WO2014093980A1 - Analysis of action potentials, transients, and ion flux in excitable cells - Google Patents

Analysis of action potentials, transients, and ion flux in excitable cells Download PDF

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WO2014093980A1
WO2014093980A1 PCT/US2013/075488 US2013075488W WO2014093980A1 WO 2014093980 A1 WO2014093980 A1 WO 2014093980A1 US 2013075488 W US2013075488 W US 2013075488W WO 2014093980 A1 WO2014093980 A1 WO 2014093980A1
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cell
cells
images
recordings
image
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PCT/US2013/075488
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French (fr)
Inventor
Fabio Cerignoli
Piyush GEHALOT
Patrick M. Mcdonough
Jeffrey H. Price
Ross J. Whittaker
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Vala Sciences, Inc.
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Priority to US14/651,644 priority Critical patent/US9939372B2/en
Priority to EP13862952.2A priority patent/EP2932238B1/en
Publication of WO2014093980A1 publication Critical patent/WO2014093980A1/en
Priority to US15/947,490 priority patent/US10359357B2/en
Priority to US16/511,050 priority patent/US10928308B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/272—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration for following a reaction, e.g. for determining photometrically a reaction rate (photometric cinetic analysis)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2823—Imaging spectrometer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • H—ELECTRICITY
    • H04—ELECTRIC COMMUNICATION TECHNIQUE
    • H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
    • H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
    • H04N23/60—Control of cameras or camera modules
    • H04N23/67—Focus control based on electronic image sensor signals
    • H04N23/676—Bracketing for image capture at varying focusing conditions
    • H—ELECTRICITY
    • H04—ELECTRIC COMMUNICATION TECHNIQUE
    • H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
    • H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
    • H04N23/90—Arrangement of cameras or camera modules, e.g. multiple cameras in TV studios or sports stadiums
    • H—ELECTRICITY
    • H04—ELECTRIC COMMUNICATION TECHNIQUE
    • H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
    • H04N5/00—Details of television systems
    • H04N5/76—Television signal recording
    • H04N5/91—Television signal processing therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • the field Includes the biological arts, such as cytometry, and particularly concerns processes, systems and Instruments for automatically measuring action potentials, calcium transients and sense flux ;n excitable cells of humans and animals.
  • Excitable cells are those cells that are able to produce and respond to electrical signals and Include neurons, muscle (skeletal, smooth, and cardiac muscle), and secretory cells. Like all ceils, excitable ceils maintain a resting membrane potential by controlling the levels of certain ions within the cell in relation to the external concentration of those same Ions, establishing an electrochemical gradient across the membrane.
  • certain stimuli can open specialized sodium channels ⁇ e.g., electrical stimuli, mechanical stimuli, or llgand binding j on the membrane of excitable cells that cause an Increase in voltage from a negative nyperpelari ed level across the membrane toward depolarization of the membrane.
  • Action potentials can propagate along neurons for long distances and cause action potentials to occur In other excitable cells loading to various effects For example action potentials In muscle cells lead to the rapid release of calcium from Intracellular stores resulting in contraction of the cell.
  • Cytometric analysis of video recordings of ceils loaded with these dyes can provide quantitative assessment of the kinetics of the action potential, calcium transient or ion flux on a cell by cell basis
  • Chemical compounds, biological molecules ⁇ Including but not limited to proteins, DMA and D A constructs.
  • RNAs, small no dransia ed RNAs such as s!R A, ms NA, or equivalent, or other molecules derived from biological materia! ⁇ , electrical stimulation, or genetic .manipulation can be applied to the cells poor to or during the recording.
  • cytometric analysts methods the effect th compound, biological molecule, electrical stimulus or genetic manipulation has on the action potential, calcium transient or Ionic flux of the ceil can be assessed quantitatively.
  • one camera may capture the light emitted from the voltage sensitive dye and a second camera may capture the light emitted Rom the caloum sensitive dye.
  • the two cameras record from the same field of view and are triggered simultaneously. Analysis of the video recordings provides a quantitative assessment of the kinetics o the action potentials and the resulting calcium transients from each ceil In the field of view,
  • RNAs and proteins may be used to obtain further information about the ceils, but many require that the cells be first fixed.
  • These labels include immunof uorescent and fluorescent sn situ hybridisation (FISH) labels, as well as their co!otmetnc counterparts' immunohistochemical (IHC) and in-color in-$3 ⁇ 4u hybridization (CiSH) labels,
  • FISH immunof uorescent and fluorescent sn situ hybridisation
  • IHC immunohistochemical
  • CiSH in-color in-$3 ⁇ 4u hybridization
  • immunoflu escence for a- actsnin may be utilized to observe and measure tne degree of contractile apparatus organisation and development in muscl cells ⁇ including cardlomyocytes) differentiated from ste ceils.
  • Immature muscle ceils eaii have less orgamaed u-acilnln patterns than more mature muscle cells. Mutations in genes produce abnormal proteins, including ion channels in muscle cells (such as cardiomyocytes). Some of these mutations, sued as mutations that cause long QT syndromes make patients prone to arrhythmias such as ventricular tachycardia and ventricular fibrillation, which can lead to sudden death. (Long QT syndrome refers to a lengthening cf the interval between the Q S and T waves in the electrocardiogram.).
  • an automated high-throughput mode Includes parallel processing of two or more wells automatically and sequentially in microtltar plates (e.g . , with 96 or 384 wells. ⁇ . g1ul0]
  • An instrument produces slmuttanoous recordings from two or mere distinct optical channels in an automated manner. Methods are executed for registering multiple optical channels and performing automated cytometric analysis of the registered recordings In order to extract measurements on a oelh by-cell basis,
  • an automated process fixes the sample after making the live cell recordings, !aoe!e th fixed sample for additional blomarkers, rescans the sample, detects and analyzes the fixed cell hiomarlrers, detects cells washed oft during the fixation and labeling process, and registers and collates the live- and fixed-cell cytometry data together.
  • FIG. 1 is schematic illustration of an instrument and light path for obtaining simultaneous recordings from two or more distinct optical channels.
  • FIG. 2 includes images illustrating an example of cardlomyoc tes loaded with calc um (Fiuo-4) and voltage sensitive (di-8-A hPPS ⁇ dyes. Recordings were made by two cameras simultaneously from the same field of view vith each camera recording from a respective fluorescent wavelength
  • FIG 3 is schematic illustration of an electrical stimulator arm for introducing electrodes into a well of a mult;wall plate.
  • FIG, 4 Is flow diagram Illustrating a process for collecting simultaneous recordings from two or more distinct optical channels using the Instrument of FIG.
  • FIG. 5 is a flow diagram Illustrating a process by which nuclea , calcium and voltage image are shade corrected, magnification corrected and registered
  • FIG. 6 is a diagram demonstrating penpbery masks and examples of subcellular mask applied to cells being Imaged.
  • FIG. ?A is a waveform diagram illustrating an excitation-contraction coupling model hi describes a rel tion hi between ctio : potential end calcium transient in cardiomyocyte contraction.
  • FIG. 78 Is a diagram illustrating curves which describe the action potentials and calcium transients of eardiornyocytes collected,
  • FIG, 8 includes diagrams illustrating measurements extracted fro curves describing tne action ote tial, calcium Uansiont. or ion flux.
  • FIG. 9 Is screen shot containing pints of multiple calcium transients sn multiple ceils from the same field of view.
  • FIG. 10 shows plots of calcium transients of about 100 cells from the same Image overlaid on top ef each other.
  • FIG. 12 compares a calcium kinetic Image cytometry average image with a post-fixat!or ; Image labeled for cardiomyocyte blomarker ; -actinsn.
  • FIG 13 Illustrates segmentation of averaged kinetic calcium and post-fixed blomarker images and also demonstrates some cells lost during fixation.
  • FIG. 14 illustrates removal of the ceils miss;ng from the post-fixation data sat from the kinetic image cytometry data set.
  • FIG. 15 is a table of data of both Kinebc and post-fixed image cytometry data collated to remove the cells missing from the latter,
  • an optical Instrument 10. such as an automated microscope system, produce simultaneous recordings from two or more distinct optical channels.
  • the Instrument 10 equipped to scan a sample 1 1 In or on a support 12 such as a muitlwell (aka, microliter) plate (any format, e,g. ; in a range from 6 to 1338 walls, though typically 96 or 384 well plates are used) containing live cells loaded with multiple fluorescent dyes in an automated narmer.
  • the Instrument 0 Includes an optical array coupled to multiple scientific cameras and Includes a light source 16, a standard microscope objective 3, multiple mirrors, preferabl dlehroic mirrors ; 19 and 20 defining the light path and optical filters 22 and 23 which may or may not be contained within automated filter wheels.
  • Light of specific wavelength or multiple wavelengths is directed from the light source 18 through the objective 13 onto the sample 1 1 to excite the fluorescent dyes within the sample cells.
  • This light is collected by the objective 18 and passes through a series of mirrors and filters which separate the distinct wavelengths of light and direct each one to a respective one of the cameras 25 27.
  • An autofoous module 30 constituted of Hardware and software components moves the objective i the z position in orde to focus the light onto a plane in the sample 11 which produces dei;necl Images in each of the cameras 25 and 27.
  • the cameras am electrically triggered by a controller 40 to start and stop recording at the same time in order to produce simultaneous recordings.
  • the cameras record to an electronic storage device 42 where the data is stored prior to processing and analysis. Two images produced simultaneously by the instrument are seen in FIG- 2,
  • the Instrument of FIG, 1 is fitted with a motorized stage 44 that holds the muiti eil plate 12 and positions the sample 1 1 to be Imaged over the object ive 18. Once imaging is completed for a given area the stage 44 moves the plate 12 to the next sample area to be imaged. The next area can be a different area of the same well or an area within a new well on the plate.
  • a user-defined map Is preprogrammed and describes the areas within eac sample plate that are to be imaged. Once started the instrument aut ma ically images each defined ares., moving from one area to the next alter recording for a defined period of time.
  • a motorized stimulator arm SO attached to the stage 44 automatically lowers electrodes 54 into the sample well 12a of the multlwail plate 2 being Imaged in order to electrically stimulate the ceils.
  • electrical pulse with a defined voltage, duration, shape, and frequency can be applied to the cells.
  • the electrodes 54 are aised to allow the stage to move to the next area to be Imaged.
  • the electrodes 54 are lowered again.
  • the application of electrical stimuli is coordinated with the triggering of the cameras so that the exact point in t e recording when stimulation Is applied can be determined,
  • the mciofked stage which holds the sample is enclosed wlfhsn an incubation chamber in order to preserve cell physiology and maintain viability.
  • Temperature, carbon dio ide, and oxygen levels are maintained at. ser defined levels within the Incubation chamber. Temperature is controlled by heater elements which ar activated and deactivated by a thermostat.
  • Carbon dioxide and oxygen levels are maintained via an electronic feedback loop which consists of carbon dioxide and oxygen sensors which control electronic valves that introduce either carbon dioxide or oxygen from gas cylinders into the Incubatio chamber as needed to maintain preset levels.
  • RG. 4 An example of a process for collecting video recordings In a high throughput manner using this instrument is illustrated in RG. 4.
  • the process which includes collecting simultaneous recordings from the example of csrdlomyocytes loaded with voltage and calcium sensitive fluorescent dyes is used for Hiasealion only. Any excitable cell and any combination of voltage or Ion sensitive dyes could be substituted; dye free imaging of the cells such as observing movement under aright field could also be incorporated.
  • the instrument is controlled by a control mechanization executed by the controller 40.
  • the controller 40 which may include, for example, a computer.
  • the video recordings are stored on the bard dbvets) of the same computer.
  • the control mechanization enables a user to define parameters for the scan, which include, without limitation, the number of fluorescent channel ' s to be imaged, the excitation wavefengfb s), light intensity, camera frame rates, duration of the recording stimulation protocol to 0 applied (voltage, duration, shape, and frequency of the electrical pulses applied to the ceils), and the plate map defining which wells on the plate recordings will e made from.
  • parameters for the scan include, without limitation, the number of fluorescent channel ' s to be imaged, the excitation wavefengfb s), light intensity, camera frame rates, duration of the recording stimulation protocol to 0 applied (voltage, duration, shape, and frequency of the electrical pulses applied to the ceils), and the plate map defining which wells on the plate recordings will e made from.
  • the instrument then begins the simultaneous video recordings by triggering two separate cameras at 1 D8. If the user has selected to apply electrical stimulation to the cells that protocol Is activated at 1 10 shortly after the recordings begin. Following completion of the stimulation protocol at 112.. the cameras stop recording at 1 14, arid the electrodes are raised at 1 16. At 1 18, If there are more wells to image the stage moves the well plate so that the next well to be Imaged Is positioned over the objective and the process is repeated,
  • the recordings are preprocessed prior to analysis to register the recordings from each camera .
  • a control routine such as that illustrated in FIG. 5 is used to correct for mirroring, X-Y shift, rotation, and magnification differences in the recordings ca tured by different cameras recording from the same field of view which are Introduced by the light path, camera position, and chromatic aberration.
  • Registration is performed using a predetermined set of parameters that are collected by imaging a muitlweii plate containing muiilspecirai deads either before or after the video recordings are produced.
  • An analysis routine analy es the images of the mu!tispectns! heads, determines the manipulations that ne d to be made in order to the register the recordings. Once the recordings are registered they are cropped so that only the areas of the Image that appear in ail channels are maintained,
  • the routine illustrated m FIG. 5 performs: Image orientation correction (e.g., correction for mirror imaging of the two cameras), correction of shade distortion (A A flat field correction ⁇ , image registration (correction fo lateral misalignment, differences in magnification, and correction for rotation between the cameras), and final cropping to make the images the same pixel size after magnification correction.
  • Image orientation correction e.g., correction for mirror imaging of the two cameras
  • a flat field correction ⁇ A flat field correction ⁇
  • image registration correction fo lateral misalignment, differences in magnification, and correction for rotation between the cameras
  • final cropping to make the images the same pixel size after magnification correction.
  • the following calibration steps are performed: [003 1. The orientation of each image with respect to the others is observed and recorded.
  • a dicbroic mirror is used to sand longer wavelength fluorescence emi sion for voltage to one camera and shorter wavelength em ss on for calcium to a other camera, resulting in a m ro orientation of one image to the other.
  • the cameras can further be mutually rotated. The orientation of the nuclear image reiative fo the other two depends on which camera Images it.
  • a shade correction calibration Is performed by measuring the shade distortion (deviation from uniform Intensity with a uniform Intensity specimen, typically a flat piece of glass, or mirror) across the image, arid collecting a series of images at known different Intensities to correction for deviation from linearity and ensure that the Intensity response curve (plot of output Intensity as a function of Input intensity ⁇ crosses the Yoniereepi at zero.
  • the routine Illustrated FIG. 5 then corrects image orientations, performs shade and linearity correction, registers the images to the nuclear image, and crops the images to the same size, Magnification correction is needed because chromatic aberration causes images of different colors tc be magnified differently.
  • shade correction AH microscopes exhibit some distortion of the Illumination intensify across the field of view. This Is called shade distortion. For example, consider a fluorescent bead in focus at the center of the camera field of view, if the stage i moved so that the sample s at the edge of the field of view, the heed's intensit s usually different (usually lo e intensity) due to shade distortion. To compensate for this, the response of eaoh pixel on the camera is modeled as
  • V is the corrected Intensity
  • b Is the value needed to obtain a Y-infercept of zero after correction, and is the slope needed to oorrect each pixel to the same intensity.
  • the values of 'a *' and 3 ⁇ 4' are calculated for every pixel on the camera's Cfv OS by linear regression when varying Lumenoor intensity for fixed exposure time.
  • the corrected Intensity Is then calculated b inverting the relationship
  • Image mglsr rabom in the dual-channel KhC images In the two distinct cameras may be linearly shifted and rotated relative to each other (X- and Y-sh!ft). Moreover, there are magnification differences between the different coiors of m.;c:ean calcium and voltage fluorescent dyes due to chromatic aberration. Finally the cameras may not be pedecfiy aligned with respect to rotation, Thus for registration, an afflne transformation Is calibrated beforehand using multicolor beads and the other images are registered to the nuclear image serves.
  • the change In intensity is then plotted against time (based on frame number and frame rate at acquisition) and the resulting plot represents the kinetics of the action potential, calcium transient; o Ionic flux depending on which dye is being analyzed. Corrections can b made prior to analysis of the kinetics to account for artifacts such as photobieaching of the dyes.
  • the fluorescent dye that is sensitive to voltage across the membrane of each cell Is mere sensitive to photobieaching than most other fluorescent dyes.
  • VYith reference to FIG. 8. measurements that describe the shape and duration of the action potential, calcium transient. : and/or Ionic flu are then automatically extracted from the plots of intensity versus time. These measurements include, but are not limited to:
  • Decay Time- AKA TDecay or T- Decay the time elapsed from the transient maximum to the 50% point on the downstroke of the action potential, calcium transient, or ion flux;
  • Full Width Half Maximum- AKA f H the time elapsed from the S0% point of the upstroke to the 50% point of the downstroke of the action potential, calcium transient, or son flux;
  • T7S-2S- AKA T-75-25 the time elapsed from the 75% point of the downstroke to the 25% point of the downstroke of the action potential, calcium transient, or Ion flux.
  • Parameters measured /horn isl derivatives such as:
  • AC/ max-do n- defines the maximum negat ve slope achieved during the downstroke of the of the action potential, calcium transient, or Ion flux;
  • AC/&tmax « up- defines the maximum positive slope achieved during the upstroke of ine transient of the action potential : calcium transient, or Ion flux;
  • FIG. 12 shows calcium and nuclear images of neonatal rat ventricular myocytes from the live cell scan and the same region after fixation and I munostaininp for the cardlomycoyte marker o-actlnln and the nuclei.
  • the two pair of images are shifted relative to each other due to misalignment during repositioning of the plate on the same microscope stage after fixing and staining.
  • the curved line (arc) show a common area between the two pairs of Images.
  • FIG. 14 demonstrates comparison of the kinetic image cytometry and the fixed Image cytometr data sets to locate the missing cells and delete them from the kinetic image cytometry data set in as a step so fcrrmng a merged kinetle-and-fixed image cytometry data sot for convenient automated analysis.
  • the nucio; demonstrate the underly ng me ged data by dis l ying the same number labels-
  • FIG. 15 illustrates an example data table of both Kinetic and post-fixed image cytometry data collated after removal of the ceils missing from the tetter are shown, along with a Hay for the measurements for the two data sate..

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Abstract

Video recordings from two or more optical channels are produced, processed, and analyzed simultaneously in order to provide quantitative analysis of action potentials, calcium transients and ionic flux in excitable cells loaded with voltage or ion sensitive dyes with distinct excitation and emission wavelengths, The specific wavelengths of fluorescent light emitted from each dye are separated and recorded. The recordings are mutually registered and cytometric analysis is performed to provide a quantitative analysis of the action potenials calcium transient, and/or ionic flux on a cell-by-cell and well-by-well basis in microtiter plates. The cells are then fixed, labeled for other biomarkers, and scanned again. The resulting fixed cell images are registered with the live cell recordings and analyzed; missing cells that were washed off are detected relative to the live recordings, and cytometry data from live and fixed cell scans is collated cell-by-cell.

Description

ANALYSIS OF ACTION POTENTIALS, TRANSIENTS, AND ION FLUX IN
EXCITABLE CELLS
PRIORITY
[Q001J Ti¾s a lication claims priority m US 61/737,863, fried 2/14/2012,
RELATED APPLICATIONS
1 0021 h s Application contains subject matter related to ids su ject matter of: US patent a lication 12,454,217, filed 05/13/2009, published as US 2010/0280837 on 11/18/2010; and, US patent a lication 12/960,313, filed 12/03/2010, published as US 2011/0318775 on 12/29/2011.
BACKGROUND
[0083] The field Includes the biological arts, such as cytometry, and particularly concerns processes, systems and Instruments for automatically measuring action potentials, calcium transients and sense flux ;n excitable cells of humans and animals.
[0004] Excitable cells are those cells that are able to produce and respond to electrical signals and Include neurons, muscle (skeletal, smooth, and cardiac muscle), and secretory cells. Like all ceils, excitable ceils maintain a resting membrane potential by controlling the levels of certain ions within the cell in relation to the external concentration of those same Ions, establishing an electrochemical gradient across the membrane. Ho e e , certain stimuli can open specialized sodium channels {e.g., electrical stimuli, mechanical stimuli, or llgand binding j on the membrane of excitable cells that cause an Increase in voltage from a negative nyperpelari ed level across the membrane toward depolarization of the membrane. Once this membrane depolarization reaches a threshold level, voltage gated sodium channels open causing a rapid depolarization of t cell membrane followed by repolarization, which is referred to as an action potential. Action potentials can propagate along neurons for long distances and cause action potentials to occur In other excitable cells loading to various effects For example action potentials In muscle cells lead to the rapid release of calcium from Intracellular stores resulting in contraction of the cell.
|00Oi iv!any fluorescent dyes which respond to changes in membrane voltage and Ion concentrations (Including hut not limited to sodium, potassium, calcium, and chloride ions) are currently known. Using these dyes, researchers can make video recordings of a magnified field of view to observe changes in the intensit of the dyes when loaded into ceiis. Changes in the intensit of these dyes correlates to the activity of the action potentials (voltage sensitive dyes), calcium transients (Intracellular calcium dyes), or ion flux across a membrane (e.g. sodium or potassiu dyes). Cytometric analysis of video recordings of ceils loaded with these dyes, which measures the change in the intensity of these dyes over time, can provide quantitative assessment of the kinetics of the action potential, calcium transient or ion flux on a cell by cell basis Chemical compounds, biological molecules {Including but not limited to proteins, DMA and D A constructs. RNAs, small no dransia ed RNAs such as s!R A, ms NA, or equivalent, or other molecules derived from biological materia!}, electrical stimulation, or genetic .manipulation can be applied to the cells poor to or during the recording. Using cytometric analysts methods the effect th compound, biological molecule, electrical stimulus or genetic manipulation has on the action potential, calcium transient or Ionic flux of the ceil can be assessed quantitatively.
SUMMARY
[00OS] it is desirable to be able to obtain images of multiple activities in excitable cells that occur simultaneously or tn sequence in order to comprehensively and efficiently asses manifold activity of excitable sells. Voltage- and son-sensi ive fluorescent dyes are available with a variety of excitation and emission wavelengths, making it possible to load excitable cells with two or more dyes and collect video recordings from multiple dyes simultaneously. For example cultured cardiomyocyies can be Simultaneously loaded with voltage and calcium sensitive dyes, each wit distinct excitation and emission spectra. Video recordings of the fluorescent light emitted by each dye can be separated and provided to individual cameras. For example, one camera may capture the light emitted from the voltage sensitive dye and a second camera may capture the light emitted Rom the caloum sensitive dye. Preferably, the two cameras record from the same field of view and are triggered simultaneously. Analysis of the video recordings provides a quantitative assessment of the kinetics o the action potentials and the resulting calcium transients from each ceil In the field of view,
£080?! An even larger number o dye labels for genes. RNAs and proteins may be used to obtain further information about the ceils, but many require that the cells be first fixed. These labels include immunof uorescent and fluorescent sn situ hybridisation (FISH) labels, as well as their co!otmetnc counterparts' immunohistochemical (IHC) and in-color in-$¾u hybridization (CiSH) labels, For example, immunoflu escence for a- actsnin may be utilized to observe and measure tne degree of contractile apparatus organisation and development in muscl cells {including cardlomyocytes) differentiated from ste ceils. Immature muscle ceils eaii have less orgamaed u-acilnln patterns than more mature muscle cells. Mutations in genes produce abnormal proteins, including ion channels in muscle cells (such as cardiomyocytes). Some of these mutations, sued as mutations that cause long QT syndromes make patients prone to arrhythmias such as ventricular tachycardia and ventricular fibrillation, which can lead to sudden death. (Long QT syndrome refers to a lengthening cf the interval between the Q S and T waves in the electrocardiogram.).
£O00§| We have realized that the wavelength separation resulting f o : use of different labels to mark different activities of excitable cells affords the opportunity to isualize those activities by simult neously acquiring Images through separate optical channels,
|000§1 Accordingly; processes, systems, and instruments are provided for producing, processing, and analyzing vid o recordings from two or more optical channels simultaneously, from a single sample, in an automated high-throughput manne in some aspects, an automated high-throughput mode Includes parallel processing of two or more wells automatically and sequentially in microtltar plates (e.g . , with 96 or 384 wells.}. g1ul0] An instrument produces slmuttanoous recordings from two or mere distinct optical channels in an automated manner. Methods are executed for registering multiple optical channels and performing automated cytometric analysis of the registered recordings In order to extract measurements on a oelh by-cell basis,
[ δΐ ΐ] in some aspects, an automated process fixes the sample after making the live cell recordings, !aoe!e th fixed sample for additional blomarkers, rescans the sample, detects and analyzes the fixed cell hiomarlrers, detects cells washed oft during the fixation and labeling process, and registers and collates the live- and fixed-cell cytometry data together.
BRIEF DESCRI PTION OF THE DRAWINGS
|0O12| FIG. 1 is schematic illustration of an instrument and light path for obtaining simultaneous recordings from two or more distinct optical channels. £0013] FIG. 2 includes images illustrating an example of cardlomyoc tes loaded with calc um (Fiuo-4) and voltage sensitive (di-8-A hPPS} dyes. Recordings were made by two cameras simultaneously from the same field of view vith each camera recording from a respective fluorescent wavelength
10014 FIG 3 is schematic illustration of an electrical stimulator arm for introducing electrodes into a well of a mult;wall plate.
£00 SJ FIG, 4 Is flow diagram Illustrating a process for collecting simultaneous recordings from two or more distinct optical channels using the Instrument of FIG.
£0Ο1δ] FIG. 5 is a flow diagram Illustrating a process by which nuclea , calcium and voltage image are shade corrected, magnification corrected and registered
£001 TJ FIG. 6 is a diagram demonstrating penpbery masks and examples of subcellular mask applied to cells being Imaged.
£0018] FIG. ?A is a waveform diagram illustrating an excitation-contraction coupling model hi describes a rel tion hi between ctio : potential end calcium transient in cardiomyocyte contraction. FIG. 78 Is a diagram illustrating curves which describe the action potentials and calcium transients of eardiornyocytes collected,
[001 S] FIG, 8 includes diagrams illustrating measurements extracted fro curves describing tne action ote tial, calcium Uansiont. or ion flux.
£0020} FIG. 9 Is screen shot containing pints of multiple calcium transients sn multiple ceils from the same field of view.
£8021] FIG. 10 shows plots of calcium transients of about 100 cells from the same Image overlaid on top ef each other.
£00221 1 le flow diagram illustrating posdkineiiC fixation and labeling of blomarkers, rescanning the same plate/cells, performing fixed Image cytometry and collating the fixed image cytometry cell-by- cel. with the transient image cytometry.
[0023] FIG. 12 compares a calcium kinetic Image cytometry average image with a post-fixat!or; Image labeled for cardiomyocyte blomarker ;-actinsn.
£0024] FIG 13 Illustrates segmentation of averaged kinetic calcium and post-fixed blomarker images and also demonstrates some cells lost during fixation. [002S| FIG. 14 illustrates removal of the ceils miss;ng from the post-fixation data sat from the kinetic image cytometry data set.
PS28| FIG. 15 is a table of data of both Kinebc and post-fixed image cytometry data collated to remove the cells missing from the latter,
DETAILED DESCRIPTION Of THE PREFERRED EMBODIMENTS
1002?] With reference to FIG. 1 , an optical Instrument 10.. such as an automated microscope system, produce simultaneous recordings from two or more distinct optical channels. Preferably, the Instrument 10 equipped to scan a sample 1 1 In or on a support 12 such as a muitlwell (aka, microliter) plate (any format, e,g. ; in a range from 6 to 1338 walls, though typically 96 or 384 well plates are used) containing live cells loaded with multiple fluorescent dyes in an automated narmer. The Instrument 0 Includes an optical array coupled to multiple scientific cameras and Includes a light source 16, a standard microscope objective 3, multiple mirrors, preferabl dlehroic mirrors ; 19 and 20 defining the light path and optical filters 22 and 23 which may or may not be contained within automated filter wheels. Light of specific wavelength or multiple wavelengths is directed from the light source 18 through the objective 13 onto the sample 1 1 to excite the fluorescent dyes within the sample cells. Fluorescent light with multiple wavelengths of light Is then emitted from the sample 11 : saca emission wavelength correlates to a specific dye. This light is collected by the objective 18 and passes through a series of mirrors and filters which separate the distinct wavelengths of light and direct each one to a respective one of the cameras 25 27. An autofoous module 30 constituted of Hardware and software components moves the objective i the z position in orde to focus the light onto a plane in the sample 11 which produces dei;necl Images in each of the cameras 25 and 27. The cameras am electrically triggered by a controller 40 to start and stop recording at the same time in order to produce simultaneous recordings. The cameras record to an electronic storage device 42 where the data is stored prior to processing and analysis. Two images produced simultaneously by the instrument are seen in FIG- 2,
2 J In some aspects, the Instrument of FIG, 1 is fitted with a motorized stage 44 that holds the muiti eil plate 12 and positions the sample 1 1 to be Imaged over the object ive 18. Once imaging is completed for a given area the stage 44 moves the plate 12 to the next sample area to be imaged. The next area can be a different area of the same well or an area within a new well on the plate. A user-defined map Is preprogrammed and describes the areas within eac sample plate that are to be imaged. Once started the instrument aut ma ically images each defined ares., moving from one area to the next alter recording for a defined period of time.
id>02§| With reference to FIG, 3, a motorized stimulator arm SO attached to the stage 44 automatically lowers electrodes 54 into the sample well 12a of the multlwail plate 2 being Imaged in order to electrically stimulate the ceils. When the ar : 50 is lowered into the well 12a ; electrical pulse with a defined voltage, duration, shape, and frequency can be applied to the cells. Follo ing completion of the recording ;n one area, the electrodes 54 are aised to allow the stage to move to the next area to be Imaged. Once the stage has moved to the next area to be imaged the electrodes 54 are lowered again. The application of electrical stimuli is coordinated with the triggering of the cameras so that the exact point in t e recording when stimulation Is applied can be determined,
C0O3QJ in some aspects, the mciofked stage which holds the sample is enclosed wlfhsn an incubation chamber in order to preserve cell physiology and maintain viability. Temperature, carbon dio ide, and oxygen levels are maintained at. ser defined levels within the Incubation chamber. Temperature is controlled by heater elements which ar activated and deactivated by a thermostat. Carbon dioxide and oxygen levels are maintained via an electronic feedback loop which consists of carbon dioxide and oxygen sensors which control electronic valves that introduce either carbon dioxide or oxygen from gas cylinders into the Incubatio chamber as needed to maintain preset levels.
|0O31} An example of a process for collecting video recordings In a high throughput manner using this instrument is illustrated in RG. 4. The process, which includes collecting simultaneous recordings from the example of csrdlomyocytes loaded with voltage and calcium sensitive fluorescent dyes is used for Hiasealion only. Any excitable cell and any combination of voltage or Ion sensitive dyes could be substituted; dye free imaging of the cells such as observing movement under aright field could also be incorporated. With reference to FIG. 4, a mttitb eil plate containing cardlomyocytes which have been loaded with a nuclear dye o.e. Hoechst) nd two or more fluorescent dyes which respond to membrane voltage fluctuations and calcine: transients and which have different and distinct excitation and emission spectra are loaded onto the stage of the Instrument Illustrated in FIG. 1 , The instrument is controlled by a control mechanization executed by the controller 40. which may include, for example, a computer. In some aspects, the video recordings are stored on the bard dbvets) of the same computer. The control mechanization enables a user to define parameters for the scan, which include, without limitation, the number of fluorescent channel's to be imaged, the excitation wavefengfb s), light intensity, camera frame rates, duration of the recording stimulation protocol to 0 applied (voltage, duration, shape, and frequency of the electrical pulses applied to the ceils), and the plate map defining which wells on the plate recordings will e made from. Once started at 100 the motorized stage moves the first well to be Imaged over the objective. If electrical stimulation Is to be applied the electrodes are lowered into the well at 102. Th Instrument then autofocuses at 104 using a nuclear signal and collects an image of the ceii nuclei at 106. The instrument then begins the simultaneous video recordings by triggering two separate cameras at 1 D8. If the user has selected to apply electrical stimulation to the cells that protocol Is activated at 1 10 shortly after the recordings begin. Following completion of the stimulation protocol at 112.. the cameras stop recording at 1 14, arid the electrodes are raised at 1 16. At 1 18, If there are more wells to image the stage moves the well plate so that the next well to be Imaged Is positioned over the objective and the process is repeated,
[0032J In some aspects, at 120 the recordings are preprocessed prior to analysis to register the recordings from each camera . A control routine such as that illustrated in FIG. 5 is used to correct for mirroring, X-Y shift, rotation, and magnification differences in the recordings ca tured by different cameras recording from the same field of view which are Introduced by the light path, camera position, and chromatic aberration. Registration is performed using a predetermined set of parameters that are collected by imaging a muitlweii plate containing muiilspecirai deads either before or after the video recordings are produced. An analysis routine analy es the images of the mu!tispectns! heads, determines the manipulations that ne d to be made in order to the register the recordings. Once the recordings are registered they are cropped so that only the areas of the Image that appear in ail channels are maintained,
| S33j The routine illustrated m FIG. 5 performs: Image orientation correction (e.g., correction for mirror imaging of the two cameras), correction of shade distortion (A A flat field correction}, image registration (correction fo lateral misalignment, differences in magnification, and correction for rotation between the cameras), and final cropping to make the images the same pixel size after magnification correction. Prior to execution of the routine, the following calibration steps are performed: [003 1. The orientation of each image with respect to the others is observed and recorded. For .example, a dicbroic mirror is used to sand longer wavelength fluorescence emi sion for voltage to one camera and shorter wavelength em ss on for calcium to a other camera, resulting in a m ro orientation of one image to the other. The cameras can further be mutually rotated. The orientation of the nuclear image reiative fo the other two depends on which camera Images it.
|0 3S| 2, A shade correction calibration Is performed by measuring the shade distortion (deviation from uniform Intensity with a uniform Intensity specimen, typically a flat piece of glass, or mirror) across the image, arid collecting a series of images at known different Intensities to correction for deviation from linearity and ensure that the Intensity response curve (plot of output Intensity as a function of Input intensity} crosses the Yoniereepi at zero.
00361 3. An Image of multicolor fluorescent beads, fluorescing at a minimum at the same colors as the fluorescent dyes to be used in the experiment Is collected at each of the emission wavelengths of the dyes to be used in the experiment. An automated registration software algorithm finds the X- and Y- lateral shifts, the differences in magnification, and the rotation of each of the other images relative to the bead Image of the nuclear color
[00373 The routine Illustrated FIG. 5 then corrects image orientations, performs shade and linearity correction, registers the images to the nuclear image, and crops the images to the same size, Magnification correction is needed because chromatic aberration causes images of different colors tc be magnified differently.
[0 3 ] After calibration, the routine of FIG. 5 -s executed as follows.
1, Image orientation, correction. For single-channel lC (kinetic image cytometry), the calcium and voltage images are aligned to the nuclear image (for two cameras, the image on one camera Is the mirror of the image on the other camera due to splitting the light with a dicbroic mirror). Using the information stored curing calibration, the osl um and voltage smages are flipped/rotated to align wi h the nuclear image,
2. Shade correction. AH microscopes exhibit some distortion of the Illumination intensify across the field of view. This Is called shade distortion. For example, consider a fluorescent bead in focus at the center of the camera field of view, if the stage i moved so that the sample s at the edge of the field of view, the heed's intensit s usually different (usually lo e intensity) due to shade distortion. To compensate for this, the response of eaoh pixel on the camera is modeled as
y ~ ax ·;·· b
where y Is raw image intensity, V is the corrected Intensity, :b Is the value needed to obtain a Y-infercept of zero after correction, and is the slope needed to oorrect each pixel to the same intensity. The values of 'a*' and ¾' are calculated for every pixel on the camera's Cfv OS by linear regression when varying Lumenoor intensity for fixed exposure time. The corrected Intensity Is then calculated b inverting the relationship
x - (y - b)/a
The values of !a! and ! ' also vary w;th excitation wavelength : and the calibration is also done for every wavelength on the lomenoof. From the linear regression, 'hot' and 'd ad' pixels would have calculated values of a » 0. Any values of ™ 0 are changed to a ~ 1. so that 'de d1 pixels remain close to 0: and 'hot' pixels remain close to saturation. An corrected pixels greater than the maximum intensit allowed by the bit dept of the image ere set to the maximum: intensify; e,g,, in an 11 -bit Image, plxes intensities greater than 2 047 are set to 2.047.
3, Image mglsr rabom in the dual-channel KhC, images In the two distinct cameras may be linearly shifted and rotated relative to each other (X- and Y-sh!ft). Moreover, there are magnification differences between the different coiors of m.;c:ean calcium and voltage fluorescent dyes due to chromatic aberration. Finally the cameras may not be pedecfiy aligned with respect to rotation, Thus for registration, an afflne transformation Is calibrated beforehand using multicolor beads and the other images are registered to the nuclear image serves. The afflne transformations compensate for linear translation, rotation and magnification (scaling) all at once for all other colors relative to the nu lear Image For every p;xel coordinate (x, ) ;n the original Image, a new set of coordinates (xf yh are calculated according la:
% a · . oy,. · 4, OI cropping. Altar registration, magnification correction creates images of different sizes in el and these images are cropped so that the resulting images stored to the hard drive are ail the same m®.
[0Q3S] The processed recordings are then analyzed using automated cytometry routines represented by F G, which identify, segment and index each ceil in the field of view to produce cell periphery masks and masks defining subcellular regions using known methods which combine data from two or more optical channels in order to define the boundaries of the whole as well as distinct regions within the cell The masks are applied to each image in each recording and the intensit of the dyes in each ceil is calculated for each frame of the recording.
|0O4S] As pe FIGS.. 7A and ?B, the change In intensity is then plotted against time (based on frame number and frame rate at acquisition) and the resulting plot represents the kinetics of the action potential, calcium transient; o Ionic flux depending on which dye is being analyzed. Corrections can b made prior to analysis of the kinetics to account for artifacts such as photobieaching of the dyes. The fluorescent dye that is sensitive to voltage across the membrane of each cell Is mere sensitive to photobieaching than most other fluorescent dyes. Thus, a straight line Is fit to the dat and the straight line parameters are used to correct for the gradual decrease in intensity caused by photobieaching The fluorescent dye that is sensitive to voltage across the membrane of each cell decreases In intensity with the increase in voltage that occurs during the action potential. Thus, the Intensity vs. tsme data Is inverted (each Intensity is subtracted bom the maximum Intensity).
|O041] VYith reference to FIG. 8. measurements that describe the shape and duration of the action potential, calcium transient.: and/or Ionic flu are then automatically extracted from the plots of intensity versus time. These measurements Include, but are not limited to:
Direct measurements scon as:
Peak heiohRhe maximum point in the curve describing the action potential, calcium transient, o ion flux;
Rise Time- AKA TRise or T-Rise. the time elapsed from the 50% point en the up stroke of the transient to the 100% point or the maximum of the action potential, calcium transient, o Ion flux;
Decay Time- AKA TDecay or T- Decay the time elapsed from the transient maximum to the 50% point on the downstroke of the action potential, calcium transient, or ion flux; Full Width Half Maximum- AKA f H - the time elapsed from the S0% point of the upstroke to the 50% point of the downstroke of the action potential, calcium transient, or son flux; and
T7S-2S- AKA T-75-25. the time elapsed from the 75% point of the downstroke to the 25% point of the downstroke of the action potential, calcium transient, or Ion flux. Parameters measured /horn isl derivatives such as:
AC/ max-do n- defines the maximum negat ve slope achieved during the downstroke of the of the action potential, calcium transient, or Ion flux;
Time to ACf max-do n - the time elapsed from the transient maximum to the maximum negative slope of the transient downstroke of the action potential calcium transient, or Ion flux;
AC/&tmax«up- defines the maximum positive slope achieved during the upstroke of ine transient of the action potential: calcium transient, or Ion flux; and
Time to AC max-yp- the time elapsed from the beginning go the transient to the maximum positive slope of the transient upstroke of the action potential, calcium transient, or ion flux.
0 2] As per FIG, 9.: the transients from the same condition or well can be plotted individually. Alternatively, many transients can be plotted on top of each other as per FIG, 10.
100 31 With reference to FIG. 11 , there are many proteins, RNA sequences and DNA sequences that can only be labeled by first fixing (killing) the cells and thus cannot be carried out during live kinetic image cytometry. To solve this problem, we use the process of fixing the cells at 200, labeling them at 202, rescanning them at 204, registering the Images at 208, and then performing image cytometry analysis at 208 by detecting the cells / nuclei lost dunng fixation and collating the kinetic image cytometry data set together with the fixed Image cytometry data set for each cell.
[0 4] An example of an averaged kinetic Image cytometry calcium video and a post- fixation Image of the same cardlcmyocytes labeled for csrdlomyocyte blomarker "U actinia are shown In FIG. 12. FIG, 12 shows calcium and nuclear images of neonatal rat ventricular myocytes from the live cell scan and the same region after fixation and I munostaininp for the cardlomycoyte marker o-actlnln and the nuclei. The two pair of images are shifted relative to each other due to misalignment during repositioning of the plate on the same microscope stage after fixing and staining. The curved line (arc) show a common area between the two pairs of Images. The same image pairs are shown segmented and registered I FIG. 13, and the rectangle and arrows sho the same regions and nuclei washed off during fixation and labeling so the fixed image, respectively. FIG. 14 demonstrates comparison of the kinetic image cytometry and the fixed Image cytometr data sets to locate the missing cells and delete them from the kinetic image cytometry data set in as a step so fcrrmng a merged kinetle-and-fixed image cytometry data sot for convenient automated analysis. The nucio; demonstrate the underly ng me ged data by dis l ying the same number labels- FIG. 15 illustrates an example data table of both Kinetic and post-fixed image cytometry data collated after removal of the ceils missing from the tetter are shown, along with a Hay for the measurements for the two data sate..

Claims

CLA MS
1 . An Instrument for producing sim l aneous video recordings from multi le optical c a nels, of a magn f ed field of view, in order to create recordings of cells ioaded with two or more fluorescent dyes designed to respond to either acti n potentials, calcium transients, or the flow of sons across a membrane. In which the instrument Includes:
an optica path Including a light source, microscope objective, mirrors, optical filters and two or more cameras arranged so as to separate fluorescent light emitted from the sample Into distinct wavelengths and then direct each wavelength to a respective camera;
a motorized stage that positions a region of th sample to be Imaged above the objective:
an autofocus module that moves the focus in relation to the sample In order to focus the Image:
an incubation chamber that contains the sample and that maintains temperature and the carbon dioxide and oxygen at preset levels: and,
a control system for recording two or more videos imultaneously from each o two or more cameras to record two or mors varying levels of fight, each of which correspond to two or more different intracellular components
2. The instrument of claim: i . further Inoiudsng a stimulator arm to automatically move a pair of electrodes Into welis of a mulcwell plate for applying a user defined electricai stimulation protocol to the cells.
3 The instrumen of claim 1 or 2. herein the autofocus module is for utilizing one of the two or more cameras to:
collect a stack of images, wherein each image in the stack is collected si a different focus position;
calculate a degree of focus or sharpness index;
derive the best focus position based on the degree of focus data, and set focus to the best focus position, wherein the images in the stack are either of a different fluorescent color (such as of a fluorescent dye labeling the D A ;n the nucleus, such as DAP!) and wherein an automatic filter chancer changes the filter after autofocus to collect the corresponding li e cell channel, or wherein the images in the stack are collected on the same fluorescent channel as the corresponding live cell channel. The Instrument of claim 3, wherein one. two or more colors (video channels) are recorded and analyzed, wherein the cells are secondarily fixed and analyzed for additional labels
5. A computer executed method wherein the recordings made by the Instrument of claim 3 are processed and analyzed to provide quantitative measurements of cellular action potentials, calcium transients, and ion flo across a membrane, the method including;
correcting for mirroring, χ~γ shift, rotation, and magnification differences between the video recordings captured by different cameras recording from the same field of view;
segmenting the videos of the cells and labeling subcellular regions of the ceils from two or mo e optica! channels In order to define the boundaries of individual cell as well as distinct subcellular regions within each cell;
generating measurements from the cell periphery and subcellular masks of the change in intensity over time of the fluorescent dyes thai are designed to respond to changes in membrane voltage or ion concentrations;
plotting the change In Intensity of the fluorescent dyes versus time; and.
extracting measurements that characterize trie shape and duration of the transsents (that can be visualized In the plots of the measurements as a function of time) of the action potential, calcium transients or son concentrations.
8. The method of claim 5, further including fixing the calls and labeling them for additional cellular molecules,
7, The method of claim 5: further including scanning the sample a second time and recording Images of various colors of the ne labels,
8, The method of claim 5. further Including registering the Images from the fixed scanning to the images and video recordings of the live scanning.
9. The method of claim 5. further includ ng segmenting the Images of the fixed scanning to detect the ceils and subcellular regions, detecting the missing, cells washed off during the process of fixing and labeling :. record measurements of the fixed cell labels in the cellular and subcellular regions, and
10. The method of claim 5. further Inducing collating the live cell and fixed cell cytometric measurements into a singl data set to study and compare the fixed call and live cell labels together.
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