EP1520250A2 - Systeme d'integration a plusieurs axes (mai) et procede afferent - Google Patents

Systeme d'integration a plusieurs axes (mai) et procede afferent

Info

Publication number
EP1520250A2
EP1520250A2 EP03716656A EP03716656A EP1520250A2 EP 1520250 A2 EP1520250 A2 EP 1520250A2 EP 03716656 A EP03716656 A EP 03716656A EP 03716656 A EP03716656 A EP 03716656A EP 1520250 A2 EP1520250 A2 EP 1520250A2
Authority
EP
European Patent Office
Prior art keywords
image
image data
optical axis
scan
scanning
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP03716656A
Other languages
German (de)
English (en)
Other versions
EP1520250A4 (fr
Inventor
Steven A. Reese
Carl S. Brown
Paul Goodwin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Global Life Sciences Solutions USA LLC
Original Assignee
Applied Precision 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
Priority claimed from US10/215,265 external-priority patent/US20030048933A1/en
Application filed by Applied Precision Inc filed Critical Applied Precision Inc
Publication of EP1520250A2 publication Critical patent/EP1520250A2/fr
Publication of EP1520250A4 publication Critical patent/EP1520250A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • 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
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T1/00—General purpose image data processing
    • G06T1/0007—Image acquisition

Definitions

  • aspects of the present invention relate generally to image acquisition systems and image processing techniques, and more particularly to a system and method of integrating image data obtained along an optical axis and in conjunction with lateral translation of an image plane.
  • a variety of autofocus techniques have been developed to enable automated and semi-automated scanning of three-dimensional (3D) objects using short depth- of-field (DOF) optics.
  • DOF depth- of-field
  • Such short DOF optical systems are routinely used in microscopes and other inspection systems, at least partially because a short DOF can provide superior image quality and resolution within the limits of the DOF.
  • NA numerical aperture
  • DOF is inversely proportional to the NA; accordingly, a high NA (i.e., high resolution) necessarily generates a narrow DOF which, in turn, increases blurring in the final image since portions of the imaged object extend above and below the plane of focus, or image plane.
  • Table 1 DOF examples fat 540 nm wavelength) for numerical apertures (NA from 0.10 to 1.40.
  • autofocusing methods may be employed in several ways.
  • autofocus techniques are typically used to collect images of two-dimensional (2D) objects mounted or disposed on non-planar surfaces.
  • autofocus is also commonly used to collect images of 3D objects; in fact, most objects of interest with respect to operation of typical image acquisition systems are 3D rather than simply 2D.
  • any 2D image of the object may be considered sufficient.
  • an image captured from a particular, predetermined, or otherwise specified focal plane is desired.
  • the 2D image obtained with an autofocus technique is ordinarily expected to contain valuable morphological and/or quantitative information about the object, which is usually 3D, as noted above.
  • each optical section generally contains information from neighboring sections (i.e., sections above and below the focal plane of a given section) due to the DOF range for a given NA, as illustrated in Table 1.
  • Images obtained from an autofocused system are typically analyzed with algorithms that are based on some form of intensity integration. For example, the pixel intensities within a particular region of interest (e.g., on a microscope slide or a microarray) can be summed or totaled to infer the quantity of a particular chemical within that region; additionally or alternatively, pixels can be counted to determine the total area of the region.
  • certain existing tests are designed to identify the simple presence or absence of a particular chemical; these tests generally rely upon an intensity integration and are configured to yield a binary result.
  • a system and method operative in accordance with some embodiments, for example, may integrate (or project) image data in the z direction as the data are acquired; significant savings in computational overhead may be achieved by projecting the image data prior to deconvolution. Lateral translation of the image plane during the scan in the z direction may provide additional efficiency, allowing large areas to be imaged in a single scan sequence.
  • an image acquisition system and method employing multi-axis integration may incorporate both optical axis integration (OAI) and time-delay integration (TDI) techniques.
  • OAI optical axis integration
  • TDI time-delay integration
  • a method of acquiring data comprises: scanning an object along an optical axis; simultaneously scanning the object along a lateral axis; acquiring image data of the object during the scanning and the simultaneously scanning; and integrating the image data concomitantly with the acquiring.
  • the scanning may comprise providing relative translation along the optical axis of the object and an image plane; similarly, the simultaneously scanning may comprise providing relative translation along the lateral axis of the object and an image plane.
  • the acquiring comprises utilizing a charge-coupled device.
  • the integrating comprises utilizing an image processor, and may further comprise deblurring the image data subsequent to the integrating, deconvolving the image data subsequent to the integrating, or both.
  • a method of acquiring data may further comprise calculating a two-dimensional projection of the object from a projection of the image data and a projection of an optical point-spread- function.
  • the scanning may further comprise selectively alternating a direction of the relative translation along the optical axis. Additionally, the simultaneously scanning may comprise synchronizing the relative translation along the lateral axis with a rate associated with the acquiring.
  • a method of acquiring image data of an object may comprise: performing an optical axis integration scan; simultaneously executing a time-delay integration scan sequence; and selectively repeating the performing and the executing.
  • the performing comprises acquiring image data of the object at an image plane positioned along an optical axis; the performing may further comprise providing relative translation along the optical axis of the object and the image plane; as noted briefly above, a direction of the relative translation may be selectively alternated.
  • the executing generally comprises providing relative translation along a lateral axis of the object and the image plane; in some implementations, the executing comprises synchronizing the relative translation along the lateral axis with a data acquisition rate associated with an imaging device.
  • the acquiring comprises utilizing a charge-coupled device; the performing further comprises integrating the image data concomitantly with the acquiring; the integrating comprises utilizing an image processor; or some combination thereof.
  • Image acquisition systems and methods in accordance with the present disclosure may comprise deblurring the image data subsequent to the integrating, deconvolving the image data subsequent to the integrating, or both.
  • a method may further comprise calculating a two-dimensional projection of the object from a projection of the image data and a projection of an optical point-spread-function.
  • FIG. 1 A is a simplified functional block diagram illustrating one embodiment of an image acquisition system operative in accordance with the present disclosure.
  • FIG. IB is a simplified functional block diagram illustrating a portion of the image acquisition system depicted in FIG. 1A.
  • FIGS. 2A-2C are simplified diagrams illustrating the general operation of embodiments of image acquisition and image data processing methods.
  • FIGS. 3A-3C are simplified flow diagrams illustrating the general operation of the methods depicted in FIGS. 2A-2C.
  • FIGS. 4 A and 4B are simplified flow diagrams respectively illustrating the general operation of embodiments of analog and digital optical axis integration.
  • FIG. 5 is an illustration of results of an optical axis integration method.
  • FIG. 6 is a simplified diagram illustrating the general operation of shifts and readout functionality for a full frame CCD camera.
  • FIG. 7 is a simplified diagram illustrating one embodiment of time-delay integration synchronized with parallel shifts.
  • FIGS. 8 A and 8B illustrate representations of images acquired using one embodiment of time-delay integration.
  • FIG. 9 is a simplified illustration of one embodiment of a CCD detector mask enabling acquisition of multi-spectral image data.
  • FIG. 10 is a simplified plot illustrating one embodiment of a multi-axis integration scan cycle.
  • FIG. 11 is a simplified side view of an object to be imaged.
  • FIGS. 12A-12C are simplified side view illustrations of an image plane traversing an object to be imaged using one digital multi-axis integration method.
  • FIG. 13 is an multi-axis integration summary.
  • image acquisition throughput often represents the rate-limiting factor in systems and methods of scanning high-content and high- throughput assays common in biomedical and other applications.
  • Image acquisition throughput can be especially problematic when an assay requires detection of fluorescent probes, for example, and when high lateral resolution (in the x and y dimensions) is required for high-content image analysis algorithms.
  • high numerical aperture (NA) lenses are generally used to maximize collection efficiency and to minimize exposure time.
  • DOF depth-of-field
  • high NA lenses have limited ability to view thick objects, and are unable to follow uneven substrates without refocus. Even in cases where the detected signal is strong or is otherwise easily acquired (such as transmitted visible light, for example) optical systems can still perform inadequately if the sample thickness is greater than can be imaged by the optical DOF; additional imaging difficulties can be introduced if the object to be imaged is not located in a plane orthogonal to the optical axis. These optical limitations often lead to the use of autofocus technology, or the need to acquire images at more than one focal plane.
  • optical axis integration techniques are more cost effective and generally provide improved performance in many scanning applications.
  • the scanning techniques set forth in detail below are very tolerant of objects having inconsistent or variable focal planes, for example, and may be used to image thick objects. Additionally, scans performed in accordance with the present disclosure may be faster than those implementing autofocus or optical sectioning procedures.
  • a system and method operative in accordance with the present disclosure employ optical axis integration (OAI) techniques as set forth in detail below.
  • OAI optical axis integration
  • the object may be scanned along the optical axis while a detector, computer, or other computational apparatus concomitantly integrates the acquired images or image data.
  • the resulting image is an integral (i.e., projection) of the image of the three-dimensional (3D) object along the optical axis. That is, an OAI image may generally be expressed as follows:
  • i' is the two-dimensional (2D) projection of a 3D image, i, along the optical axis (z direction).
  • the OAI image, i'(x,y) may be expressed as: (x,y) jTpsf(x -x',y -y,z)dz dx'dy' 6
  • Equation 6 shows that an OAI image, i'(x,y), may be expressed as the convolution of the integral of the object along the optical axis with the integral of the
  • Equation 6 is also illustrative of the relationship between the projection of the object, the projection of the image, and the projection of the
  • Equation 8 The best method of solving Equation 8 for o'(x,y) involves Fourier Transforms, and is a well known procedure. Applying a Fourier Transform to both sides of Equation 8 and applying the convolution theorem (see, e.g., Bracewell, 1986) results in the following relationship:
  • Equation 10 describes an efficient method of calculating a 2D projection of an object from a projection of the image and a projection of the optical PSF.
  • a single-step solution may work well with good quality images; for lower quality images, however, an iterative solution of Equation 10 may yield a more reliable result. See, e.g., the constrained iterative technique described by Agard et al. (David A. Agard and John W. Sedat, Nature, volume 302, 1984, pages 676 et seq.).
  • a system and method may implement, incorporate, or comprise OAI techniques in either of two forms: digital; or analog.
  • OAI for example, a series of images may be collected along the optical axis and then digitally summed to form i'(x,y). This summation may occur during or after the scan, i.e., it may not be necessary to save individual optical sections as discrete images or collections of image data.
  • i'(x,y) may be generated by scanning the object along the optical axis while the image data are accumulated within a charge-coupled device (CCD) or other detector.
  • CCD charge-coupled device
  • the integration may be performed in the CCD chip and generally may result in only a single image, i.e., a single image may represent the entire depth of the object in the z direction along the optical axis.
  • Analog OAI may have particular utility with respect to operations involving scanning microarrays, for example, with CCD cameras or other detectors.
  • a system and method employing analog OAI may eliminate or substantially reduce reliance upon sophisticated, time-consuming, and processor intensive autofocus procedures.
  • analog OAI may provide a number of advantages over digital OAI and autofocus, especially for automated scanners.
  • the analog OAI embodiments require substantially lower data collection and processor overhead; exhibit lower read noise; and exhibit lower photon noise for equivalent exposure times.
  • advantages of the analog OAI embodiments may include the following: faster scan times; lower total exposure requirements; minimization or elimination of problems related to determining an arbitrary plane of focus; and integration of the 3D object yields or allows full quantitation of the object, i.e., the information content of the OAI image is higher than that achieved with autofocus systems, and accordingly, fewer structures associated with the object of interest are missed.
  • advantages of digital OAI embodiments may include a potential for achieving substantially larger photon counts; accordingly, 3D images may be made available for advanced image analysis such as 3D deconvolution, volumetric measurements, and the like.
  • OAI images generally may benefit from the quantitative deblurring procedure; similarly, deconvolution performance may be improved because Equation 10 deals with images in 2D rather than 3D.
  • deconvolution performance may be improved because Equation 10 deals with images in 2D rather than 3D.
  • many forms of image analyses based upon images obtained from autofocused systems will work equally well (or better) with projected images.
  • a basic object detection operation may benefit from OAI image processing techniques; in that regard, it will be appreciated that images with minimal DOF (i.e., autofocus images) are less likely to contain a specific object of interest than the corresponding projection image.
  • analyses that use intensity integration may also benefit from application of OAI techniques, at least because the z dimension (i.e., along the optical axis) is already integrated into the OAI result.
  • assays that integrate intensities within 3D structures e.g., nucleus, cytoplasm, and endoplastic reticulum
  • 3D structures e.g., nucleus, cytoplasm, and endoplastic reticulum
  • FIG. 1 A is a simplified functional block diagram illustrating one embodiment of an image acquisition system operative in accordance with the present disclosure
  • FIG. IB is a simplified functional block diagram illustrating a portion of the image acquisition system depicted in FIG. 1A.
  • FIGS. 1 A and IB are provided by way of example only, and that the specific arrangement of components is susceptible of numerous modifications; the exemplary scale, orientation, and interrelationship of the various components may be altered in accordance with system requirements. Additionally, as will become apparent from examination of the following description, some or all of the functionality of some components depicted as discrete elements may be combined or incorporated into other components.
  • System 100 generally comprises a microscope 110 operably coupled to a precision movable stage 120 and to an image acquisition component 140; stage 120 may be configured and operative to support a microarray, microscope slide, or other similar structure (reference numeral 190) upon which a specimen or object 199 to be imaged is disposed.
  • microscope 110 may comprise, or be operative in conjunction with, an illumination source 111 for illuminating stage 120, slide 190, or both with light of a predetermined or selected frequency or spectral bandwidth; in that regard, illumination source 111 may provide light in the visible, infrared, or ultraviolet wavelengths.
  • illumination source 111 may be incorporated within housing 112 of microscope 110, i.e., on the opposite side of stage 120 and slide 190 than depicted in FIG. 1 A.
  • an additional source of illumination (not shown) to be used in conjunction with, or in lieu of, source 111 may be accommodated or maintained in housing 112.
  • any such illumination source disposed within housing 112 may be suitably dimensioned and positioned neither to interfere with optical components of microscope 110 nor to obstruct the optical path through microscope 110 (to image acquisition component 140).
  • stage 120 may be movable relative to optics (e.g., objective
  • stage 120 may be movable in both the x and y directions (where the y axis is normal to the plane of FIGS. 1A and IB).
  • both the x axis and the y axis may generally be referred to herein as "lateral" axes, and may describe a plane orthogonal to the optical axis (described below) of system 100.
  • stage 120 may incorporate or comprise one or more structures and mechanisms configured and operative precisely to position slide 190 laterally in the x and y directions relative to the structure of stage 120 itself.
  • precise 2D lateral positioning i.e., x and y coordinates
  • precise 2D lateral positioning i.e., x and y coordinates
  • object 199 relative to the optical path of microscope 110 may be achieved through movement of stage 120 relative to microscope optics, movement of slide 190 relative to stage 120, or both.
  • stage 120 may also be movable along the z axis (the optical axis).
  • microscope optics may also facilitate positioning an object on slide 190 in the proper location in 3D space (i.e., x, y, and z coordinates) relative to the optical path and the focal point of objective 119.
  • one or more optical components of microscope 110 such as objective 119 may be movable in the z direction, either in addition to, or as an alternative to, selectively moving stage 120 along the optical axis.
  • objective 119 may be movable along the x axis, the y axis, or both.
  • Microscope optics may generally be configured and operative in conjunction with image acquisition component 140; in that regard, component 140 generally comprises a camera, charge-coupled device (CCD), or other detector 141 operably coupled to an image processor 142 or other appropriate electronics.
  • System 100 may additionally include control electronics 150 operative to control, for example: operational parameters, functional characteristics, or other configurable aspects of image processor 142 and detector 141; two- or three-dimensional motion of stage 120, objective 119, or other components; power output, spectral bandwidth, frequencies, or other parameters for source 111 and any other illumination source incorporated into system 100; data storage; and the like.
  • electronics 150 may comprise one or more microprocessors, microcontrollers, or other programmable devices capable of executing computer readable instructions; additionally, electronics 150 may also comprise or be operably coupled with data storage media or networked devices such as file servers, application servers, and the like.
  • data storage media or networked devices such as file servers, application servers, and the like.
  • image data acquired by detector 141 may be summed, manipulated, saved, or otherwise processed by hardware, software, or both resident at image processor 142; in some embodiments, functionality of processor 142 may be influenced or controlled by signals transmitted from electronics 150 as noted above. Alternatively, the functionality of image processor 142 and electronics 150 may be incorporated into a single device, for example. Specifically, image processor 142 may be operative in accordance with instruction sets to compute solutions or approximations for the equations set forth herein.
  • FIGS. 2A-2C are simplified diagrams illustrating the general operation of embodiments of image acquisition and image data processing methods
  • FIGS. 3A-3C are simplified flow diagrams illustrating the general operation of the methods depicted in FIGS. 2A-2C.
  • FIGS. 2 A and 3 A generally illustrate one conventional approach to image processing operations.
  • a series, or stack, of 2D images is acquired in sequential x,y planes (i.e., optical sections) along the z axis.
  • the resulting image, i(x,y,z) is expressed mathematically at Equation 2 above, which is computationally expensive to solve.
  • the deconvolution operation depicted at block 312 is executed with respect to the entire stack of optical sections, and is accordingly inefficient and processor-intensive; since each optical section includes data from other sections (due to DOF range, for example), the deconvolution operation processes more data than required.
  • the deconvolved 3D image is projected into 2D image, o'(x,y), as indicated at block 313.
  • FIGS. 2B and 3B generally illustrate a significantly improved approach to image processing operations as contemplated herein.
  • a series, or stack, of 2D images may be acquired in sequential x,y planes (i.e., optical sections) along the z axis (block 321).
  • the resulting image, i(x,y,z) is expressed mathematically at Equation 2 above, which is computationally expensive to solve.
  • the stack of optical sections may be projected into 2D image, i'(x,y), prior to deconvolution; this image is expressed mathematically at Equation 8 above, which is a substantially simplified, 2D version of Equation 2.
  • FIGS. 2C and 3C illustrate one embodiment of an image acquisition and processing methodology employing OAI techniques for efficient production of 2D images of 3D objects.
  • an OAI image may be captured directly from the object (block 331), for example, by integrating x,y image data across a selected or predetermined range along the optical axis in the z direction; in that regard, relative translation of the object and the image plane may be provided during the OAI scan as set forth above.
  • the OAI image, i'(x,y) like the projected image illustrated in FIG. 2B, is expressed at Equation 8 above.
  • the OAI image may be generated as the image data are obtained; the integration in this instance may be said to occur concomitantly or substantially simultaneously with the data acquisition. Accordingly, image data for a stack of discrete optical sections need not be acquired; this results in a significant reduction in data processing overhead. Efficient deconvolution performance at block 332 may be achieved in part because the OAI image is expressed in 2D rather than in 3D.
  • FIGS. 4 A and 4B are simplified flow diagrams respectively illustrating the general operation of embodiments of analog and digital optical axis integration. It will be appreciated that the exemplary embodiments illustrated in FIGS. 4 A and 4B may generally correspond to the integration operation depicted at block 331 and described above with reference to FIG. 3C; the illustrated OAI techniques may enable generation of an OAI image, i'(x,y), such as expressed at Equation 8.
  • a target object to be imaged may be identified and positioned (blocks 411, 421) relative to microscope optics substantially as set forth above; with respect to positioning at blocks 411 and 421, only the x and y coordinates of the object may be considered, i.e., the object may be accurately aligned with the optical path.
  • the operations depicted at blocks 412 and 422 are intended to represent any type of initiation or enablement of data capture or data acquisition; specifically, such initiation is not intended to be limited to physical movement or manipulation of a camera shutter, for example, nor to imply other structural limitations.
  • the functionality represented at blocks 412 and 422 may include, ter alia, providing power to a CCD, for instance.
  • the operations depicted at blocks 415 and 424 are not intended to imply structural limitations, and generally encompass numerous techniques for disabling data capture functionality, for example, such as by closing a camera shutter.
  • the object may be moved along the optical axis through a predetermined or specified range (i.e., distance in the z direction) as indicated at block 413.
  • a predetermined or specified range i.e., distance in the z direction
  • the movement represented at block 413 may generally be measured relative to microscope optics or an image plane, for example, as set forth in detail above. Specifically: the object itself may remain stationary while the optics are moved; the optics may remain stationary while the object is moved; or some combination of system components may be moved in cooperation.
  • an analog method of OAI may comprise obtaining image data (block 414) continuously during the z axis scan (i.e., relative translation of the object and the image plane along the optical axis).
  • the OAI image may be generated by scanning the object along the optical axis while the image data are accumulated within a CCD or other detector 141.
  • the integration or projection may be performed during (concomitantly or substantially simultaneously with) data acquisition, and the image data written to memory at block 416 generally may represent a single image, i'(x,y), such as depicted in the center of FIG. 2C.
  • the method of FIG. 4A therefore, may result in or facilitate generation of a single 2D image representing the entire depth of the object in the z direction.
  • the method of FIG. 4A may generally correspond to the operation depicted at block 331.
  • the digital OAI embodiment of FIG. 4B may obtain image data (block 423) in discrete steps.
  • acquired image data generally represent a plurality of 2D (x,y) sections; image data representing discrete optical sections are written to memory (block 425) and summed or otherwise manipulated (block 426) as acquired at each z position.
  • the integration or projection may occur as image data for each optical section are captured, i.e., concomitantly with the data acquisition. Accordingly, generation of a 3D image, i(x,y,z), such as illustrated at the left side of FIGS. 2 A and 2B, for example, may not be necessary.
  • the method may progress to the next optical section in sequence, moving the object, the microscope optics, or both, so as to position the focal plane at the next sequential position along the z axis of the object; this event is represented at block 427.
  • the method may end (block 428), resulting in or allowing generation of a single, two-dimensional OAI image, i'(x,y), representing the entire depth of the object in the z direction.
  • the method of FIG. 4B may generally correspond to the operation depicted at block 331.
  • FIG. 5 is an illustration of results of an optical axis integration method. It will be appreciated that the OAI images illustrated in FIG. 5 may be enabled by the procedures set forth and described above with reference to FIGS. 4 A and 4B. Time-Delay Integration
  • time-delay integration generally represents a method of continuous scanning which may be implemented in conjunction with CCD cameras or other imaging devices.
  • CCD cameras for example, incident light is creates electric charge at individual charge-coupled wells on the device surface. Charged electrons are then transferred sequentially down the columns of the chip (parallel shifts) while the row that reaches the bottom of the chip is transferred to an accumulator called the serial register. The serial register is then shifted horizontally and processed by an A/D converter.
  • precision motion control may be employed to synchronize motion of the object being imaged or motion of the camera or other imaging device (as set forth above with reference to FIG. 1A) with motion of the charged electrons across the CCD or imaging device surface.
  • Relative translation of the object and the image plane along the lateral axis may be controlled such that a particular portion of the imaged object tracks down the chip as the electrons representing that particular portion of the image are shifted down the chip.
  • such relative translation may comprise motion of the object, motion of the image plane, or both. Accordingly, the object may be continuously imaged as it passes down the chip.
  • TDI methodologies may facilitate or enable efficient scanning of fluorescent DNA microarrays, for example, and may have utility in various other applications related to scanning myriad biological specimens.
  • FIG. 6 is a simplified diagram illustrating the general operation of shifts and readout functionality for a full frame CCD camera.
  • FIG. 6 provides a simple demonstration of slow-scan CCD camera read operations. Individual pixel electrons are shifted in parallel (e.g., down the columns to successive rows) to a predetermined portion of the chip (e.g., the bottom of the chip in FIG. 6). Image data at the bottom row are shifted off of the chip onto the serial register, which is, in turn, shifted horizontally to the readout amplifier to create a voltage that is digitized to form a digital image.
  • FIG. 6 embodiment is provided for illustrative purposes only, and that various CCD cameras or other imaging devices may be characterized by alternative operational features, particularly with respect to the exemplary geometry.
  • the operation of some CCD cameras may execute parallel shifts oriented at 90 or 180 degrees from those depicted in FIG. 6.
  • FIG. 7 is a simplified diagram illustrating one embodiment of time-delay integration synchronized with parallel shifts.
  • FIG. 7 illustrates a precise motion control TDI implementation which may be employed in fluorescence imaging systems, for example, or in numerous other imaging applications.
  • a given location on the specimen (“object" in FIG. 7) may move (either relative to the CCD chip, for example, or relative to the image plane of the system) in synchrony with the parallel shifts such that NP (i.e., the parallel shift velocity) is equal to VY (i.e., the image shift velocity).
  • NP i.e., the parallel shift velocity
  • VY i.e., the image shift velocity
  • synchronous motion between the object and the CCD row may be effectuated substantially as set forth in detail above with reference to FIG. 1A.
  • Relative motion of slide 190, stage 120, various optical components, or some combination thereof, for instance may accurately position the object depicted in FIG. 7 in a suitable location for imaging during the scan.
  • the degree of synchronicity, the velocities imparted to the mechanical components, the precision with which the object must be positioned for a desired imaging quality, mechanical settling time, backlash, and the like may vary in accordance with system configuration, and may be application dependent.
  • FIGS. 6 and 7 Upon examination of FIGS. 6 and 7, it will be readily apparent that relative translation of the object and the image plane along the lateral axis as set forth above may be synchronized with the rate at which data are acquired and read from the detector. Accordingly, it is noted that the capabilities of the CCD camera or other imaging device (such as detector 141) and other components of the optical system (such as, ter alia: the maximum resolution and the time required to focus the imaging device; and NA and DOF of the optics) may affect the behavior and performance of the system employing the FIG. 7 TDI embodiment.
  • FIGS. 8 A and 8B illustrate representations of images acquired using one embodiment of time-delay integration. In the TDI system used to generate the exemplary images, the period of time for reading the entire CCD chip is approximately 0.4 seconds.
  • FIG. 8 A is a representation of an image of a dirty blank slide scanned using a TDI technique.
  • Nertical bands indicate the locations of individual TDI "strips," or portions of the imaged area.
  • the sample, the imaging device, or both may then be moved horizontally and a new TDI strip may then be acquired. Multiple strips may then be assembled to create the final image.
  • FIG. 8B depicts a comparison of a region of a fluorescent microarray sample imaged first with individual panels and then with one embodiment of a TDI method. It is noted that the individual panel borders are visible both horizontally and vertically in the "standard scan" image on the left side of FIG. 8B, while only the vertical bands are visible in the TDI scan on the right. Both of these images were acquired without calibrating the illumination and collection efficiencies across the field (i. e. , flat-field calibration).
  • TDI may be employed to image objects in applications involving limited or minimal signal intensity.
  • the motion control characteristics of TDI allow for longer exposure per picture element (pixel) for a given total image collection time, facilitating increased imaging quality even in instances where signal intensity ordinarily would be a deleterious factor.
  • signal intensity may be limited or otherwise impeded either by the nature of the sample, by the illumination intensity, by the physical characteristics or operational parameters of the indicators used in conjunction with the sample, or by some combination of the foregoing.
  • one application in which the above-mentioned factors are especially problematic is in the imaging of fluorescently labeled biological specimens.
  • TDI methodologies may be used in conjunction with known fluorescence imaging technology to minimize the attendant effects of weak signal intensities.
  • some TDI systems and methods may employ a single, wide detector; in that regard, a broad detection apparatus may be used to collect a single "strip" that represents the final image.
  • detector 141 a CCD camera, or other imaging apparatus accommodates a sufficiently broad imaging surface, for example, the multiple strips illustrated in FIGS. 8 A and 8B may be acquired in a single scan.
  • one of the difficulties associated with scanning biological specimens, especially for fluorescence characteristics is generally due to the fact that a finite limit exists with respect to the intensity of light emanating from an illuminated sample.
  • adjusting the scan rate i.e., the relative movement of the sample across the CCD imaging surface
  • the readout speed of the imaging device enables a system and method of TDI imaging to control the exposure time for each pixel in the acquired image.
  • TDI embodiments may incorporate an ability to address multiple wavelength data.
  • data spanning multiple wavelengths may be acquired in at least two different ways: sequential scanning; and simultaneous scanning.
  • sequential scanning a single, monochromatic detector may be employed; in this embodiment, multiple wavelengths may be selected through filters, for instance, or through other wavelength selection components or methods.
  • an instrument or system operative in accordance with the present disclosure may scan the sample (or a strip of the sample) through a selected or predetermined filter to acquire image data at a desired wavelength, change the filter (using a filter wheel, for example, or similar apparatus), and scan the same sample (or strip of sample) through the new filter to acquire additional image data at a different desired wavelength.
  • the foregoing operations may be selectively repeated in accordance with system requirements to acquire a plurality of scan images at a desired number of selected wavelengths. In this manner, a final image may be constructed of the data acquired during the plurality of sequential scans.
  • a TDI system and method may measure and control the actual velocities and positions of the sample during each of the plurality of scans; precise control of scan speeds, and accurate measurements thereof, may prevent or allow for correction of chromatic shift in the portions of the image that are derived from the component scans. Accordingly, systems and methods of TDI as described herein may selectively synchronize the movements as set forth above responsive to such different conditions.
  • multi-spectral image data may be acquired from a single sample using multiple detectors, for example, each with its own spectral response. In this manner, a multiple wavelength image may be collected in a single scan.
  • a multi-spectral scan may be accomplished with a single detector or imaging device equipped with a specially designed color mask.
  • FIG. 9 is a simplified illustration of one embodiment of a CCD detector mask enabling acquisition of multi-spectral image data.
  • the mask may be arranged in alternating columns of red (R), green (G), and blue (B), for example, as depicted in FIG. 9. Each of these respective columns in the mask may facilitate the respective acquisition of red, green, and blue spectral image data.
  • the alternating pattern may be oriented in such a way as to be parallel to the scan direction of the imaging device, i.e., where the scan occurs in rows, or 90 degrees from the scan direction illustrated in FIG. 7, the mask may be arranged to accommodate the operational characteristics of the imaging device.
  • a single, multi-wavelength, simultaneous scan may be implemented in conjunction with a mask such as illustrated in FIG. 9.
  • positioning methodologies in various TDI embodiments may employ constant relative velocity of the object to be imaged and the imaging surface of the imaging device. As set forth above, constant relative positioning of the object to be imaged may be accomplished through precise motion of the slide, the stage, the optics, or some combination thereof.
  • TDI methods employing one or more incremental positioning strategies; in that regard, relative motion of the object to be imaged may be synchronized to the readout (or output) capabilities of the CCD camera or other imaging device. In this manner, long exposures may be accommodated.
  • the velocity of the object may become so slow as to become non-constant, depending upon the output bandwidth and readout rate of the imaging device.
  • the sample or object may be translated (relatively) a distance corresponding to one camera row, maintained at that location for the duration of an exposure, and subsequently translated a distance corresponding to the next camera row.
  • the foregoing procedures may be selectively repeated in accordance with system requirements or as desired.
  • relative movement of the object, the slide, the stage, the optics, or some combination thereof may begin and cease at each row shift in the camera or imaging device.
  • the high degree of synchronization provided by such an approach may yield excellent resolution and registration between wavelengths.
  • Rotational and positional errors in precision motion stage systems may be measured and corrected, for example, using optically determined coordinate calibration or other methods. In particular, such errors may be minimized dynamically through precise modification of stage movements. Specifically, such coordinate calibration may also be used in conjunction with TDI techniques dynamically to correct rotational and positioning errors during a given TDI scan.
  • a system and method employing multi-axis integration may incorporate both OAI and TDI techniques.
  • TDI is described in co-pending United States nonprovisional application Serial No. 10/215,265, filed August 6, 2002, entitled “TIME-DELAY INTEGRATION IMAGING OF BIOLOGICAL SPECIMENS.”
  • TDI methodologies as set forth in detail above enable a system and method of image data processing simultaneously to collect image data over a given lateral area and to read data out of the camera or detector. That is, data are acquired and read from the detector simultaneously.
  • a system implementing TDI methods may efficiently acquire image data over a large area (relative to the focal plane of the detector) with substantially reduced overhead as compared to systems which do not implement TDI.
  • an OAI image may be generated through integration of data acquired during a z translation (i.e., parallel with the optical axis as illustrated in FIG. IB); image data may be read from the CCD camera or other detector 141 before another integration begins.
  • the readout may require up to one second, during which time many aspects of the scan must stop.
  • This inefficiency due to downtime may be avoided if the z translation is coordinated with the lateral translation (i.e., motion in the x and y directions, orthogonal to the optical axis) attendant with the TDI scan as noted above. That is, data gathered by detector 141 may be read continuously during the z translation (resulting in an integration of intensities along z) and simultaneously with lateral movement (such as along the y axis) which presents new areas of the sample to detector 141; in some embodiments synchronizing such lateral movement with the readout capabilities of the CCD camera or other imaging device (i.e., detector 141), additional computational efficiency may be achieved.
  • the lateral translation i.e., motion in the x and y directions, orthogonal to the optical axis
  • the camera or detector 141 generally operates to acquire image data in cooperation with continuous and simultaneous z- and y-motion.
  • a system and method incorporating MAI techniques may generally acquire a plurality of OAI images, each of which may include data captured from a different lateral location in x-y space on slide 190 or stage 120.
  • the lateral scan may employ a raster or serpentine pattern, for example; in some embodiments, a serpentine lateral scan pattern may enable an MAI system and method to operate at optimum efficiency.
  • z-axis motion may be suitably controlled to assure an appropriate scan profile; accordingly, the resulting OAI image may not be biased with information from any particular z location in the sample or the object to be imaged.
  • the period of the scan profile may be computed as a multiple of the time required to read a whole frame of data from detector 141 as set forth above in detail with reference to FIGS. 6 and 7.
  • FIG. 10 is a simplified plot illustrating one embodiment of a multi-axis integration scan cycle.
  • the z position of the image plane along the optical axis is represented on the ordinate axis
  • the y position of the optical axis relative to the stage 120 or slide 190 upon which the object to be imaged is disposed is represented on the abscissa. It will be appreciated that similar results may be obtained by providing lateral movement along the x axis, either in addition to, or as an alternative to, motion along the y axis.
  • FIG. 10 is a simplified plot illustrating one embodiment of a multi-axis integration scan cycle.
  • the z position of the image plane along the optical axis is represented on the ordinate axis
  • the y position of the optical axis relative to the stage 120 or slide 190 upon which the object to be imaged is disposed is represented on the abscissa. It will be appreciated that similar results may be obtained by providing lateral movement along the x axis, either in addition to
  • the z location of the image plane may be moved continuously along the optical axis between a minimum and maximum position (i.e., in and out of focus).
  • the velocity of this z translation may be determined or calculated as a function of the required scan distance (i.e., the distance between the minimum and maximum z locations of the image plane), as well as a desired scan time period. Acceleration may be made as large as possible such that, for example, the position-versus-time curve of the image plane may have a triangular wave pattern.
  • a system operating in accordance with the analog MAI scan pattern illustrated in FIG. 10, for example, may facilitate uniform exposure at each z location within the scan.
  • the y axis may be simultaneously scanned at constant velocity such that the image traverses the entire detection surface of detector 141 while the z translation completes a cycle. Accordingly, each row of detector 141 may be exposed to each z position four times as indicated by the points marked __ in FIG. 10, i.e., every z position may be scanned four times during a single MAI cycle. In such an embodiment, the exact starting location or position (in the z dimension) of the object to be imaged may be irrelevant with respect to the quality and the efficiency of the scan, since the object will cross each z position four times during a particular MAI cycle.
  • Digital MAI In accordance with digital MAI methodologies, the y and z axis translations may be executed incrementally while rows of image data are read from the detector.
  • the row height is the binned CCD row height.
  • n z may also be used for certain scanning techniques.
  • various of the foregoing equations are susceptible of modification in accordance with system requirements, for example, or in accordance with individual limitations of system components.
  • operational characteristics of one or more stepper motors or servos implemented in stage or optics component motion control may dictate the values for n z or n y in some instances.
  • some circumstances may require or benefit from dynamic computation or adjustment of n z and n y ; one such circumstance may include, for example, acquisition of image data at a plurality of wavelengths during multi-spectral scans.
  • motion control of various system components may be provided or accommodated by electronics 150 as set forth above with reference to FIG. 1.
  • Dynamic configuration of MAI scan motion parameters may be readily implemented by one or more microprocessors or microcontrollers associated with, incorporated into, or operably coupled to electronics 150.
  • FIG. 11 is a simplified side view of an object to be imaged
  • FIGS. 12A- 12C are simplified side view illustrations of an image plane traversing an object to be imaged using one digital multi-axis integration method.
  • the z and y directions are indicated by the coordinate axes in FIG. 11.
  • the 3D object to be imaged is represented by the 2D grid and designated by rows 1-4 in the z dimension and columns a-d in the y dimension; it will be appreciated that the object to be imaged may extend in the x direction, i.e., normal to the plane of FIG. 11, as well.
  • a smaller representation of the FIG. 11 object is reproduced in FIGS.
  • FIGS. 12A-12C a side view of the image plane is represented by the darkened grid squares, and the arrows indicate the direction of image plane motion relative to the object to be imaged; as with the object depicted in the figures, it will be appreciated that the image plane generally extends in the x direction, i.e., normal to the plane of FIGS. 12A-12C.
  • the arrows depicted in the drawing figures are not vectors; in that regard, the arrows are only intended to indicate the direction of relative motion for the image plane from one scan position to the next, and are not intended to convey information regarding the velocity of such motion.
  • the shading in the representation of the object to be imaged is provided to indicate the number of times a particular location of the object has been imaged by the image plane.
  • the partially exploded view i.e., the separation between the object and the image plane) is provided for clarity.
  • the image plane at scan position 2 in FIG. 12 A may be acquiring data at portions a2 and b2 of the object.
  • the locations (on the object) of data acquired at earlier or previous scanning positions in the sequence are indicated above the image plane in FIGS. 12A-12C.
  • these data depicted above the image plane may have been read from the imaging device (such as detector 141 in FIG. 1 A, for example) during the synchronous z and y motion.
  • the image plane may reverse direction; accordingly, z translation may be omitted at this point in the sequence.
  • the image plane may remain at the same z location for scanning operations which occur at scan positions 4 and 5. While the image data acquired at position 5 will be at the same z location as those acquired at scan position 4, the image plane has continued traversing in the y direction relative to its y location at scan position 4.
  • each z position is traversed by the image plane three times during the scan sequence; in conjunction with relative motion in the y direction, the image plane may acquire image data from each location al-d4 on the object twice.
  • image data are acquired for location al when the image plane is at scan positions 1 and 8; similarly, image data are acquired for location a2 when the image plane is at scan positions 2 and 7; and so forth.
  • n z integral multiples of n z may be employed in certain embodiments effectively to increase the z scan frequency.
  • Alternative computations may also be used to calculate n z as a function of n y , additional factors, or some combination thereof in some embodiments.
  • digital MAI scans techniques may employ a variety of alternative z scan frequencies depending upon system requirements, component capabilities, multi-wavelength scan strategies, and so forth.
  • Motion parameters such as lateral velocity and z scan frequency may be dynamically adjusted or selectively altered, for example, by control electronics 150 or similar programmable electronic components. In some embodiments, for example, electronics 150 may selectively adjust motion control parameters in accordance with feedback, data, or other instructions transmitted by image processor 142.
  • image plane dimensions may affect the total amount of data acquired during an MAI scan, in general, and when the image plane is at specific scan positions, in particular.
  • image data for additional object locations may be acquired, for instance, when the image plane is at scan positions 1-3 and 9-12.
  • FIG. 13 is an MAI summary.
  • the FIG. 13 summary represents the image data acquired during the digital MAI scan described above with reference to FIGS. 12A-12C.
  • One or more data processing hardware or software components for example, integrated with image processor 142 or associated with electronics 150 depicted in FIG. 1A may organize or otherwise manipulate image data acquired during the MAI scan.

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Abstract

Ce système, et le procédé afférent, permettant une acquisition d'image par intégration à plusieurs axes (MAI), peut faire intervenir à la fois des techniques d'intégration optique (OAI) et d'intégration de retard temporel (TDI). Ces systèmes MAI, et les procédés afférents, permettent d'intégrer des données d'image dans le sens Z, une fois les données acquises, et de projeter ces données d'image avant déconvolution. Une translation latérale du plan d'image lors du balayage dans le sens Z permet d'imager de grandes zones en une seule séquence de balayage.
EP03716656A 2002-03-13 2003-03-13 Systeme d'integration a plusieurs axes (mai) et procede afferent Withdrawn EP1520250A4 (fr)

Applications Claiming Priority (7)

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US36476202P 2002-03-13 2002-03-13
US364762P 2002-03-13
US10/215,265 US20030048933A1 (en) 2001-08-08 2002-08-06 Time-delay integration imaging of biological specimen
US215265 2002-08-06
US43169202P 2002-12-06 2002-12-06
US431692P 2002-12-06
PCT/US2003/008182 WO2003079664A2 (fr) 2002-03-13 2003-03-13 Systeme d'integration a plusieurs axes (mai) et procede afferent

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US7283253B2 (en) * 2002-03-13 2007-10-16 Applied Precision, Llc Multi-axis integration system and method
WO2004113922A2 (fr) * 2003-06-19 2004-12-29 Applied Precision, Llc Systeme et procede mettant en oeuvre des techniques photocinetiques dans des applications d'imagerie de biologie cellulaire
JP5497386B2 (ja) * 2009-09-11 2014-05-21 浜松ホトニクス株式会社 画像取得装置

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WO2003079664A2 (fr) 2003-09-25
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EP1520250A4 (fr) 2009-07-01

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