Method and apparatus for automated assay of biological specimens

What is claimed is:

1. An apparatus for automated assay of biological specimens positioned on a plurality of slides of a group, comprising:

operator interactive optical means actuated by an operator for viewing the biological specimen on each of a plurality of slides at a first low magnification and for producing an interactive location signal corresponding to locations of operator selected fields of the viewed image for each of the slides;

means for storing the location signals of the located biological specimens for each of the slides of the group;

means for holding each of the slides of the group and to shift successive selected fields from each of the slides of the group into position for automatic scanning;

automated optical means responsive to said interactive location signal for viewing said selected fields of each of the slides of the group at a second magnification, larger than the first low magnification;

automated means for recognizing each new slide of the group and automatically focusing the automated optical means on each of the slides of the group;

calibrating means for automatically measuring at least one known measurable attribute of a calibration material on at least one slide of the group and for computing a calibration value form said measured attribute value and said known attribute value of said calibration material, said calibration material being located on at least one of the slides of the group;

means for generating an automatic video signal from scanned images of the selected fields;

processing means for processing the automatic video signal for each of the slides of the group to measure said at least one measurable attribute value and correct said at least one measured attribute value in accordance with said computed calibration value for the biological specimens;

said processing means reads the recorded locations, scans the identified locations of the fields and stores image fields corresponding thereto; and

said processor means includes means for determining which of a plurality of stored image fields from said automated optical system are selected for biological assay processing.

2. An apparatus for automated assay of biological specimens according to claim 1 further comprising means for automatically adjusting an effective spectral characteristic of light being processed into said automatic video signal.

3. The apparatus in accordance with claim 1 wherein said biological specimen comprises a tissue section sample.

4. The apparatus in accordance with claim 1 wherein said biological specimen comprises a cell sample.

5. An apparatus in accordance with claim 1 wherein said processing means includes:

storing means for storing said computed calibration values and for correcting each of the measured attribute values calculated for each slide of the group in accordance with said computed calibration value.

6. A method of analyzing a plurality of tissue section cell samples, fixed to microscope slides, by means of an image field producing apparatus comprising the steps of:

automatically shifting the slide to position the slide at a substantially known location to bring an opaque target thereon into view;

automatically focusing the image producing means by using the opaque target at the substantially known location;

providing first and second light sensing means for sensing light and producing images for said image field producing apparatus;

adjusting a light source to balance the light sensed by the first and second light sensing means;

automatically adjusting the light intensity in response to a light level sensed by said image producing means;

optically sensing a calibration material on a calibration slide;

optically sensing the tissue section cell samples on the microscope slides;

automatically measuring said at least one known measurable attribute of said calibration material;

computing a calibration value from said at least one measurable attribute of said calibration material and said known quantity of said attribute;

automatically measuring and recording said at least one measurable attribute of the tissue sections cell samples fixed on said tissue sample slides; and

correcting the value of the at least one measurable attribute recorded for said tissue section cell samples in accordance with said calibration value.

7. A method in accordance with claim 6 comprising mounting said calibration slide and slides having tissue samples fixed thereto in a slide holder prior to said optical sensing steps.

8. An apparatus for automated assay of biological specimens positioned on a plurality of slides of a group, comprising:

operator interactive optical means actuated by an operator for viewing the biological specimen on each of a plurality of slides at a first low magnification and for producing an interactive location signal corresponding to locations of operator selected fields of the viewed image for each of the slides;

means for storing the location signals of the located biological specimens for each of the slides of the group;

means for holding each of the slides of the group and to shift successive selected fields from each of the slides of the group into position for automatic scanning;

automated optical means responsive to said interactive location signal for viewing said selected fields of each of the slides of the group at a second magnification, larger than the first low magnification;

automated means for recognizing each new slide of the group and automatically focusing the automated optical means on each of the slides of the group;

calibrating means for automatically measuring at least one known measurable attribute of a calibration material on at least one slide of the group and for computing a calibration value form said measured attribute value and said known attribute value of said calibration material, said calibration material being located on at least one of the slides of the group;

means for generating an automatic video signal from scanned images of the selected fields;

processing means for processing the automatic video signal for each of the slides of the group to measure said at least one measurable attribute value and correct said at least one measured attribute value in accordance with said computed calibration value for the biological specimens; and

wherein said interactive optical means is a separate and distinct apparatus from said automated optical means.

9. An apparatus for automated assay of biological specimens positioned on a plurality of slides of a group, comprising:

operator interactive optical means actuated by an operator for viewing the biological specimen on each of a plurality of slides at a first low magnification and for producing an interactive location signal corresponding to locations of operator selected fields of the viewed image for each of the slides;

means for storing the location signals of the located biological specimens for each of the slides of the group;

means for holding each of the slides of the group and to shift successive selected fields from each of the slides of the group into position for automatic scanning;

automated optical means responsive to said interactive location signal for viewing said selected fields of each of the slides of the group at a second magnification, larger than the first low magnification;

automated means for recognizing each new slide of the group and automatically focusing the automated optical means on each of the slides of the group;

calibrating means for automatically measuring at least one known measurable attribute of a calibration material on at least one slide of the group and for computing a calibration value form said measured attribute value and said known attribute value of said calibration material, said calibration material being located on at least one of the slides of the group;

means for generating an automatic video signal from scanned images of the selected fields;

processing means for processing the automatic video signal for each of the slides of the group to measure said at least one measurable attribute value and correct said at least one measured attribute value in accordance with said computed calibration value for the biological specimens;

storing means for storing said computed calibration values and for correcting each of the measured attribute values calculated for each slide of the group in accordance with said computed calibration value; and

wherein said calibrating means measures at least one measurable attribute of the calibration material only on one slide of the group of slides and the storing means stores the computed calibration value for use by the processing means for correcting the measured attribute values for the remainder of the slides of the group.

10. An apparatus in accordance with claim 9 wherein said processing means includes:

means for optimizing the light intensity of the light source for each recognized new slide of the group.

11. A method for automated assay of biological specimens positioned on a plurality of slides of a group, comprising the steps of:

providing a single slide having calibration material thereon in a group of slides;

substantially simultaneously staining said slides of said group under substantially the same staining conditions;

viewing by an operator an image of the biological specimen on each of said plurality of slides at a first low magnification;

selecting by operator interaction fields of interest from said viewed image;

producing an interactive location signal corresponding to locations of operator selected fields of the viewed image for each of the slides;

storing the location signals of the located biological specimens for each of the slides of the group;

holding each of the slides of the group and shifting, successive selected fields from each of the slides of the group into position for automatic scanning;

automatically viewing said selected fields of each of the slides of the group at a second magnification larger than the first low magnification using automated optical means responsive to said interactive location signal of said selected fields;

automatically recognizing each new slide of the group and automatically focusing the automated optical means on each of the slides of the group;

calibrating by automatically measuring said single slide of the group of slides for at least one measurable attribute of said calibration material and computing a calibration value from said measured attribute value and said known attribute value of said calibration material;

storing the computed calibration value for use by the processing means for correcting the measured attribute values for the remainder of the slides of the group;

generating an automatic video signal from scanned images of the selected fields; and

automatically processing the automatic video signal for each of the slides of the group to measure said at least one measurable attribute value and to correct said at least one measured attribute value in accordance with said computed calibration value for the biological specimens.

12. An apparatus for analyzing biological specimens on a plurality of microscope slides comprising:

a slide holder for holding the plurality of microscope slides;

means for producing digital image fields from microscope slides placed in proximity thereto;

means for moving said slide holder to place the biological specimens on the said slides in proximity to said image field producing means without the removal of said slides from said slide holder during image field production; and

means for automatically measuring at least one attribute of one of a plurality of digital images produced by said image field producing means from said biological specimens on said plurality of slides, said digital images including digital images from each of said plurality of microscope slides;

focusing means responsive to said digital images from said image producing means for automatically focusing said image producing means on the surface of each of said microscope slides having a biological specimen fixed thereto, said focusing means including, means for producing an image field including a target image and for focusing said image producing means on a target on a surface of the microscope slide having the biological specimen fixed thereto;

a light source;

means for moving said image producing means to a predetermined position with respect to said target;

means for adjusting the light source in response to a light level sensed by said image producing means at said predetermined position;

first light sensing means for sensing a first portion of the light spectrum;

second light sensing means for sensing a second portion of the light spectrum; and,

and adjusting means to balance the light sensed by the first and second light sensing means.

Description Submit all comments and votes

BACKGROUND OF THE INVENTION

This invention relates to a system for performing an assay of biological cell samples, and more particularly, for providing an automated method and arrangement of measuring attributes of the image fields and cells of slide mounted tissue samples.

The diagnosis and/or prognosis of a patient's condition frequently includes the removal of a cell sample, such as a tissue mass, from the patient. Although an attending physician may have good intuition regarding the patient's diagnosis and/or prognosis, confirmation of the diagnosis with histological examination of the cell sample removed from the patient is necessary. The histological examination entails cell staining procedures which allow the morphological features of the cells to be seen relatively easily in a light microscope. A pathologist, after having examined the stained cell sample, makes a qualitative determination of the state of the tissue and reaches a conclusion regarding the prognosis for the patient. While this diagnostic method has a long history, it is somewhat lacking in scientific rigor since it is heavily reliant on the subjective judgment of the pathologist and it is extremely time-consuming.

The alternative to the strictly qualitative and time-consuming human analysis is automated cell analysis where the pathologist uses specialized equipment to perform the analysis. Flow cytometry equipment is one type of automated apparatus for cell analysis. With flow cytometry, mass tests are performed in gross on a specimen cell population without a researcher being able to exclude or include certain data of the population. The specimen is measured "as is" without really knowing what cells are being measured and how many. Important single cell data or data from relatively small groups of cells are lost in the overall averaging of a specimen. Further, relatively large amounts of a specimen have to be used to provide a required level of accuracy. Again, small changes in individual cells or small cell populations cannot be discerned.

Commercially available general purpose flow cytometers are very expensive and can handle only liquid blood specimens or tissue disaggregated specimens. Additionally, flow cytometers are incapable of working on standard tissue sections or using conventional microscope slides which are the preferred specimen forms of pathology laboratories.

Although the automation of cell analysis using microscope slide cell samples is exceedingly difficult, such has been automated to a human-machine interactive level. One such method and apparatus is described in U.S. Pat. No. 4,471,043 to Bacus, for Method And Apparatus For Image Analysis Of Biological Specimens. Cell samples are attached to slides and an operator adjusts the system optics to view desired image fields of the cell sample. The operator then selects and classifies particular cell objects of the sample. After such operator action, the automated equipment quantitatively measures particular attributes of the selected and classified cell objects and records a digital representation of the optical image. The measured attributes can be reported on a per object basis or on an accumulated basis, and the stored image representations can later be read from memory for review.

The automation of analysis of slide cell samples as described in U.S. Pat. No. 4,471,043 has provided many advantages over both the historical pure human analysis and the automated flow cytometry analysis. Large amounts of human operator time and judgment are still required to complete a tissue section assay. A need exists, however, for improvements in the automation of analysis of slide cell samples, and particularly, for slide tissue samples.

SUMMARY OF THE INVENTION

An apparatus and method for automated assay of biological specimens positioned on microscope slides comprises an interactive optical subsystem for viewing the biological specimen on the slide and for producing an interactive video signal corresponding to the viewed image. An automated optical subsystem includes a single high power microscope objective for scanning a rack of slides, portions of which having been previously identified for assay in the interactive optical means. The system also includes a processor subsystem for processing the interactive and automatic video signals from the two optical subsystems. The processor receives the automatic video signal and performs biological assay functions upon it.

It is a principal aspect of the present invention to provide an apparatus for automated assay of biological specimens having a low magnification interactive optical subsystem for interactively scanning the fields of microscope slides having biological specimens positioned thereon.

It is another aspect of the present invention to provide an apparatus for automated assay of biological specimens which apparatus can process a plurality of slides automatically without operator intervention.

It is a still further aspect of the present invention to provide an apparatus for automated assay of biological specimens having both a low magnification interactive optical portion and a high magnification and the automated optical section for high power automated assay.

Other aspects and advantages of the present invention will become apparent to one of ordinary skill in the art upon a perusal of the following specification and claims in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an apparatus for assaying biological specimens embodying the present invention;

FIG. 1A is a perspective view, having portions broken away, of an automatic optical input subsystem of the apparatus for assaying biological specimens shown in FIG. 1;

FIG. 2 is a block diagram of the apparatus shown in FIG. 1;

FIG. 3 shows a slide holder and associated control equipment of the apparatus shown in FIG. 1;

FIG. 4 is a block diagram view of focus and light control portions of the apparatus shown in FIG. 1;

FIG. 5 is a plan view of a tissue section microscope slide for use with the apparatus of FIG. 1;

FIG. 6 is a graphical representation of the optical properties employed in biological specimen assay;

FIGS. 7 and 8 are schematic views of the biological specimen preparation prior to assay;

FIGS. 9 and 10 are flow diagrams of the control procedures invoked in the assay of a plurality of biological specimens;

FIG. 11 represents an image field of an optically unfiltered tissue section;

FIG. 12 represents the image field of FIG. 11 when optically filtered by a red filter having a passband centered about 620 nanometers; and

FIG. 13 represents the image field of FIG. 11 when optically filtered by a green filter having a passband centered about 500 nanometers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The preferred embodiment disclosed herein is used for the assay or quantitation of biological specimens specifically estrogen and progesterone in tissue samples. The tissue sample staining and measuring techniques for the estrogen/progesterone assay are described in detail in U.S. Pat. No. 5,008,185, issued Apr. 16, 1991 to Bacus which is hereby incorporated by reference. The tissue sample assay is performed using a two-color optical system to enhance the optical characteristics of stained tissue samples. It will be apparent to those skilled in the art that many inventive features of the disclosed embodiment may be employed for other types of cell analysis e.g., DNA quantification and that other types of optical apparatus e.g., single color could be employed.

An apparatus for assaying biological specimens, and embodying the present invention and generally identified by numeral 10 is shown in perspective view in FIG. 1 and in block diagram form in FIG. 2. The apparatus 10 comprises an interactive optical input system 11a primarily for use in low power scanning of microscope slides of biological specimens to select fields for later analysis. An automated assay processing system 11b also comprises a portion of the apparatus for scanning up to eight slides at once at relatively high magnification for performing biological assays on the slide.

A processor system 11c receives signals from the optical units for later image processing.

The interactive optical system 11a comprises an optical microscope 12, which may be of any conventional type, but in this embodiment, is a Riechart Diastar. An optical conversion module 14 is mounted on the microscope 12 to enhance the optically magnified image of cell samples viewed with the microscope 12. The optical conversion module 14, as may best be seen in FIG. 2, includes a beam-splitting prism 80 which conveys approximately 90% of the light into optical conversion module 14 and passes the remaining 10% to a microscope eyepiece 76. The light transmitted into module 14 is fed to a dichroic beam-splitter 82 which reflects a portion of the light to a television camera 20 via a red filter 18 and a mirror 81. The remaining portion of the light is filtered by a dichroic beam-splitter 82 and fed to a television camera 26 through a green filter 24. The dichroic beam-splitter 82 selectively passes light having wavelengths greater than approximately 560 nanometers to the filter 18 and having a wavelength of less than 560 nanometers to the filter 24. Thus, the dichroic beam-splitter 82 acts as a first color filter before the light reaches the color filters 18 and 24. Red filter 18 is a 620.+-.20 nanometer bandpass optical transmission filter which provides a high contrast image to the camera 20. As shown in FIG. 2, the camera 20 then generates an NTSC image signal which is fed through an optical signal switch 90a to an image processor 90 of an image processor module 28 (FIG. 2). Green filter 24 is a 500.+-.20 nanometer narrow bandpass optical transmission filter which provides a high contrast image to a camera 26. The camera 26 then feeds an NTSC image signal through the optical signal switch 90a to an image processor 92. Both of the image processors 90 and 92 contain analog to digital converters for converting the analog NTSC signals to a digitized 384 by 485 array pixel image. The center 256 by 256 array of pixels from this digitized image is then stored within frame buffers internal to the image processors 90 and 92. The visual image represented by the 256 by 256 array of pixels is referred to as an image field.

During assembly of the apparatus of FIG. 1, and from time to time thereafter, if necessary, the optical elements of conversion module 14 are adjusted so that each camera 20 and 26 receives the same optical image and each pixel of the digitized pixel arrays produced by processors 90 and 92, presents the same region of a viewed optical field.

Each of the image processors 90 and 92 is a Model AT428 from the Data Cube Corporation, and includes six internal frame buffers. The image processors 90 and 92 are connected to a system bus 34 of a computer 32. The frame buffers of image processors 90 and 92 are mapped into the address space of a microprocessor 36 in computer 32 to provide easy access for image processing. Additionally, an image monitor 30 is connected to image processor 92 and displays a cell sample image field stored in a predetermined one of the frame buffers. The storage of an image field representation into the predetermined frame buffer is described later herein.

The automatic optical conversion module 11b, as may best be seen in FIG. 2, includes a beam-splitting prism 80a which conveys approximately 90% of the light into optical conversion module 14a and passes the remaining 10% to a microscope eyepiece (not shown). The light transmitted into module 14a is fed to a dichroic beam-splitter 82a which reflects a portion of the light to a television camera 20a via a red filter 18a and a mirror 81a. The remaining portion of the light is filtered by a dichroic beam-splitter 82a and fed to a television camera 26a through a green filter 24a. The dichroic beam-splitter 82a selectively passes light having wavelengths greater than approximately 560 nanometers to the filter 18a and having a wavelength of less than 560 nanometers to the filter 24a. Thus, the dichroic beam-splitter 82a acts as a first color filter before the light reaches the color filters 18a and 24a. Red filter 18a is a 620.+-.20 nanometer bandpass optical transmission filter which provides a high contrast image to the camera 20a. As shown in FIG. 2, the camera 20a then generates an NTSC image signal which is fed through the optical signal switch 90 to the image processor 90 of the image processor module 28 (FIG. 2). Green filter 24a is a 500.+-.20 nanometer narrow bandpass optical transmission filter which provides a high contrast image to a camera 26a. The camera 26a then feeds an NTSC image signal through the optical signal switch 90a to the image processor 92.

The microprocessor 36 of computer 32 is an Intel 80486 microprocessor which is connected to the system bus 34. The optical switch 90a, under control of the microprocessor 36, selects the signal from interactive unit 11a or automatic unit 11b to be fed to the image processors 90 and 92. A random access memory 38 and a read only memory 40 are also connected to the system bus 34 for storage of program and data. A disk controller 42 is connected by a local bus 44 to a Winchester disk drive 46 and to a floppy disk drive 48 for secondary information storage.