Amnis Corporation

Neopath, Inc.

Tripath Imaging, Inc.

James K Riley

Keith Frost

William E Ortyn

Keith L Frost

Tuan H Phan

David Abraham Basiji

Kim J Hansen

David Joseph Perry

James A Stephanick

William C Lindow

Richard Bauer

Seho Oh

Michael G Meyer

Chih-Chau L Kuan

Andrew David Silber

Michael J Seo

Yuhui Cheng

Wayne A Biggs

A multi-channel imaging system generates an ensemble of images for each field of view of an object. Each image in the ensemble is intended to contain information from only one source among a plurality of sources for the object. However, due to crosstalk, at least a portion of the signal from a first source appears in a channel intended for a second source. Because the accuracy of the correction will be degraded if the images in an ensemble are spatially misaligned with respect to one another, the spatial offset between images is determined and a correction is applied to substantially eliminate the offset. Then, a correction to the signals is determined to substantially reduce the contributions to the signal in a channel from the signals in other channels. The signal processing can be employed to process the output signals for each of a plurality of different disclosed imaging systems.

Method and apparatus for correcting crosstalk and spatial resolution for multichannel imaging

In automated computation-based interpretation of images, the accuracy and reliability of the detection and delineation of objects, known as 'object segmentation,' is implemented so as to provide efficient performance. In a multi-step process, objects are first detected and captured into regions of interest (ROIs). Sets of pixels belonging to respective objects are then identified. Preferably object detection is achieved using both a two-dimensional (2D) low pass filter and a 2D edge enhancement filter. Two different gradient based edge enhancement filters are disclosed. One embodiment of the invention defines a (ROI) by first determining the center of objects by executing a plurality of decimations on the filtered image data, and then establishing object boundaries. In a second embodiment the ROI is defined by generating an amplitude histogram of the filtered image data, and for histograms exceeding a threshold determining by pixel which rows are to be included in the ROI.

Computational methods for the segmentation of images of objects from background in a flow imaging instrument

A multichannel imaging system generates an ensemble of images for each field of view of an object. Each image in the ensemble is intended to contain information from only one source among a plurality of sources for the object. However, due to crosstalk, at least a portion of the signal from a first source appears in a channel intended for a second source. Because the accuracy of the correction will be degraded if the images in an ensemble are spatially misaligned with respect to one another, the spatial offset between images is determined and a correction is applied to substantially eliminate the offset. Then, a correction to the signals is determined to substantially reduce the contributions to the signal in a channel from the signals in other channels. The signal processing can be employed to process the output signals for each of a plurality of different disclosed imaging systems.

In an instrument generating images from the fluorescent emissions of a plurality of fluorescent dyes carried by objects in a flow stream, spectral dispersion is used to expand the images of the objects along one axis of a two-dimensional photosensor array according to emission wavelength. The dispersion is unable to completely separate the emissions of a plurality of dyes because the emission spectra of the dyes overlap in wavelength. The method of the present invention accomplishes accurate estimation of the intensity of the light received from each of a plurality of dyes by decomposing the two dimensional spectrally dispersed images into a set of characteristic parameters using either an iterative curve fitting optimization method or a linear algebraic method.

Spectral deconvolution of fluorescent markers

Frequency domain velocity measurements and time domain velocity measurements are made using light from cells or other objects. An optical grating is used to modulate the light from an object so that it has a frequency proportional to the velocity of the object. Depending upon the embodiment, the pitch of the optical grating is uniform or varying. The modulated light is detected and various signal processing techniques, such as a Fast Fourier Transform function, are used to indicate the velocity of the object. Preferably, the velocity measured is applied in determining a timing signal employed for synchronization of an image of the object and an detector signal in an optical analysis system that uses a time delay integration detector to determine characteristics of the object in response to light from the object.

Measuring the velocity of small moving objects such as cells

Frequency domain velocity measurements and time domain velocity measurements are made using light from cells or other objects. An optical grating is used to modulate the light from an object so that it has a frequency proportional to the velocity of the object. Depending upon the embodiment, the pitch of the optical grating is uniform or varying. The modulated light is detected, producing an analog signal that is then digitally sampled. Time domain signal processing techniques are used to determine the velocity of the object from the digital samples. Preferably, the velocity measured is applied in determining a timing signal employed for synchronization of an image of the object and an detector signal in an optical analysis system that uses a time delay integration (TDI) detector to determine characteristics of the object in response to light from the object.

A system for collecting images at multiple wavelengths for image analysis of biological specimens. The process separates contrasts for images of a biological specimen prepared with a chromogen. Light wavelengths matched with each chromogen optimize separation for each image. Computing attenuation coefficients and extinction coefficients for each chromogen provides for determination of the concentration of each chromogen. A chromogen separator allows for subtracting predetermined chromogens to generate new images and aid in identification of cell morphology. The system can perform the image analysis of the new images using image processing techniques such as segmentation, feature calculation and object classification. At each stage of image processing, data from each of the new images may influence the processing of the other images.

Method and apparatus for deriving separate images from multiple chromogens in a branched image analysis system

Improving accuracy of frequency response measurements of linear systems using modulation transfer function test compensation. An optical imaging system illuminates and images an input test pattern. A processor measures a modulation transfer function. The processor determines a compensating factor for error in the input test pattern duty cycle. The driving function for the measurement is a test pattern having a periodic waveform approximating a square wave with an error in duty cycle. A windowed fast fourier transform on a greyscale image of the test pattern generates odd harmonics. Adjusting amplitude values of the odd harmonics corrects for the error in resolution target duty cycle.

Modulation transfer function test compensation for test pattern duty

Measurement of the optical transfer function of an optical system imaging a bar pattern. A one dimensional fast Fourier transform processes sampled image data from the bar pattern. A model of the system utilizes robust measurements of the period, duty cycle and center of each stripe in the pattern. A signal alias free optical transfer function is estimated from the plurality of one dimensional frequency representation of the signal. An idealized bar pattern synthesized from the measured parameters of period, duty cycle and the center of each stripe in the pattern generates an ideal optical transfer function. The noise reduced optical transfer function, OTF, is estimated from OTF of the signal and OTF of the synthesized bar pattern.

Method and apparatus for alias free measurement of optical transfer

A computer receives image data from a star-shaped optical target in the object plane and calculates angle-dependent boundary sharpness. The horizontal, x-direction, amplitude derivative and the vertical, y-direction, amplitude derivative are computed over a portion of each star pattern image from a Z panning sequence. A microscope slide stage, carrying the target, is moved vertically from a level just below where the target is in focus to a level just above where the target is in focus. For each small increment of vertical motion, Z panning, an image of the star pattern is captured for analysis. Computations are performed on the differentiated images to search for evidence of elongation of the point spread function and variation with stage Z position of the angle of long axis of such an out-of-round point spread function. The presence of a distorted point spread function that varies along the optical axis indicates astigmatism.

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Redmond, WA, United States of America

James K Riley