front perspective view of an incubator imaging system 2 shown with the incubator door 6 open.
  • Incubator imaging system 2 comprises an incubator 4 having an incubator door 6 which when closed creates an incubation chamber for sustaining an interior environment in the interior chamber of the incubator 4 sufficient to support the growth of cells.
  • An imaging system is disposed inside the incubator 4 optionally in a microscope housing 10, and together the imaging system and incubator 4 make up incubator imaging system 2.
  • the imaging system is preferably housed inside microscope housing 10, though a variety of housings and non-housing type support systems can support the imaging system inside the incubator 4.
  • the imaging system has a stage 20 for supporting a cell culture sample to be imaged inside the incubator 4.
  • An optional display screen 8 provides data on the interior conditions of the incubator.
  • the display screen 8 can also be used for real time focusing for clear image acquisition.
  • the collected image from the optical sensor can be displayed on the display screen 8 and the z-adjustment device for the microscope stage 20 can be adjusted to improve focus.
  • the conditions inside the incubator 4 can also be displayed on the display screen 8, as well as, optionally, one or more incubator identifier, experiment details, alarms, and other information. The same or different information can also be provided to a remote computing device for remote observation, monitoring or control of the incubator imaging system.
  • Incubator 4 can also be provided with one or more shelf 12 to incubate cells at controlled and monitored incubator conditions.
  • Optional input controller 14 can provide an interface for a user to manually change the view on display screen 8, or to control the conditions inside the incubator or the microscope imaging system.
  • Input controller 14 can be any manual control device known, including but not limited to one or more buttons, switches, dials, or tactile or digital or haptic input devices such as a touch-sensitive device or touchscreen.
  • the display screen 8 can also be a touchscreen to provide input control.
  • An illumination matrix 18 positioned above the microscope stage 20 is a matrix or array of individual light sources.
  • the imaging system uses the illumination matrix 18 to provide illumination to the cell sample on the stage 20 inside the incubator.
  • the individual light sources in the illumination matrix 18 can provide a variety of illumination patterns in different configurations, luminosities, wavelengths, etc.
  • the illumination matrix 18 can also be capable of providing illumination to the sample in an asymmetric pattern to enable imaging at a variety of angles. Different illumination patterns can be created by illuminating different parts or areas of the illumination matrix, and combining two or more images with the illumination matrix illuminated with two or more patterns can provide a single composite image that is of higher quality than a single image taken with a single illumination.
  • One example of two different illuminations is a first illumination and image capture with the centre of the illumination matrix lit and the outside of the illumination matrix dark, and a second illumination and image capture with the centre of the illumination matrix dark and the outside of the illumination matrix lit.
  • Other examples include varying the angle of illumination to the cell sample by illuminating one half of the illumination matrix and capturing a first image, then illuminating the other half of the illumination matrix and capturing a second image.
  • four images can be taken with each image illuminated by one of the quadrants of the illumination matrix separately. It is evident that with illumination matrix control of the plurality of light sources a very wide variety of illumination patterns can be created to vary the illumination of the cell sample during imaging.
  • Each single point light source in the illumination matrix 18 is preferably a programmable light emitting diode (LED) comprising a plurality of single LEDs which can be selectively illuminated, allowing flexible pattern control and a variety of illumination angles without requiring any moving parts in the imaging system.
  • the multiplexed illumination in which patterns of LEDs in the illumination matrix are used enable illumination of live cells at a variety of illumination conditions wherein multiple images can be captured in rapid succession, each under different illumination. This provides a set of images that can be computationally combined to create a single high quality image.
  • the illumination matrix 18 can have a variety of configurations to be able to produce a wide variety of symmetric and asymmetric patterns.
  • variability can include the number of LEDs, spacing between LEDs in the array, color of LEDs, size of the matrix, shape of the matrix (e.g. square, circular, polygonal, etc), and lattice arrangement of LEDs (e.g. square, hexagonal, radial lattice, etc).
  • the matrix design can also be variable in, for example, LED type with the same or different LEDs in the matrix, LED voltage, LED color or colors, additional filters, and variable chromaticity such as monochromatic or polarized.
  • the illumination matrix can also be variable in its three- dimensional configuration, and can be flat or alternatively have overall curvature toward or away from the objective.
  • the illumination matrix can also be configured to emit light outside of the visible spectrum, and paired with an appropriately sensitive sensor to detect the light interacting with the live cells.
  • the term "light” as used herein refers to any electromagnetic radiation that can be reasonably used to image the live cell culture and includes visible light as well as light outside the visible spectrum.
  • the illumination matrix can also have an optional optical filter placed between the light source and the sample.
  • the filter can be, for example: a polarization filter; a wavelength filter such as a band-pass, a band-stop, high-pass, or low-pass filter; a diffusion filter; a filter that masks parts of the illumination; or a combination thereof.
  • the distance of the illumination matrix 18 to the objective lens should be such that part of the illumination matrix 18 can fall outside the range allowed by the numerical aperture of the objective lens.
  • the numerical aperture (NA) of an optical system is a number that characterizes the range of angles over which the objective lens can accept light.
  • Light outside of the numerical aperture which is emitted from the outer edges of the illumination matrix will only enter the objective lens by scattering on the sample. This is also referred to as dark field illumination.
  • light emitted from the central area of the illumination matrix will be within the numerical aperture of the objective, also referred to as brightfield illumination.
  • separate brightfield and darkfield condensers are used to illuminate the sample.
  • by changing the illumination pattern in the illumination matrix brightfield illumination and darkfield illumination images can be captured and processed computationally without additional hardware.
  • Conditions inside the incubator can be controlled with a variety of environmental regulators, such as gas supplies and associated supply line controls, temperature regulators such as heaters and coolers, and other environmental controllers.
  • a temperature control range of room temperature (ambient) or about 22 °C to 55°C is desirable, in particular for mammalian cells.
  • the incubator can be configured to provide a broader range of temperature control for different growth conditions. Temperature can be controlled by, for example, one or more incubator jacket heaters, positive temperature coefficient (PTC) heater, resistance heater, peltier heater, or other type of controllable heater.
  • PTC positive temperature coefficient
  • Humidity control can be provided in the incubator up to and greater than 90% using a variety of known humidity control devices including but not limited to an attached passive water tank, water vapor supply line, water bath, or mister. Humidity can be monitored with a humidity sensor to maintain the desired humidity inside the incubator.
  • Various gas supplies can be attached to the incubator to provide inflow of gas or other fluid medium to the interior chamber and to control the environmental conditions in the chamber to maintain the gaseous environmental conditions at a desired state.
  • gas or other fluid medium can be attached to the incubator to provide inflow of gas or other fluid medium to the interior chamber and to control the environmental conditions in the chamber to maintain the gaseous environmental conditions at a desired state.
  • CO2 or nitrogen (N2) gas can be injected into the incubator to expel oxygen gas (O2) and provide hypoxic growth conditions for live cells in the incubator.
  • the percentage of O2 in the incubator can be adjusted by controllably combining CO2 or N2 or a combination thereof with ambient air or compressed air.
  • CO2 can be adjusted in the range of, for example, 0.04% to 21 %
  • O2 can be provided in a range of 0.2% (hypoxic conditions) to 21 % (ambient conditions).
  • the gas supply is preferably of medical grade purity or of sufficient purity to support cell culture.
  • the gas composition inside the incubator can also be adjusted over time using a pre-set protocol or manually, and gas sensors can be further used detect the proportion of the gas of interest in the air inside the incubator.
  • a CO2 tank can be used to boost up CO2 levels up to about 20- 21 %, and ambient air or ambient air supplied by an air gas tank can provide down to 0.04% CO2.
  • Other environmental conditions can also be tracked by appropriate sensors and sensor output monitored and recorded by the controller and associated data storage, and the environmental conditions regulated by controller signals to environmental control devices such as thermal regulators and gas and humidity supply lines and valves. Regular sensor readings inside the incubator can provide an accurate tracking of cellular response to environmental conditions when used in correlation with imaging obtained by the imaging system.
  • a controller is a hardware device combined with software that manages or directs the flow of data in the incubator imaging system.
  • the controller can be positioned inside or outside the incubator 4 cabinet and is connected to one or more environmental control systems that control one or more environmental conditions inside the incubator.
  • the controller can also be connected to one or more sensors that detect the internal environment inside the incubator 4 and optionally one or more sensors outside of the incubator to provide a feedback and/or confirmation of environmental control inside the incubator.
  • the controller can control the environmental controls inside the incubator, including but not limited to gas flow, temperature, humidity, and other internal environmental conditions.
  • the controller is also connected to at least one data storage device for storing data collected by the controller and/or the sensor.
  • the controller can also be provided with one or more general purpose input/output connectors so that the user can connect a peripheral device to the controller and operate the peripheral device with the controller.
  • peripheral devices which could be connected to the controller are one or more shakers, fans, additional light sources, or robotic devices.
  • the data storage device has at least one computer readable medium in the form of volatile memory, such as random access memory (RAM), and/or non-volatile memory, such as read only memory (ROM), and may also include other removable/non-removable, volatile/non volatile data storage media.
  • volatile memory such as random access memory (RAM)
  • ROM read only memory
  • the data storage device is accessible to and by the controller and is also optionally connected to a transceiver for receiving and transmitting data.
  • the system memory can also contain data such as data and/or program modules such as operating system and application software that are accessible to and/or are operated on by the processing unit for example computer code, computer readable instructions, data structures, program modules, and other data that can be accessed by the controller.
  • the data storage device can be a hard disk, a removable magnetic disk, a removable optical disk, magnetic cassette or other magnetic storage devices, flash memory cards, CD-ROM, digital versatile disks (DVD) or other optical storage, random access memories (RAM), read only memories (ROM), electrically erasable programmable read-only memory (EEPROM), and the like.
  • Any number of program modules can be stored on the data storage device, including by way of example, an operating system and application software. Each of the operating system and application software (or combination thereof) may include elements of the programming and the application software.
  • Data can also be stored on the data storage device. Data can also be stored in any of one or more databases known in the art. The same or different data storage device can also be used to collect image data collected by the optical sensor.
  • the controller, the one or more data storage device, or both are also optionally connected with a wired or wireless connection to an external data transmission source such as an internet connection.
  • the controller may also produce one or more control outputs to the environmental controls in response to input from the display screen or controller knob, or from a wired or wireless control signal, or from a control protocol in memory, to change the internal environment inside the incubator.
  • Feedback obtained by the controller from one or more environmental sensor which senses the internal conditions inside the incubator can also be used to adjust the environmental controls such as a gas supply line, heater/cooler, humidity or ventilation, or other supply system or environmental control system in the incubator to maintain a desired set point for any particular environmental control.
  • the controller can also be connected to the internet and be an Internet of Things (loT) device.
  • LoT Internet of Things
  • FIG. 2 is a front view of an incubator imaging system 2 shown with the incubator door 6 closed.
  • the incubator shown can be of variable size.
  • a version sized for small footprint benchtop applications can have external dimensions (width x depth x height) of 15"x 12"x 19" (38cm x 30.5cm x 28.25cm), internal chamber dimensions of 8"x12"x16" and an incubator chamber volume of 25L.
  • incubator imaging systems can be fit on a standard laboratory benchtop and may also be stacked to optimize space.
  • the incubator imaging system can be much larger and sit on a laboratory floor, optionally housing more than one imaging system inside.
  • Incubator door 6 can be opaque or optionally have one or more transparent feature or window to enable viewing from the outside of the incubator.
  • Optional display screen 8 can be any display screen and/or optionally a touchscreen and can report interior conditions, protocol in process, and can also provide options to change the incubator interior conditions or protocol via one or more optional controller devices 14.
  • FIG 3 is a front view of an incubator imaging system 2 shown with the incubator door 6 open.
  • Incubator imaging system 2 shown has an interior microscope housing 10 for housing and protecting the optical system components.
  • Incubator door 6 seals against the body of the incubator to provide a controllable compartment for establishing non-ambient environmental conditions.
  • Display screen 8 displays information on the status of the incubator imaging system.
  • Shelves 12 and 12b can provide additional storage capacity for incubating cell cultures.
  • Interior shelving inside the incubator provides additional space away from the microscope but still subject to incubator conditions to grow cells and move them to microscope when visualization is desired. The environmental conditions for cell growth prior to visualization can thereby also be tracked.
  • the incubator can have no shelves or one or more shelves.
  • External access port 28 provides a locus for access to the incubator from the outside.
  • the external access port 28 can be used, for example, for cables, devices for growth media manipulation, tubing, and any other devices useful for incubator access.
  • Figure 4 is a front view of an imaging system for the incubator imaging system.
  • Stage 20 supports the placement of a cell culture container for observation by the imaging system.
  • Common cell culture containers include cell culture plates, slides, and dishes.
  • One or more hold assist devices can protect the cell culture from movement relative to the stage 20.
  • one or more stage clips 22 can be used to secure a cell culture dish.
  • Translational x/y-adjustment devices 26a, 26b can be provided to adjust the x and y location of the cell culture container relative to the optical components of the system, and specifically the distance from the cells to be imaged to the optical window. Adjustment in the up/down direction can be accomplished using, for example, z-adjustment wheel 42, or any other similar mechanism capable of moving the stage up and down relative to the objective lens. Alignment in the z direction changes the focus as it changes the distance between the sample and the objective.
  • the x/y/z adjustment devices enable adjustment of the stage or sample resting on the stage relative to the cover glass, illumination matrix, and optical path of the light entering the microscope.
  • optical tube 32 houses and sets the distance between the optical components of the optical system.
  • An optical sensor positioned at the end of the optical tube 32, which can be a camera assembly, receives and records light images leaving the tube lens.
  • a secondary illumination system 46 for example a fluorescence illumination system, in the light path, can provide secondary illumination to the cell culture sample from below.
  • Lower flange mounting 48 provides additional support to the optical system.
  • FIG. 5 is a top perspective view of an imaging system for the incubator imaging system.
  • Stage 20 is shown with two stage clips 22a, 22b and a hole in the centre of the stage 20.
  • the illumination matrix positioned above the stage illuminates the cell culture which sits on the stage, and light then travels through the hole or optical window 38 in the centre of the stage to the optical system.
  • a cover glass covering the optical window 38 in the stage 20 can be used to protect the optical system below the stage x/y-adjustment devices 26a, 26b are used to position the cell culture sample relative to the light path.
  • Optional z-adjustment device 42 shown here as a wheel, adjusts the position of the cell culture sample up and down.
  • Optical tube 32 receives light from the illuminated cell culture through the objective lens and directs the light to a sensor assembly or optical assembly. Cover glass covers optical window 38 to provide some protection to the optical assembly under the optical window 38.
  • FIG. 6 is a cross-sectional view of the imaging system for the incubator imaging system.
  • Stage 20 supports the cell culture sample for illumination and imaging.
  • the illumination matrix is mounted above the stage to illuminate the sample from above.
  • a cover glass 24 is shown at the centre of the stage, covering the optical window and protecting the imaging components from inadvertent spills and general incubation conditions such as heat, humidity, gasses, etc.
  • Z-adjustment wheel 42 shown provides up and down adjustment of the stage normal to the light path, and retention ring 40 provides stability to the stage and upper optical system components.
  • Lower flange mounting 48 at the bottom of the optical tube 32 can be of a variety of sizes to match the optical sensor for light capture and provides stability to the light path and optical system from movement during movement of the incubator components.
  • an objective body comprising an objective lens 30 positioned below the cover glass receives the light in the light path.
  • the working distance, or distance from the objective lens 30 to the cells being imaged must be sufficiently small to provide good imaging of the cells. Typically, the higher magnification the objective is, the smaller the working distance.
  • An optional emission filter 34 can be positioned in the light path, here shown at a 4° angle to the lens tube.
  • Tube lens 36 provides magnification of light inside the tube along the light path. In some configurations the objective and tube lens can be integrated into a single component. The combination of the objective lens 30 and tube lens 36 provides an inverted optical arrangement, single field of view magnification.
  • Optical sensor 44 can be a camera assembly or any other device capable of receiving and storing optical data received from the light path.
  • the optical sensor can be, for example, a complementary metal-oxide-semiconductor (CMOS) or charge-coupled device (CCD) imager or other device sensitive to electromagnetic radiation or fields.
  • CMOS complementary metal-oxide-semiconductor
  • CCD charge-coupled device
  • the camera can optionally be a black and white camera sensor.
  • Raw data obtained from the optical sensor can be stored in one or more data storage devices or on a cloud storage system.
  • a secondary illumination system 46 can provide an alternative or secondary illumination source and image data stream to provide enhancement to the imaging data obtained from primary illumination from the illumination matrix.
  • the optional secondary illumination system can provide additional detail to the final image, in particular a different type of light illumination to the sample, such as, for example, fluorescence illumination to a cell sample that has been fluorescently tagged.
  • the fluorophores in a fluorescently tagged sample will be excited by fluorescent illumination and the resulting fluorescence can be captured by the optical sensor and used to create the composite image.
  • the incubator imaging system shown has the secondary illumination system under the objective, however it is understood that the secondary illumination can also be provided from above the sample.
  • During secondary illumination light is emitted from the secondary illumination system 46 which is filtered by excitation filter 52 to provide single or near-single wavelength secondary illumination at an excitation frequency to the sample.
  • a dichroic mirror 54 in mirror housing 50 then directs light at the excitation frequency toward the sample.
  • a sample comprising a fluorophore sensitive to the excitation frequency will absorb the light directed by the dichroic mirror 54 and emit at a different emission frequency or fluorescence frequency, with some of the light emitted from the excited fluorophores directed back toward the dichroic mirror 54 through the optical tube 32.
  • green fluorescent protein is often used during cell culture as a reporter for gene expression, and locational isolation and imaging of sites of gene expression in the cell culture provide enhanced optical detail.
  • the dichroic mirror 54 acts as a beam splitter and wavelength specific filter, transmitting fluoresced light at the emission frequency through to the optical detector. Excitation light is transmitted through the specimen, with only emission light reaching the objective.
  • the emission filter 34 filters out all light other than light at or around the emission frequency, and light not filtered by the emission filter 34 proceeds to be captured by the optical sensor.
  • the interior conditions of the incubator can be changed, such as but not limited to temperature, gas composition, humidity, etc.
  • the image capture conditions can be varied widely including but not limited to: image capture frequency; telemetry reporting frequency; image capture type or illumination pattern such as fluorescence, darkfield, brightfield, or other symmetric or asymmetric illumination pattern; or optical sensor settings.
  • One practical example of the usefulness of varying the image capture conditions is based on the observation that fluorescence imaging can be considered invasive for some cells, and also that the fluorescence tags themselves can be susceptible to photobleaching, meaning they will stop working after being exposed to too much light, or for too long.
  • One method of conserving image quality over the growth and observation period for the cells can be to obtain more darkfield images, which are less intrusive to a growing cell culture than brightfield or other illumination, until some trigger is observed or desired.
  • the trigger can be, for example, time, or some image analytics-based trigger like, for example, detection of cell budding, confluency, migration. Image recognition of these cellular phenomena can dynamically change image acquisition settings such as capture rate, wavelength detection, exposure time, illumination pattern, etc.
  • the imaging system is capable of limiting light exposure to live cells such that moments during the growth cycle that matter more can be captured with limited cell disturbance.
  • image capture frequency can be limited during slower growth phases and increased during active growth phases to limit image capture frequency for more significant periods of cell growth.
  • Triggers that are time-based or based on cell culture analytics can also be used not only to change internal growth conditions, but also other variables of the incubation protocol. In particular, a variety of parameters can be changed according to time, conditions, or image analytics. These include but are not limited to the type of images to be captured in the collection of images, the image capture frequency, the type of notifications sent to the user, and the update frequency for the telemetry.
  • One potential application related to incubator conditions and imaging control is to only include acquire fluorescence imaging after the cell culture reaches a certain size. This automatic trigger could be used to limit exposure and reduce photobleaching during times that are deemed visually less interesting for image capture.
  • Figure 7 is a graph of an example oxygenation protocol that can be used with the incubator imaging system.
  • the incubator can be programmed to vary its internal conditions during a cell culture growth experiment, in particular for gas levels, temperature, and humidity.
  • Live analytics obtained through imaging can monitor cell growth patterns such as, for example, confluency and migration rate, and correlate cell growth patterns with recorded experimental conditions. Logging of the conditions of incubation in a particular experiment or for a multitude of related experiments enables prediction, replication, and optimization of cell growth conditions. Protocols and actual growth conditions as monitored through internal sensors can also be logged to satisfy policies and reporting standards. Recording of actual growth protocols and conditions can alleviate the recording and reporting burden on laboratory technicians.
  • the incubator can also provide dynamic control and reporting such that the control system can modulate, for example, temperature, CO and O over time for any desired specialized environment.
  • An environmental protocol designer can also be provided to pre-program the system to control, fluctuate, change, and record CO2, O2, other gases, temperature, or any other controllable environmental condition, over time for any desired experiment.
  • Setting and monitoring environmental conditions can be done before and/or during live cell imaging, and data collected to compare cell growth with environmental conditions and environmental changes.
  • the present system can further be provided with remote programmability, as well as live monitoring and logging of gases such as CO2 and O2 that affect cell growth, as well as temperature levels over time to record a complete history of experiments.
  • Optical data capture and analysis in combination with environmental control can also provide additional details for assisting with optimizing cell growth and protocol design.
  • Environmental system control protocols can be set and changed for changing environmental conditions at any time, providing the ability to monitor visual data provided by live cell image and adjust environmental conditions with feedback of live cell image data.
  • control of environmental conditions can be provided by automatic feedback by analysis from the imaging system.
  • the imaging system detects when the number of cells in the optical field reaches above a certain number, the environmental condition of oxygen percentage can be lowered in the incubator to slow or halt cell growth. In this way, the environmental control conditions inside the incubator can be automatically changed by the integrated imaging system based on the live cell image.
  • the present system can also allow researchers to monitor their cell cultures remotely, for example from their computers and cell phones. Primary researchers can also be enabled to monitor the progress of cell cultures for every researcher in their group via an administrator portal.
  • artificial intelligence Al
  • a central and optionally cloud-based analytics platform can collect and aggregate data developed into individual reports for individual experiments, but can also be used to aggregate large data sets.
  • Figure 8 illustrates a set of screen shots from data obtained by the incubator imaging system for display on a smartphone.
  • scientists can be provided with a remote interface showing visual results of an experiment in progress or completed for remote review.
  • the interface can also provide analytical results such as cell count, confluency, density, or other culture analysis. Protocols can also be accessible for view and potential modification from the remote device.
  • Figures 9a-9c are single images taken of the same sample at different illuminations.
  • Figure 9a is an image taken of a sample under brightfield illumination
  • Figure 9b is an image taken of the same sample under darkfield illumination
  • Figure 9c is an image taken of the same sample under fluorescence illumination. As shown, the image obtained is dependent on the illumination conditions of the image capture.
  • Figure 10 is a composite image of the individual images shown in Figures 9a-9c.
  • a set of images are captured by the optical sensor on the imaging system, where the set of multiple images are captured in rapid succession, with each image obtained at a different illumination of the illumination matrix.
  • Algorithmic combining of the separate images provides a final high quality image. Compilation of the set of images into a single image provides a high quality final image of the live cell culture that is better than any of the individual images.
  • a variety of techniques and algorithms can be used to combine images to provide the final composite image.
  • the pixel illumination at each of the pixels in the captured image can be averaged to product a single average composite image. By averaging in this way, random noise can be reduced.
  • textured contrast in the cell features is accentuated, making cellular structures stand out.
  • An image can also be captured from the optical sensor in the absence of illumination to capture the background, which can be subtracted in the composite image to eliminate any background artifacts.
  • Figure 11 a is an example darkfield (left) and brightfield (right) illumination pattern of the illumination matrix.
  • the illumination matrix is shown here as a 16x16 matrix, however it is understood that the illumination matrix can be comprised of more or fewer individual light sources, and arranged in other than x/y axis or square orientation. Another matrix orientation may be where the individual light sources are in a hexagonal close-packed pattern or a radial pattern.
  • Figure 11 b is an example set of four asymmetric illumination patterns in an example square illumination matrix. Shown are four approximate half-circles on a 16x16 LED matrix, in which the white boxes are illuminated LEDs and the dark boxes are unlit LEDs in the illumination matrix during an illumination event.
  • illumination conditions can be changed to provide the desired single as well as composite image.
  • the image capture conditions of any individual image can be varied, and the images that make up the final composite image can have different capture conditions. For each different illumination pattern produced by the illumination matrix there are a wide variety of variables that can be changed to adjust the output or collected image.
  • pattern or, luminosity, wavelength, shape of illumination, number of different illuminations, exposure time of the camera, time delay between illuminations and image acquisition can all be changed at the illumination matrix.
  • Other variables in the capture conditions that can be changed for each composite image include but are not limited to, time delay between image capture, illumination exposure times across individual images, number of images captured, brightness across the set of illumination events, asymmetric pattern, wavelength of illumination, and camera properties such as exposure time, gain, and brightness.
  • One method of changing the brightness of an individual image is to vary the number of LEDs lit in the illumination event. Brightness of the individual LEDs can also be changed, for example, by changing the current to each LED or by changing the pulse width modulation.
  • the illumination intensity can also be same or different for each captured image in a composite image set.
  • a high dynamic range composite image can be obtained by combining a low illumination image with a high illumination image.
  • image processing is used to combine images and generate the live cell image based on the image set.
  • image processing can include pixel averaging, which can result in random noise being reduced in the image.
  • An image can also be captured from the optical sensor in the absence of illumination to capture the background, which can be subtracted in the composite image to eliminate any background.
  • Hot pixels from the optical sensor can also be adjusted in the final image by taking a long exposure high-gain background image and identifying the hot pixels, then eliminating them from the final image.
  • Differential phase contrast imaging can also be used to obtain phase information using at least two images taken under different illumination conditions.
  • Time lapse imaging of cell growth can also be compiled by viewing multiple individual and/or composite images over time.
  • Figure 12 is an example method of use of the incubator imaging system.
  • a cell culture sample is positioned on the microscope stage inside the incubator 102. The cell culture can be pre-incubated inside the incubator or other incubator prior to image collection, or can begin incubation on the microscope stage.
  • environmental conditions inside the incubator are established 104.
  • Environmental conditions inside the incubator are monitored throughout incubation and environmental condition data is stored locally on a data storage device, remotely on a remote data server, or in both locations.
  • a set of images of the cell culture are obtained 106 under variable image capture conditions, and these images are combined 108 either immediately or at a later time to provide a high quality image of the cell culture during incubation. Multiple image sets can be obtained over time to provide a time course and/or video of culture growth or change over time.

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Abstract

Un système d'imagerie d'incubateur pour l'imagerie de cellules vivantes comprend une chambre d'incubation pour maintenir un environnement interne, un dispositif de commande environnemental pour commander l'environnement interne dans la chambre d'incubation, un capteur environnemental pour détecter un état environnemental dans la chambre d'incubation, et un dispositif de microscopie avec une matrice d'éclairage ayant une pluralité de sources de lumière. Le système d'imagerie incubateur peut capturer des images de cellules dans une variété de conditions d'éclairage pendant la croissance cellulaire en faisant varier les conditions de capture d'image.
PCT/CA2020/051336 2019-10-07 2020-10-06 Système d'imagerie d'incubateur WO2021068060A1 (fr)

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Cited By (3)

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CN113136336A (zh) * 2021-05-08 2021-07-20 冰山松洋生物科技(大连)有限公司 一种co2培养箱防止湿度过饱和的调节装置
CN114525203A (zh) * 2022-02-21 2022-05-24 江苏京牧生物技术有限公司 一种饲料添加剂中有益菌的菌落密度测定设备
CN114574545A (zh) * 2022-03-30 2022-06-03 山东中科先进技术有限公司 一种基于多尺度超分辨率的小目标监测方法

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US8859263B2 (en) * 1996-11-01 2014-10-14 University Of Pittsburgh Method and apparatus for holding cells
US20180346868A1 (en) * 2015-03-31 2018-12-06 Thrive Bioscience ,Inc. Cell culture incubators with integrated imaging systems

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US8859263B2 (en) * 1996-11-01 2014-10-14 University Of Pittsburgh Method and apparatus for holding cells
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113136336A (zh) * 2021-05-08 2021-07-20 冰山松洋生物科技(大连)有限公司 一种co2培养箱防止湿度过饱和的调节装置
CN114525203A (zh) * 2022-02-21 2022-05-24 江苏京牧生物技术有限公司 一种饲料添加剂中有益菌的菌落密度测定设备
CN114574545A (zh) * 2022-03-30 2022-06-03 山东中科先进技术有限公司 一种基于多尺度超分辨率的小目标监测方法
CN114574545B (zh) * 2022-03-30 2023-11-24 山东中科先进技术有限公司 一种基于多尺度超分辨率的小目标监测方法

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