INSTRUMENTATION
Advisory Panel Jonathan W . Amy Glenn L. Booman Robert L. Bowman
Jack W . Frazer G. Phillip Hicks Donald R. Johnson
Howard V. Malmstadt Marvin Margoshes William F. Ulrich
Image-Analyzing Microscopes Philip G. Stein National Bureau of Standards, Washington, D. C. 20234
Image-processing technology, through expanded data gathering and storage capabilities, now has the potential to solve difficult problems in metallurgy, polymer and fiber chemistry, and cell biology. Many studies involving the counting and sizing of particles or fibers can be automated through the use of image-processing techniques Λ/Γ ANY PROBLEMS in metallurgy, poly-
-*-"-*- mer and fiber chemistry, and cell biology are being tackled today using image processing techniques. A typi cal measurement of the area of voids in a metallic specimen, for example, would require making a photomicro graph, printing it, and measuring the resulting photo. Some laboratories use electromechanical scanners to make these measurements on the print, or directly from the negative. Elemen tary tests may be done with logic in the scanner. More sophisticated prob lems are attacked by making a digital tape recording of the print and process ing it with a general-purpose computer. Λ different approach to this type of problem has recently been embodied in several instruments, known as imageanalyzing microscopes. At the very least, they can eliminate the time-con suming and inaccurate photographic steps by scanning the specimen directly in the image plane of the microscope. Further sophistication could lead to improvement of the data gathering process with on-line computer control of the scanning and other microscope functions. Each of these instruments contains a scanning microphotometer, which di vides the image plane of the microscope into a number of resolution elements and measures the luminous flux trans mitted through (or reflected from) each element. Data are then processed by special or general purpose computers connected directly to the microscope. Some instruments include variablewave-length illumination and a motor-
driven specimen stage which allows po sitioning of more than one specimen field under the microscope objective. All of these systems share one basic consideration that colors their design: the amount of data is immense. In the SPECTRE I I system at the National Institutes of Health, a single scan at one wavelength produces 2 19 informa tion bits. A single scan in this case, resolving at or near the limits of the optical microscope, covers only a 50X 50-μΓη square on the specimen. A 10- X 10-mm specimen would require 40,000 such scans at each wavelength of interest to acquire all of the data.
For this reason, most of the instru ments do not store the raw data. They may, in fact, have provisions for re cording or transmitting the data to a computer, but in normal operation they rely on the specimen itself to act as a data storage medium. By repeated reference to the actual image, very little storage of the basic density-position data is required. Gathering the Data
The heart of any image-analyzing microscope is the scanner. It parti tions the image into a number of equal
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970 ·
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Instrumentation
areas and measures the light through each. The size of the area measured is a function both of the microscope ob jective and the scanning aperture. An aperture of lO-^m diam at the image plane, for example, corresponds to a 0.25-/ΛΠΙ diam spot on the specimen (with a 40X objective). To take full advantage of the resolution implied by this aperture-objective combination, one must use large aperture, oil-immer sion objectives and condensers, and be extremely careful with the incident il lumination. All image-analyzing micro scopes require the operator to set up the condenser system manually, and the resultant measurements are somewhat dependent on how well this is done. For less critical problems, smaller mag nifications and apertures obviate many of these problems. The user must also make some trade-off between resolution and contrast. At high resolution, the image contrast is reduced, and more amplification is required in the scanner light-measuring circuit. This, in turn, results in increased noise. Again, lowerresolution studies are relatively easy.
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DEVELOPMENTS IN APPLIED SPECTROSCOPY* VOLUME β Edited by E. L. Grove, Illinois Institute of Technology Research Laboratories, Chicago, III. A new v o l u m e in t h i s i m p o r t a n t c o n t i n u i n g series, Volume 8 presents b o t h t h e o r e t i c a l a n d a p p l i e d princi ples of e m i s s i o n , a t o m i c absorp t i o n , x-ray, nuclear particle, R a m a n and i n f r a r e d , nuclear magnetic resonance, a n d electron spin reso nance spectroscopy. In a d d i t i o n , t h e book reports recent a d v a n c e s in t h e areas of c o m p u t e r ap plications; i n s t r u m e n t a l applica tions to b i o m e d i c i n e toxicology; spectra characterizations; a n d air a n d water p o l l u t i o n . New f i n d i n g s on trace e l e m e n t analyses, silicate analyses, Môssbauer t e c h n i q u e s , reference spectra, and retrieval syst e m s are also i n c l u d e d . Proceedings of the 20th Annual MidAmerica Symposia on Spectroscopy, held in Chicago, Illinois, May 12-15, 1970 325 PAGES OCTOBER 1970 $20.00 SBN 306-38308-X
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Figure 1 is a block diagram of an image-analyzing microscope. Clearly, the technical details of the equipment following the scanner depend on the scanner design. Breaking these into broad categories, they are TV scan ners (raster), electronic scanners (using a random-scan tube), and mechanical scanners. The TV scanner is most amenable to use with special purpose computers. The data are scanned in a raster fash ion, completing one line of X for every increment in Y until the entire field is covered. This is done at a fixed rate. For vidicon-type camera tubes, this is important, as the tube photocathode integrates all of the light reaching each position. When a given spot is interro gated by the beam, the information is erased and a new integration period begins. For the data to be uniformly noisy (or quiet), the integration time at each point must be the same. There fore, use of a vidicon for random scans is not recommended. Since each line is being scanned at a fixed rate, an object in the field will be transformed into a time series of volt ages representing the transmission at each point. If we set a threshold (Fig ure 2) which says "anything darker than this level is an object," we then get a pulse whose width is determined by the object size, the scanning rate, and the threshold setting. This is the basic principle behind the TV-type special computers.
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ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
VOLUME 2 Edited by Roger S. Porter, Head, Polymer Science and Engineering Program, University of Massachusetts, Amherst, Massachusetts a n d Julian F. Johnson, Department of Chemistry, University of Connecticut, Storrs, Connecticut V o l u m e 2 brings research workers in t h i s d y n a m i c area a b r o a d , new perspective on current activities. T h e work covers a wide range of materials, i n c l u d i n g : s t u d i e s of p o l y f i n s , polymer single crystals, c o p o l y m e r s , polyblends, elastomers, epoxy resins, a n d a variety of t h e r m o s e t t i n g polym e r s ; special reports on low m o l e c u lar w e i g h t organic c o m p o u n d s , org a n o m e t a l i c s , inorganic salts, a n d metal alloys; d e s c r i p t i o n s of related t h e r m a l t e c h n i q u e s , such as t h e r m a l d e p o l a r i z a t i o n , microscopy, t h e r m a l mechanical and thermogravimetric t e c h n i q u e s , as well as i l l u s t r a t i o n s of t h e varieties of i n s t r u m e n t a t i o n used in analytical c a l o r i m e t r y i n c l u d i n g high-pressure o p e r a t i o n , a u t o m a t i o n , and data i n t e r p r e t a t i o n ; and the use of t h e r m o a n a l y t i c a l e q u i p m e n t , i n c l u d i n g a p p l i c a t i o n s of c o m b i n e d differential s c a n n i n g c a l o r i m e t r y a n d mass spectroscopy. Proceedings of the Symposium on Analytical Calorimetry at the meeting of the American Chemical Society, held in Chicago, Illinois, September 13-18, 1970 460 PAGES SEPTEMBER 1970 $19.50 SBN 306-30366-3 * Place your continuation order today for books in this series. It will ensure the delivery of new volumes immediately upon publication; you will be billed later This arrangement is solely for your convenience and may be cancelled by you at any time. VISIT US AT BOOTH #9 AT T H E EASTERN ANALYTICAL SYMPOSIUM
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Instrumentation
the data at each different setting, thus generating "local" thresholds in the image. This is cumbersome, though. It is better to approach those problems requiring this degree of sophistication with a different instrument. Other Ways to Do It
Ana lysis-TV Microscopes
From this point, there are many ingenious ways to process the pulse data using logic circuits. Some of the possibilities are: (1) Using a TV display of the image, intensify or otherwise identify all points above the threshold enabling the operator easily to find a proper threshold setting. (2) Similarly intensify only the leading or trailing edge of an object. (3) Restrict the image processing to a rectangular subportion of the screen, and indicate the area to the operator. The operator may continuously adjust this ''window." (4) Reject pulses shorter than a certain minimum length, thereby ignoring much dirt and some artifacts of the specimen as being too small. (5) Save the previous scan line in a memory and compare it with the current one. If an object was present on the last line and is still present, do not count it again. With these and other processing techniques, it is possible to automate many problems involving counting and sizing of particles or fibers. By rotating the image 90°, either mechanically or optically, measures of out-of-roundness or form factor are also easily generated. The data may be transferred bv an
automatic sequencing programmer to a desktop calculator; statistics developed about the field, and measurements which are numerically derived from two or more pulse variables can be calculated. In addition, the data may be transferred to a general purpose computer through an appropriate interface, and further computations done. Why Not?
The most serious problem with instruments of this type lies in their assumption of data uniformity. The threshold control must be set manually by an operator, and so must the illumination. This is indeed true of any image-gathering device of this type, but the TV system relies very heavily on this setting for the repeatability of the data. In addition, the logic must depend on an implicit requirement of spatial uniformity of both specimen and illumination. The threshold remains uniform over the field, even though the data may not be. Another problem is that, even with the most sophisticated logic now available, it is not possible to recognize donuts or other multiply-connected shapes as just one object. Whether this is of any consequence, of course, depends on the problem. It would be possible to connect a computer in such a way that it could adjust the threshold and interpret
Random-access scanners may use a moving specimen stage, vibrating mirrors in the light path from objective to image plane, or an image-dissector scanning tube. Of these, only the image dissector shares with the vidicon the problem of photocathode nonuniformity, but it has a great speed advantage over the mechanical scan methods. In all cases though, the output of the scan is transmitted directly to a general-purpose digital computer. In some instruments, this computer can directly control the scan pattern. It can also be connected to make large motions of the specimen stage (such as from field to field), change the color, intensity, and bandwidth of the illumination, and change the focus. None of these systems has a storage photocathode, so there is no need to scan every point in the field at a fixed rate. The transmitted or reflected intensity data are gathered directly by the computer, and the image processing may be done either directly or by transmitting the data to a large, high-speed machine. This approach is far more complicated and expensive than the purchase and operation of a simple TV microscope. It is also much more flexible, permits a great deal of investigation into the interaction of the machine and the properties of the data, and allows unlimited sophistication of the imageprocessing techniques employed. These might involve, even on the most basic level, continuously variable local thresholds, depending on the data themselves, thresholds of sharpness (rate of change of light) rather than simple intensity, and programs that follow boundaries with the scan to resolve ambiguities about shapes such as the donut. Since the data are available in their raw form, the computer might select a proper wavelength for a study on the basis of contrast, or it might adjust the integration time at each point in the image to improve statistics while wasting little or no time on background areas. The computer also might instruct the scanner to look at a field with very low resolution (perhaps every tenth line), and locate all of the large objects. The scan would then return to those areas where data were found to scan them in detail.
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Instrumentation Artificial Intelligence I t is also possible, in some special cases, to use t h e information about geometry and spectral transmission or reflection to identify a n d classify isolated specimens. T h i s hopefully would lead to t h e ability to distinguish normal a n d abnormal specimens a u t o matically. This development depends strongly on processing t h e image in a p r o g r a m m a b l e fashion, making it easy to change classification techniques. A v e r y general facility as in Stein et al. (1) could be used t o simulate t h e o p eration of any n u m b e r of specialized image-gathering and image-analyzing microscopes. I t could be used, t h e r e fore, as a design tool for single-purpose instruments. Finally, t h e computer can produce a display for t h e operator which shows not only the raw d a t a plus a threshold, b u t also t h e result of t h e d a t a processing b y a n y algorithm available to t h e p r o g r a m m e r . I n this way, t h e imageanalyzing techniques themselves m a y be itératively improved.
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Literature Cited (1) P . G. Stein, L. E . Lipkin, H . M . Shapiro, Science 166, 328-33 (1969).
^DEKASTALTIC™ PUMP Suggested Source Material (1) R. C. Bostrom, H . S. Sawyer, W. E . Toiles, Proc. IEEE 47, 1895 (1959) ; P. H. Neurath, B. L. Bablouzian, T. H . Warms, R. Serbagi, A. Falek, Ann. Ν. Y. Acad. Sci. 128, 1013 (1966). (2) M. I,. Mendelsohn, Β . Η. Mayall, J. M. S. Prewitt, R. C. Bostrom, W. G. Holcomb, Advan. Opt. Electron Microsc. 20, 77 (1968). (3) M. Ingram, P. E . Norgren, K. Pres ton, Jr., Ann. Ν. Y. Acad. Sci. 157, 275 (1969). (4) G. L. Wied, P. H. Bartels, G. P . Bahr, D. G. Oldfield, Acta Cytol. 12, 180 (1968). (5) W. Prensky, J. Cell Biol. 39, 157a (1969). (6) K. Preston, Jr., P . E . Norgren, Ann. N. Y. Acad. Sci. 157, 393 (1969). (7) L. E . Lipkin, W. C. Watt, R. A. Kirsch, Ann. Ν. Υ. Acad. Sci. 128, 984 (1966). (8) L. E . Mawdesley-Thomas, P. Healey, Science 163, 1200 (1969). (9) S. A. Rosenberg, K. S. Ledeen, T. Kline, Science 163, 1065 (1969). A publication of the IT. S. National Bureau of Standards. N o t subject to copyright. Discussion or description of specific instruments herein should not be construed to indicate that these instru ments have been tested or otherwise eval uated by the National Bureau of Stan dards, and no endorsement or criticism of any specific manufacturer is intended or implied.
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