Methods for immunoploidy analysis - Patent Review 5281517

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FIELD OF THE INVENTION

The invention relates generally to the measurement of cell object features and other parameters by image analysis, and is more particularly directed to quantitative measurement methods and apparatus for DNA analysis of small cell populations.

BACKGROUND OF THE INVENTION

The present invention is directed to quantitative testing apparatus and methods which may be used for a wide range of diagnostic and prognostic evaluations of various cells, antigens, or other biological materials taken from the human body. However, for purposes of illustration and ease of understanding, the invention will be disclosed in conjunction with its preferred use, which is the quantitative measurement of cellular DNA for the purpose of cancer diagnosis and prognosis. More specifically, the present invention is directed to methods and apparatus for interactive image analysis which are adapted to analyze and quantify the DNA in different classes of specimen cells taken from a human body.

The current state of the art in the pathology laboratory for measuring the DNA content of a cell is by visual observation. A pathologist observes through a microscope primarily the shape and texture of suspected cancer cells and then classifies the cells into a normal category or into one of several abnormal or cancer categories. Such evaluations are very subjective and can not differentiate and precisely quantify small changes in DNA within individual cells or in very small populations of abnormal cells. These small changes may represent an incipient stage of cancer or a change in cell structure due to treatment of the cancer by chemotherapy or radiation. Such small changes are, therefore, important in the diagnosis and prognosis of these diseases.

However, the advantage in diagnosis and/or prognosis of abnormal ploidy distributions that a pathologist viewing a specimen under a microscope has is the discerning expertise of a skilled person in classifying cells as normal or abnormal. There is an innate human ability to make relatively quick infinite gradations of classification, i.e., almost normal, slightly abnormal, etc. On the other hand, the classification and measurement of cell features and parameters manually by a pathologist on a cell-by-cell basis is extremely tedious and time consuming. Statistical analysis of such cell data taken by hand is relatively difficult because each record has to be entered and then processed. For different records, taken at different times, and under varying conditions broad statistical categorizations may be unreliable.

The alternative is automated cell analysis where the pathologist uses specialized equipment to perform the analysis. In automatic cell analysis, such as that accomplished by a flow cytometer, 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 results from these tests and the sample is consumed.

Although there are commercially available general purpose flow cytometers, they are very expensive and can handle only liquid blood specimens or tissue disaggregations. These cytometers are incapable of working on standard tissue sections or using conventional microscope slides which are the preferred specimen forms of pathology laboratories. Additionally, a flow cytometer does not allow for the analysis of morphological features of cells such as texture, size and shape of cell nuclei and alterations in the nuclear-to-cytoplasmic ratios of cells.

The methods and apparatus illustrated in the referenced Bacus applications have solved these and other problems relating to the analysis of various features and parameters of cell objects. Bacus discloses a measurement method and apparatus which can acquire accurate quantitative data concerning a plurality of individual cells very quickly by an interactive process with a pathologist or an operator.

The Bacus apparatus provides means for displaying on a video monitor an image of a group of cells from a field of a microscope slide. The image is further digitized and stored in a memory of the apparatus. From the digitized image, a processor means identifies each possible cell object automatically by a pattern recognition technique. An interactive program allows the operator to point to each object or cell in succession and make morphological decisions for classification and measurements concerning each. For quantitative DNA analysis, the measurement is of the optical density of the cell object and the classification is by a pathologist as to whether the cell appears normal or cancerous. The decisions include whether to accept or reject a particular cell for further measurement and processing. The cell object, if selected, can then also be classified into one of several classifications for later statistical analysis. The apparatus further has means which permit the classification and storing of more than one image.

When the apparatus is used for DNA analysis, tissue and cell specimens are applied to a slide which is then stained with a specific stain that combines proportionately with the DNA and essentially renders invisible the remainder of the cell so that the image analysis apparatus can measure the optical density of the DNA which is concentrated in the nucleus of the cell. The stain associates with the DNA to provide a detailed nuclear structure and pattern which may be visually observed and interpreted by the pathologist using the apparatus for classification. The amount of DNA in the malignant cells is substantially greater than that for normal cells because the malignant cells are usually dividing and replicating rapidly or the malignant cells have abnormal numbers of chromosomes or have defective chromosomes.

The Bacus apparatus can not only detect minute alterations in the nucleus by providing a real and accurate measurement of the DNA mass in picograms but also can measure and quantify the amount of DNA and relate it to stored statistical analyses to aid in diagnosis. More specifically, the invention allows an iterative analysis of specimen population cells and provides a histogram or other statistical display of the population distribution of the cells with respect to their DNA content and with respect to a standard DNA for normal cells so that subtle shifts in population distribution can be readily understood. To this end cell nuclei images are not only acquired and stored but the data therefrom can be integrated with other statistical data to provide multivariable analysis, discrimination of cells, histograms, and scattergrams of cells or cell populations.

While the methods and apparatus described above are extremely advantageous and advance the art of aneuploidy analysis by image processing, they are not as sensitive as they could be. With the progress in measuring the quantity and distribution of DNA in a cellular population, there has come the need to further refine and sensitize that analysis and characterization process. One area in which sensitivity of the above described process can be improved is in the operator classification of cell types.

The previous apparatus of Bacus relies mainly on the pathologist or operator to make a subjective judgement concerning the classification of cell types, and whether they are to be classified at all. This is a principal advantage of the apparatus where the expertise of the pathologist in discerning cell types is automated and measurement of specified parameters of the chosen cells is accurately made. However, it has been learned that different cell types which are really quite different structurally appear morphologically similar under the microscope.

This is particularly true when the nuclear DNA has been enhanced by Feulgen staining. Such nuclear staining is for the purpose of enhancing the optical characteristics of the nuclei of the cells which contain the DNA, but that necessarily de-emphasizes the visual characteristics of the cytoplasm in the rest of the cell outside of the nucleus. The result is to allow easier image analysis and precise measurement of the DNA of the nuclear material, but at the same time this enhancement causes the loss of the visual morphological characteristics of the cytoplasm which a pathologist might use to distinguish one type of cell from another. Additionally, there are different cell types, which it is advantageous to classify separately, but which provide no or only faint visual clues as to their differences.

Thus, there is the need to alert a pathologist classifying the cell populations for DNA analysis with the Bacus instrument about the different cell types, whether by optical enhancement or otherwise. A more definitive mechanism would be the use of some demonstrable marker on the cells themselves by which the pathologist can objectively separate the various cell types. There are known in the art many optical enhancement or marking techniques for cell populations, including those described in the above referenced Bacus applications. For example, since the advent of monoclonal antibody production, numerous antibodies have been developed which are specific for cellular components, either in the cytoplasm, nucleus or on the cell membrane. Some have already been used to type cells in pathology to assist in the definition of the cell of origin of a number of tumors where subjective morphology is equivocal.

Among the most notable of these antibodies are antibodies to Leukocyte Common Antigens, which identify inflammatory cells, and antibodies to a family of cytoplasmic structural proteins called cytokeratins which identify cells arising from epithelial structures. Other antibodies to cytoplasmic components such as intermediate filaments can be utilized to identify cells which provide structural support, the so called stromal cells. In addition, numerous antibodies exist which are more specifically related to individual tumor types.

However, further optical enhancement of the cytoplasm for different types of cells is problematic in view of the current DNA staining technique. There are many difficulties, the least of which is that an optical enhancement factor for the cytoplasm should be compatible with the present imaging techniques using computer analysis of optical density and be required to provide such compatibility without impairing the sensitivity of the imaging techniques for the present nuclear staining. Chemical compatibility with the present Feulgen staining technique also presents a major hurdle. Optical enhancement of the cytoplasm after Feulgen staining of the DNA is substantially unavailable because the Feulgen process is destructive of the cell cytoplasm and changes the way it appears normally. However, prior optical enhancement of the cytoplasm is equally as difficult because the Feulgen staining process is caustic with the use of highly acidic reagents which can easily destroy other optical enhancement factors. Moreover, if done prior to Feulgen staining, the optical enhancement process of the cytoplasm cannot affect the nuclear material in a manner such that the result of the subsequent Feulgen staining will be changed.

SUMMARY OF THE INVENTION

The invention provides methods and apparatus for the measurement of selective features and parameters of cells in a population by the optical identification of their type. More specifically, the invention measures the DNA content of selected cells of a subpopulation which is selected from a larger population based on optically marking certain cells in the population.

In a preferred embodiment the optical marking of the cell types is effected by binding an optical enhancement factor, such as a chromogen, to a specific protein in the cytoplasm of a cell in order to type a cell. Particularly, a monoclonal antibody specific to the cytoplasmic protein binds to the protein site and is magnified by an enzyme development technique. After certain types of cells in the population have been tagged with a protein specific optical enhancement, a Feulgen staining process is used to stain the nuclear DNA in all of the cells. An imaging apparatus is then used for the computerized image analysis of the cell population. The apparatus provides means for displaying on a video monitor an image of the cell population from a field of a microscope slide. The image is further digitized and separated into two separate images where in the first the DNA stained areas are visible and in the second the optically enhanced cytoplasm areas are visible. The two image areas are combined and those cells which contain optically enhanced cytoplasm areas are marked so that the operator can visualize those specific cells.

From the digitized DNA areas, the imaging apparatus identifies each possible cell object automatically by a pattern recognition technique. An interactive program allows a pathologist to point on the video monitor to each identified object or cell in succession to make decisions for classification and measurements concerning each. The marked cells can be specifically excluded from a subpopulation by the classification process or specifically included. They may further be identified as to DNA content in a separate classification.

By combining the marking or identification of certain types of cells by an immunohistochemical technique with DNA Feulgen staining, the ability to perform DNA content analysis with a greater degree of accuracy and sensitivity is enhanced. This greater sensitivity provides at least two more avenues of diagnostic and prognostic utility for human tumors. In one method, the immunologic marking can be used to mark which of the cells of a particular population are not derived from the tumor, leaving the remaining cells which are not marked immunologically to be analyzed for DNA content. This method is advantageous where a moderate number of inflammatory cells are present in a tumor. Thus, using an antibody to leukocyte common antigen, the immunological marking can identify these inflammatory cells so they can be excluded from the DNA assay. Alternatively, when the tumor cells are relatively rare and non-tumor cells make up the majority of the cells available for analysis, using immunohistochemical marking which specifically identify tumor cells provides a much easier and more sensitive determination of DNA mass for a cell population. In this case, antibodies to cytokeratin are utilized to identify epithelial derived tumors such as carcinomas. The analysis will then be focused on these cell types while discarding cells negative for cytokeratin as being inflammatory or support cells.

One specific embodiment of the invention includes staining of the cell population with an alkaline phosphatase technique utilizing a monoclonal antibody against a specific cytoplasmic antigen. The resulting stain is substantially specific to the cytoplasm and does not stain the nucleus of the cells. A Feulgen staining process using Thionin is then performed to stain the DNA in the nucleus of each cell. The alkaline phosphatase staining method is used because of its compatibility with the Feulgen staining technique. The alkaline phosphatase staining is specific to the cytoplasmic antigen binding the chosen monoclonal antibody and does not harm the nuclear material so that it may receive the Feulgen stain in a subsequent step. The alkaline phosphatase staining is accomplished first before the destruction of the cytoplasm by the Feulgen staining technique. The chromogen chosen for the staining technique is a fast red dye which is advantageous for two reasons. In the first instance the fast red dye which is precipitated is not susceptible to being washed out by the Feulgen stain process and thus will remain for the optical visualization. The second reason is that the chromogen provides excellent optical separation from the blue Thionin dye used in the Feulgen staining process.

Accordingly, a general object of the invention is to provide a new and improved method and apparatus for analyzing cells or other biological materials by using image analysis techniques.

Another object of the invention is to provide new and improved methods and apparatus for making a quantitative ploidy analysis of cells using image pattern recognition equipment.

These and other objects, features, and aspects of the invention will become apparent upon reading the following detailed description when taken in conjunction with the appended drawings wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial representation of an image analysis system constructed in accordance with the invention;

FIG. 2 is a functional block diagram of the image analysis system illustrated in FIG. 1 which is adapted to perform the methods for the quantitation of nuclear DNA in accordance with the invention;

FIG. 3 is a schematic block diagram of the image acquisition apparatus illustrated in FIG. 2;

FIG. 4 is a functional system diagram illustrating the major operations of the system control illustrated in FIG. 2;

FIGS. 5 and 6 are top perspective and cross-sectional views, respectively, of a slide particularly adapted for use in the image analysis system illustrated in FIG. 1 and having separate areas for calibration cell objects and specimen cell objects;

FIG. 7 is a pictorial view at the microscopic level of the binding effects of a monoclonal antibody;

FIG. 8 is a graphical representation of the % of light transmission as a function of light wavelength for the two stains and the two color filters used in accordance with the invention;

FIGS. 9, 10, and 11 are pictorial representations of images of a cell population showing an unfiltered image, a red filtered image, and a blue filtered image, respectively;

FIG. 12 is a functional flow chart of one preferred method of quantitating DNA for human carcinoma in accordance with the invention;

FIG. 13 is a pictorial representation of the image monitor 37 during the selection process, illustrating the marked cells;

FIG. 14 is a pictorial representation of the many optical fields on the slide illustrated in FIGS. 5 and 6;

FIG. 15. is a pictorial representation of the calibration screen which appears on the instruction monitor illustrated in FIG. 1;

FIG. 16 is a pictorial representation of the analysis screen which appears on the instruction monitor illustrated in FIG. 1;

FIG. 17 is a system flow chart of the analysis system screen architecture of the image analysis system illustrated in FIG. 1;

FIG. 18 is a functional flow chart of the main menu of the main screen illustrated in FIG. 17;

FIG. 19 is a functional flow chart of the calibrate menu of the calibrate screen illustrated in FIG. 17;

FIG. 20 is a functional flow chart of the adjust blue boundary menu of the adjust blue boundary screen illustrated in FIG. 17;

FIG. 21 is a functional flow chart of the adjust red boundary menu of the adjust red boundary screen illustrated in FIG. 17; and

FIG. 22 is a functional flow chart of the analysis menu of the analysis screen illustrated in FIG. 17;

FIGS. 23A-23D illustrate different histograms for aneuploidy analysis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The apparatus illustrated in FIGS. 1 and 2 and the methods described herein can be used to develop histograms, and other statistical data, of cell populations for the diagnosis and prognosis of malignancies and other diseases. Specifically, the quantity and distribution of nuclear DNA in separate or combined classifications of cell populations is available. To illustrate the utility of such statistical analyses reference is directed to FIGS. 23A-23D.

Referring now to FIG. 23A there is shown a normal ploidy histogram having a typical cell number versus mass distribution for healthy, non-dividing cells. The number of cells is provided on the ordinate axis and their nuclear DNA mass on the abscissa. If the cell population shown in the figure is not dividing, the DNA content should be peaked around a normal peak G0/G1 which is the diploid amount, 7.18 picograms/cell. This relative mass of DNA is labelled as 1.0 to normalize the abscissa of the histogram. FIG. 23B also shows a normal cell population which is dividing, such that there is a significant G0/G1 peak at 1.0 and a second peak G2 at 2.0. The peak at 2.0 is normal because some of the cells are in division and have double the normal diploid amount of DNA. The saddle S between the two peaks is relatively low and does not indicate any malignancy.

Comparing the histogram in FIG. 23C with the first two, it is seen that this cell population is skewed from normal by having a higher first peak around 1.5 and second peak around 3.0. Further, the saddle S is more pronounced and can be rough in cell count. This histogram may show a malignancy because of the abnormally high DNA content for many of the cells. This high DNA content is likely indicative of the increased ploidy amount of malignant cells which are rapidly dividing.

Likewise, in FIG. 23D it is shown that the G0/G1 peak occurs at 1.0 with a normal diploid amount of DNA but has a relatively large trailing saddle from 1.0 to 2.8. A normal G2 second peak is not noted and is indicative of an abnormal cell population. The shape of the histogram is likely due to abnormal DNA amounts in cells and clones of cells indicative of malignancy. Therefore, from the shapes and changes in cell distribution histograms, diagnostic and prognostic information can be obtained.

DNA analysis of human cells has been shown to have both diagnostic and prognostic utility for human tumors. As with any test, its usefulness is dependent on both the accuracy and sensitivity of the technique employed for the analysis. If a tumor specimen were composed only of the tumor cells, the accuracy and sensitivity of the illustrated technique would be a function of the DNA staining and the accuracy of the measuring instrument. However, tumors are most commonly composed of a mixture of cell types. In addition to the tumor cells one finds the normal tissue from which the tumor arose, supportive and structural elements and a variety of inflammatory cells and cells which are part of the repair and defense process of the host. These cells vary in amount from tumor to tumor and may indeed numerically overshadow the tumor cells in many cases.

If non-tumor cells are included in the histograms of the DNA analysis illustrated, several errors can occur:

1. An insufficient number of tumor cells may be identified resulting in a tumor inappropriately being assigned a normal DNA content;

2. In tumors with a normal DNA content, the normal cells will exaggerate the peak on the histogram where the resting tumor cells appear and artifactually lower the percentages of proliferating tumor cells;

3. If the non-tumor cells themselves are proliferating, they will give an artifactual elevation to the assessment of proliferating activity in the tumor.

Thus, an improvement to the DNA analysis could be made with a mechanism to appropriately eliminate irrelevant cells. Among the potential mechanisms are to attempt to distinguish tumor cells from non-tumor cells by cell size and shape characteristics, either quantitatively or by subjective morphologic assessment by a pathologist. The quantitative method is not useful in that tumor cells themselves can vary significantly in size and shape and there is substantial overlap between these parameters in tumor cells and those seen in the non-tumor type cells. The subjective morphologic method is more useful in that it takes into account multiple diagnostic criteria. The present apparatus takes advantage of this by allowing the pathologist to use his subjective skills to separate the tumor cells from the non-tumor cells for DNA analysis. One problem is that the pathologist traditionally uses characteristics of both the nucleus and the cytoplasm to make these subjective judgments. However, when dealing with the previously disclosed method of analysis, only the nucleus is stained making any morphologic assessment more difficult. The invention solves this problem by the optical enhancement or the marking of selected cells, which exhibit a certain characteristic or type to identify them immediately.

In the implementation shown, the system is a computerized image analysis system designed to measure a number of cell object features and parameters from their image on a typical glass slide. The instrument includes a sophisticated digital image processing system controlled by software to perform quantitative analysis on individual cells for nuclear DNA content by Feulgen staining as well as measurement of other nuclear features. The imaging system couples the ability of a pathologist to identify and classify cells to be studied with the capability of computer enhanced, high resolution digital video image processing to quantify optical and stain density accurately. Further, the system optically marks certain types of cells such that the pathologist in making his classifications can include or exclude them from the study to improve the sensitivity of the process.

In general, a pathologist first prepares a needle aspirate preparation of fresh tissue. The sample is first stained with a alkaline phosphatase technique using a monoclonal antibody specific against an antigen in the cellular cytoplasm. The nuclear DNA in the sample is then stained by the Feulgen technique using Thionin as the dye or optical enhancement factor. After fixation and staining, the preparation is ready for analysis.

The operator has the option of classifying the cells morphologically into any one of six categories or rejecting inappropriate cells or debris. The cell data are processed by a system control and the cellular elements are characterized by a quantitative DNA analysis for each cell class. The information when compared with either a standard cell calibration or published data allows a pathologist to accurately quantify and classify abnormalities that might ordinarily be described only verbally from the image.

The addition of quantitative data enables pathologists to perform their work in a more standardized and reproducible manner. The system is of value in classifying lesions that may be malignant and in providing prognostic information for known malignancies based on DNA content. The image identification system is more advantageous than common flow cytometry methods of evaluating DNA content. Flow cytometry permits an operator to classify neoplastic cells based only on cell markers. The pathologist, however, never sees the cells that the instrument has examined. In addition, the cell preparation must be used in a short time frame and is consumed in the course of the study. Although a permanent section of a tumor under study may be examined at the same time, there is no guarantee that the same cells are examined in both areas. Also the quantity of tumor available may not be large enough to permit a flow cytometric examination.

In the invention, the quantitative DNA analysis is performed rapidly for the measurement of DNA and ploidy distribution pattern in a cell population under study. The pathologist advantageously selects the cells which are to be used in the population measurements. The measurement of DNA content is useful and believed to be relevant in diagnosing and determining prognosis for a variety of tumors that involve the breast, colorectum, and prostate. The system takes advantage of the skill of the pathologist and the selected cell marking to visually identify and classify abnormal cells, and then uses the computer aided imaging analysis to analyze quantitatively those particular cells selected for the parameters desired. Such instrument advantageously extends and augments the recognition and diagnostic skills of the pathologist.

With reference to FIGS. 1 and 2 of the drawings, the invention is embodied as an apparatus 11 (FIG. 1) which functionally operates as a digital image analysis and processing system 13 (FIG. 2). The apparatus 11 comprises a high resolution microscope 15 with which an operator can view magnified specimens on a support which, in a preferred embodiment is a glass slide 14. The microscope 15 includes adjustment or positioning means 70 for focusing its optics 16 on the slide 14 and a platform 51 movable incrementally in X and Y directions via positioning means 12 and 17 in order to view different areas thereof. Positioning means 12, 17 and 70 are the form of mechanical adjustment verniers which are conventional for instrument quality microscopes.

The specimens in the field under study are further viewable by the imaging system 13 via image acquisition apparatus 18 (FIG. 2). The apparatus 18 receives the light intensities of the image of the field and converts them into two analog signals (Red, Blue) which can be sampled and processed by the image analysis system 13. The image analysis system 13 is controlled by a system control 22 in the form of a digital processor such as a personal computer.

An operator, such as a pathologist or laboratory technician, can interactively communicate with the system control 22 via a keyboard 36, and interacts further with the system to quantitate nuclear DNA and classify cell objects by the viewing of two displays or monitors. A first display, image monitor 37, is a conventional RGB video monitor which displays through the system control 22 and the image acquisition apparatus 18, the same image field as seen through the microscope 15. A second display, instruction monitor 62, is another conventional RGB video monitor and is used to provide the operator with interactive prompts, messages, information, and instruction screens from a system program executed by the system control 22.

The keyboard 36 is preferably a conventional AT type keyboard which has on the left-hand side a plurality of function keys F1-F10, in the middle a plurality of alphanumeric keys including the special keys of ENTER, SHIFT, CONTROL, and ALTERNATE, and on the right-hand side cursor control keys including up, down, left and right arrow keys, a numeric keypad, a numeric lock key, and an escape key. A keyboard interface 35 translates the keystrokes of the operator into numerical codes recognized by the system control 22 for specific key indications. A printer 38 is provided for producing reliable hard copy output of the statistical data and reports produced by the apparatus 11.

A functional schematic of the apparatus 11 is illustrated in FIG. 2 as image analysis and processing system 13. The image processing system 13 is used to analyze a plurality of specimen cell objects on the support or glass slide 14 of the microscope 15. Suitable high resolution microscope optics 16 receive light from a variable intensity source 19 and transmit the light through the slide 14.

Because the source 19 transmits light through the cell objects on slide 14, the optical density of each pixel of the image will convert the light into a different intensity depending upon its percentage of transmission. Areas with no cell objects in them will appear relatively light or intense and areas having nontransmissive objects will appear darker. In general, unmodified cell objects are relatively transparent and their features difficult to distinguish. Staining the cell objects allows an optical enhancement of the features stained so they will appear darker than other features and their background.

The optical image of each of the cell objects on the slide 14 passes through an optical image splitter 25. On one side of the splitter 25, the image acquisition apparatus 18, or other detector, converts the optical images point by point into two scanned electronic signals (Red, Blue) representing a monochromatic representation of the optical intensity of each point in the image on the other side of the splitter 25, a true color image of the field is provided to the operator by viewing optics 24.

FIG. 3 illustrates the optical filtering and splitting of the image performed by the image acquisition apparatus 18. The focused image formed by the light intensities is transmitted substantially vertically through the slide 14 (not shown) and enters the beam splitter 25 mounted in a holder 53. The first true color image passes vertically therethrough. A second true color image is further transmitted by the beam splitter 25 perpendicular to the vertical path through the focusing lens 154 to image acquisition apparatus 18. The image acquisition apparatus 18 comprises a plurality of optical elements including a second image splitter 156, mirrors 158, 160 and 162, and two monochromatic optical filters 164 and 166. The image acquisition apparatus 18 further includes dual video cameras 168 and 170 which each receive a portion of the split image. After the second true color image is split from the microscope optics, it passes into the second beam splitter 156 where along one path the image is reflected by mirror 158 through filter 164 and imaged by camera 168. Along a second path, the image is reflected from mirror 160, to mirror 162, and then through a second filter 166 to be imaged by camera 170. The filters 164 and 166 are narrow bandpass filters substantially blocking all light frequencies outside their pass band. The images from cameras 168 and 170