Image analysis – Image transformation or preprocessing – Changing the image coordinates
Reexamination Certificate
2002-04-24
2004-07-13
Johns, Andrew W. (Department: 2621)
Image analysis
Image transformation or preprocessing
Changing the image coordinates
C382S300000, C382S218000
Reexamination Certificate
active
06763149
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to a method and apparatus for improving the accuracy of quantitative images generated by multichannel imaging instruments, and more specifically, to correcting errors introduced by crosstalk between channels, with application to a broad range of imaging instruments and particularly, to flow imaging instruments using time-delay-integration image detectors.
BACKGROUND OF THE INVENTION
The parallel advancement of the technology of video microscopy and techniques for preparing and staining biological samples has enabled those working in areas such as fundamental biological science, diagnostic medicine, and drug discovery to gather an ever-increasing amount of information from a single biological specimen. In the fields of cell biology and clinical cytology, for example, specimens may be stained with absorption dyes to define cell morphology, and with fluorescent dyes that attach to molecules bound to specific proteins or nucleic acid chains. Microscopes equipped for exciting and imaging the fluorescent dyes and concurrently imaging cell structures are routinely used for studying complex processes that modify cells on the gross structural level and also at the molecular level. In recent years, computational analysis of images captured from multiparameter microscopes has shown promise for automating large investigative studies such as those conducted by drug discovery and development companies and for automating complex cellular diagnostic tests for clinical medicine. Optimal use of such technology can be attained only if the signals used for image generation are accurately scaled to information about the cells being studied. However, such information can be degraded during the capture process. Specifically, interference can be introduced into a channel dedicated to a first signal due to leakage of a signal intended for a second channel. This type of signal degradation is generally referred to as channel-to-channel crosstalk.
An advancement to computer-based multiparametric imaging is disclosed in commonly assigned U.S. Patents, both entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS, U.S. Pat. No. 6,249,341, issued Jun. 19, 2001 (filed Jan. 24, 2000), and U.S. Pat. No. 6,211,955, issued Apr. 3, 2001 (filed Mar. 29, 2000), the complete disclosure, specification, and drawings of both of which are hereby specifically incorporated herein by reference. The technology disclosed in these applications extends the methods of computer vision to the analysis of objects either flowing in a fluid stream or moving relative to the imaging instrument on a rigid substrate, such as a glass slide. Instruments based on the inventions of the patent applications cited above deliver improved sensitivity at high spatial resolution through the use of time-delay-integration (TDI) electronic image acquisition, a method wherein signal integration is accomplished by shifting charge packets through an imaging array in synchrony with the motion of the target object being imaged.
The TDI-based flow imaging technology, with its ability to substantially improve signal-to-noise ratio, is of exceptional utility for multiparametric imaging. Each of the channels of a TDI flow imaging instrument can be dedicated to a single light source in the target objects. One such light source, for example, is the fluorescent dye attached to a molecule selected for its specificity for binding to a target protein. Each of a plurality of channels can be dedicated to a particular different dye, and all of the dyes addressed by the instrument may be present in close proximity on a single target cell. Because the dyes may have emission spectra broader than the passbands of the channels that collect their signals, channel-to-channel crosstalk can prevent the accurate estimation of the intensity of the signal from each dye.
Accordingly, it would clearly be desirable to develop a method and apparatus that simultaneously offers speed and accuracy in eliminating such channel-to-channel crosstalk. Preferably such crosstalk reduction can be achieved in conjunction with the TDI-based flow imaging method and apparatus noted above, which are intended for real time collection and processing of images from objects moving in high concentration, at high speed, through the instrument. Accordingly, the crosstalk reduction of the present invention is preferably applicable in real time and in synchrony with the collection of images of the moving targets that include indicators attached to the targets.
SUMMARY OF THE INVENTION
The present invention is directed to enabling an accurate reconstruction of information about objects imaged by an instrument using multiple channels, each channel being generally optimized to receive signals of a type differentiated from other signal types by predefined characteristics. These predefined characteristics may include, but are not limited to wavelength, a modulation of a signal received from a source, a scatter angle, a Doppler shift, and a phase shift (e.g., with respect to a reference phase). The present invention applies to, but is not limited to, instruments for collecting information from electromagnetic waves in all portions of the spectrum, by acoustic waves, by particle flux, and by measurement of object characteristics such as conductivity, chemical reactivity, size, shape, and mass.
One example of an application of the present invention is its use in a multiple-wavelength optical imaging instrument. In such an instrument, each channel is made sensitive to electromagnetic radiation of wavelengths bounded by an upper and lower limit, defining different wavebands for each channel. Typically these limits are determined by the characteristics of one or more filters disposed in a path between a light source and a photodetector servicing a channel. The images in each channel are detected, producing signals that are processed by the present invention to correct errors in alignment between the channels and a reference and then, to correct for crosstalk between the channels.
Thus, the present invention is directed to a method and apparatus that not only corrects for crosstalk between channels, but also ensures that signal data in each channel is properly aligned with signal data in other channels, so that the benefit from the crosstalk correction is not degraded by signal misalignment.
In one preferred embodiment, a method is provided for correcting signal misalignment between individual channels in a multichannel imaging system, such that data in a first channel is substantially aligned with data in other channels. The method also includes the step of reducing erroneous contributions to signal data from a source intended to provide signal data for other channels.
Preferably, the signal data are used to produce an image for display. Accordingly, a preferred embodiment is directed to a method that includes the step of spatially aligning images input in an image ensemble from a plurality of channels, such that each image in the image ensemble is substantially aligned with other images in the image ensemble, and the step of applying spectral crosstalk corrections, to remove the channel-to-channel crosstalk from the image ensemble output.
In one embodiment, the step of spatially aligning images includes the step of utilizing two classes of information, including a first and second class of constants. The first class of constants includes horizontal and vertical spatial offsets, which are derived from an on-line calibration image. The second class of constants is accessed during the step of spatially aligning images, but is not modified. Preferably the second class of constants includes at least one of channel start columns for each image, and inverted source coefficients.
The horizontal and vertical spatial offsets are preferably generated based upon a comparison of each image in an image ensemble with a calibration image. The comparison with a calibration image can be performed when a system for generating the multichannel signal i
Frost Keith L.
Riley James K.
Alavi Amir
Amnis Corporation
Anderson Ronald M.
Johns Andrew W.
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