The evolution of cytometers (2004)

Microscopy is difficult when cells are on the fly; It's lucky the cytometer's now quicker than the eye. This hasn't been the case throughout the gadget's evolution, But lasers and computers have provided a solution To problems of illuminating cells, at least a myriad Per second, and collecting ample data, in that period, To pick out some to keep, and destine others for rejection. For modern labs, the instrument's a natural selection! The first microscopes were as likely to be gentlemen's toys as scientists' tools. By the beginning of the 20th century, the professionals had largely taken over from the amateurs, and it had become possible to draw at least some quantitative conclusions from observations made using microscopes. Hemocytometers allowed an observer to derive a reasonably accurate count of the number of cells or other particles in a unit volume of specimen, although precision of counts was limited by both counting statistics and dilution errors. Using eyepiece reticles, grids, etc., one could also measure the size of microscopic objects, at least in two dimensions. It was not, however, until some time later that the tools developed by chemists and physicists for spectroscopy and photometry or radiometry were adapted for use with the microscope, thus producing the first true optical cytometers. I have written at length about the development of cytometers on numerous occasions, most recently in the 4th edition of Practical Flow Cytometry (1); in this briefer exposition, influenced by the title suggested by the Editor of this journal, I will emphasize the lineage of the instruments. I will apologize in advance to the many colleagues whose contributions I cannot discuss in the allotted space, and thus hope to avoid possible derogatory comments about my own lineage. I am pleased that Leonard Ornstein, a pioneer in the development of both static and flow cytometric apparatus and techniques, appears to share my perspective on the early history of cytometry, as evidenced by an informative and entertaining reminiscence he published in 1987 (2). It is probably fair to say that the evolution of cytometers from microscopes began in the 1930s in Stockholm. By this time, conventional histologic staining techniques of light microscopy had suggested that tumors might have abnormalities in DNA and RNA content. Torbjörn Caspersson (3), working at the Karolinska Institute (Stockholm, Sweden), began to study cellular nucleic acids and their relation to cell growth and function. He developed a series of progressively more sophisticated microspectrophotometers, which made fairly precise measurements of nucleic acid and protein content based on the intrinsic ultraviolet (UV) absorption of these substances near 260 and 280 nm. Caspersson's early apparatus now seems hopelessly primitive. Cadmium spark sources were used for ultraviolet illumination; photocurrent measurements were done with string electrometers, unless the signal was strong enough to permit use of a vacuum-tube amplifier. However, even this primitive apparatus got results, and attracted the attention of other researchers; many of the advances in analytical cytology from the 1940s on were made by people who had made the pilgrimage to Stockholm. Ornstein (2) documents the influence of Caspersson's work in establishing the role of DNA as the genetic material. It was during the 1950s that analytical cytology acquired its name, coined by Francis O. Schmitt of the Massachusetts Institute of Technology (MIT; Cambridge, MA); the first and second editions of a book entitled Analytical Cytology, edited by Robert Mellors, of the Memorial Sloan-Kettering Cancer Institute (New York, NY), appeared in 1955 and 1959 (4). The book included chapters on the fluorescent antibody method, on histochemistry, and on phase, interference, and polarizing microscopy; Arthur Pollister, Ornstein's mentor at Columbia University (New York, NY), and Ornstein contributed several chapters, including one on the theory and practice of absorption measurements that is well worth reading even today. Microspectrophotometers were first made by putting a small “pinhole” aperture, technically known as a field stop, in the image plane of a microscope, restricting the field of view to the area of a single cell, and placing a photodetector behind the field stop. If a 40× objective lens is used, measuring the transmission through, or the absorption of, a cell 10 μm in diameter requires a 400 μm diameter field stop; with a smaller field stop, it becomes possible to measure the transmission through a correspondingly smaller area of the specimen. A 40 μm field stop allows measurement of a 1 μm diameter area of the specimen, and. By moving the specimen in precise incremental steps in the x and y directions (i.e., in the plane of the slide) in a raster pattern, and recording the information, it becomes possible to measure the integrated absorption of a cell, and/or to make an image of the cell with each pixel corresponding in intensity to the transmission or absorption value. This was the first, and, until the 1950s, the only approach to scanning cytometry. By the 1960s, Zeiss (Oberkochen, Germany) had commercialized a current version of Caspersson's apparatus, and others had begun to build high-resolution scanning microscopes incorporating a variety of technologies. By the mid-1950s, it had become apparent that malignant cells were likely to contain more nucleic acid than normal cells, and Mellors proposed construction of an automatic scanning instrument for screening cervical cytology (Papanicolaou or “Pap”) smears. Tolles et al. (5), at Airborne Instruments Laboratory (Mineola, NY), described the “Cytoanalyzer” built for this purpose. A “Nipkow disc” containing a series of apertures rotated in the image plane of a microscope, producing a raster scan of a specimen with approximately 5 μm resolution. A hardwired analyzer extracted nuclear size and density information; cells were then classified as normal or malignant using these parameters. The Cytoanalyzer was, to make a long story short, right more of the time than it was wrong, but its false-positive and false-negative rates were too high for it to be suitable for clinical use. The results were encouraging enough for the American Cancer Society and the National Cancer Institute to continue funding research on cytology automation. Recording and storing cell images was a nontrivial task in the 1960s, when mainframe computers occupied entire rooms, required kilowatts of power and heavy-duty air conditioning, and cost millions of dollars, which bought a processor with 160 kb of memory and a 6.4 μs instruction cycle (equivalent clock speed 160 kHz). Input and output and data storage typically used tape drives; large random access storage media had not yet arrived on the scene. However, when minicomputers became available in the middle of the decade, there were at least a few groups of analytical cytologists ready to use them. The TICAS system, assembled in the late 1960s by George Wied of the University of Chicago, Gunter Bahr of the Armed Forces Institute of Pathology, and Peter Bartels of the University of Arizona (Tucson, AZ), interfaced Zeiss's commercial version of the Caspersson microspectrophotometer to a minicomputer, with the aim of automating interpretation of Pap smears (6). The use of stage motion for scanning made operation extremely slow; it could take many minutes to produce a high-resolution scanned image of a single cell, and there were no computers available to capture the data. Somewhat higher speed could be achieved by using Nipkow discs or galvanometer-driven moving mirrors for image scanning, and limiting the tasks of the motorized stage to bringing a new field of the specimen into view and into focus; this required some electronic storage capability, and made measurements susceptible to errors due to uneven illumination across the field, although this could be compensated for. My colleagues and I at National Institutes of Health (NIH) built “Spectre II” (7), which incorporated a galvanometer mirror scanning system (8) developed by Kendall Preston, an Airborne Instruments alumnus then working at Perkin-Elmer (Norwalk, CT), and a Digital Equipment Corporation (Maynard, MA) LINC-8 computer. While this system had sufficient computer power to capture high-resolution cell images (0.2 μm pixels), data were recorded on 9-track tape and transported (by “sneakernet”) to a mainframe elsewhere on the NIH campus for analysis (9). Since the late 1940s and early 1950s had already given us Howdy Doody, Milton Berle, and the Ricardos, it might be expected that, somewhere around that time, someone would have tried to automate the process of looking down the microscope and counting cells using video technology. Most of the imaging cytometers developed then were not based on video cameras, for a number of reasons, not the least of which was the variable light sensitivity of different regions of a camera tube, which made quantitative measurements difficult. However, it was recognized that the raster scan mechanism of a cathode ray tube could be used on the illumination side of an image analysis system, with the “flying spot” illuminating only a small segment of the specimen plane at any given time (10). The CYDAC system, a flying spot scanner built at Airborne Instruments Laboratory, was used in studies of the automation of differential leukocyte counting (11) and chromosome analysis (12) by Mortimer Mendelsohn (later the first president of the Society for Analytical Cytology), Brian Mayall (later the founding editor of this journal), Judith Prewitt, and their colleagues, then at the University of Pennsylvania. Somewhat simpler tasks of cell or particle identification, characterization, and counting than those involved in Pap smear analysis and differential white cell counting had attracted the attention of other groups of researchers at least since the 1930s. During World War II, the U.S. Army became interested in developing devices for rapid detection bacterial biowarfare agents in aerosols (a continuing preoccupation); this would require processing a relatively large volume of sample in substantially less time than would have been possible using even a low-resolution scanning system. The apparatus built in the Chemistry Department of Northwestern University by et al. in of this achieved the rapid specimen by the air containing the sample into the of a of air that through the of a through the system light into a producing from a The instrument could on the of μm in and is recognized as been the first flow used for of By the late 1940s and early 1950s, the including the use of were to the detection and counting of cells in automation for a for its when by a observer using a and a the bacterial the early cell had any for different of cells or for quantitative of instrument were measuring would now as side although in than smaller particle and signal were not for cell counting was developed in the 1950s by that cells, which are by a are relatively of as to he an apparatus in which cells one by one through a small two with a cell through, the of the in to the volume of the cell, producing a The was in clinical for cell it was that it could more accurate measurements of cell size than had been available the early 1960s, working with Germany) a that a measurement to the light measurement used in cell If a fluorescent as were to the white cells would be more than the white cell count could then be from the and the cell count from the It was also that could be used to cells from However, it is not that the which would have a new of in flow cytometry, was the time, the results with the Cytoanalyzer in to automate reading of Pap smears at the Corporation to into producing an this would be some of image for the to who had developed a optical He some of would be required in the of light scanning and computer storage and processing to the using image and that a different approach would be from in that cell size and nucleic acid content could a of cervical cells were normal or to Caspersson's in and He then built a flow that used a transmission measurement at to cell size and a 260 absorption measurement to nucleic acid content of this which incorporated a computer system, could measure as many as cellular A on cervical cytology the system had some to normal from cells it could also produce from different of cells in with a of and that flow might be for differential leukocyte The first commercial flow cytometric differential in the early was Ornstein was a in its with the (2). The used light and absorption measurements made at different in different flow cytometers to were used to and by the of to high and high of by their content. was based on detection of in using A single as light for flow the staining that were well by for as of lineage of cells, the apparatus, which was with a of at least in due to the of flow cytometry. The and of image differential which than the could to of flow for as long as the would be by its on which was by the of cell and and optical flow cytometers could of cells second, a high enough data to be for clinical static cytometers a A system with stage motion could be to a cell on a the field of view of the objective (7), the cell to be or by it was, not possible to cells with known from a flow this could be it would be difficult to any cell arrived at using a flow the of cervical or might be This was in the when both working at the National Laboratory and at cell built as to their flow cytometers. system used a to cells from its relatively sample was based on then recently developed by at University through the cytometer's measurement system a the sample was into and those that cells with measurement were at the The were then into a by an as it down the the early the at the in of flow their with capability, measurements with measurements of volume and to the contributions of and light at several The cytometers were interfaced to Digital Equipment Corporation made at were to the other most or of the in their own measurement was to flow in the late 1960s as a of both quantitative and By that time, et al. at and and in had built flow cytometers to measure cellular DNA analysis of abnormalities in cells and of cell cycle in both and normal The instrument incorporated the now in with the optical of illumination and light at right to each other and to the of sample who had to NY), the an flow that was the first commercial to an Germany) built around a microscope with was by Leonard and colleagues at that flow and cell could a and for cells for developed a series of to a to by their apparatus with was not to permit to their objective of cells from the system, based on the and intensity of staining by the second version which used a was more than This was commercialized as the in by a at now by which by had become a large and of the development of flow cytometers through a in The first in this the in It used an and could measure and was to flow in apparatus built at MA) during an to a The first instrument illuminating from a single the second used both could over cells second and, using hardwired and cells less than of the The system incorporated and side which influenced by work done at also built a flow system for detection of and in it could from large and could a few The cytometers were but influenced the and of later instruments. By the time the Society for Analytical into in and (a of that bought were producing flow cytometers that could measure and light and in at least two several cells second, and with cell was also the by of its optical could make higher precision measurements of DNA content than could flow cytometers. DNA content analysis was attention as a of the of and other and, at least in due to the results of a in at the University of had begun to as for the of development of cells of the and system. and had a in the process although it was that a to be done in the area of fluorescent development to the of The late 1960s and early the development of a number of image differential leukocyte the based on work by the and a system based on research by the The incorporated field microscopes and cells with or in smears on later a which field image that were to DNA content measurement in cells, using and detection of using cytometers incorporating both field and microscopes in research but were less precise and less adapted for and even less than the early flow cytometers. these although system, which incorporated both illumination and illumination for could cells with light This of cell or was also as an to flow However, by the most development in static in the was the of the of microscopy to be in by and and a conventional microscope, a fairly large area of the specimen at any given a field stop the area in a or scanning microscope, the image is by the of from and the of this light are in different of with the that it become possible to high-resolution images of of a specimen, and to from a of microscopes are by flow cytometers at are of the cytometric the early commercial of allowed researchers who had not developed and built their own apparatus to of flow and in the to in a relatively small of and got done in any one was by the problems and/or clinical and also by the of and/or from one to This process some attention from of in by Peter and and in a video history by which was and influenced by a is available from the the on on the of with the aim of with in the in a in than in a flow the this and the of the instrument was the built by the at the Institute for Chemistry in This apparatus could at ultraviolet it was used to measure as intrinsic protein and and to the of Germany) as a DNA and as an RNA The flow cytometers developed at were by at other the Institute University the University of at and the University of an instrument was to flow cytometric analysis of also provided the for the growth of for flow that at National Laboratory flow was as a for The developed by and others at was by and been by since The processing recently into commercial from Corporation and mirrors work done beginning in the by et al. at the University of on flow to the development of progressively more apparatus for processing and for imaging cells in for use in Digital processing was used in in the early by with sufficient speed and were not these those built at could a large enough to the for the that became in flow cytometers during the instrument first described by and in cells in flow a in air the of a and measurements with sufficient sensitivity to early commercial version of this apparatus was by a later version was made by and an even later available from as the is now by in the small as the and the to reasonably high measurement precision and due at least in to their use of microscope which provided more light than was available in flow cytometers. the and late and its flow cytometers and in which measurements were made in flow and in which microscope were used to light This made it possible to use than lasers for the and power of and to into an clinical a system, was at clinical as were a small but analytical apparatus an and an instrument that included By the with the of for clinical had out the a analyzer using a with a lens for light which could make more measurements using a than were possible using more power in The was by the which included a relatively both were by the which both a and a with from a the there been a of and and these and relatively sources have been incorporated into flow cytometers. The use of lasers in was first described at the by the at least had incorporated sources into instruments. light at have also into as have lasers at now available as for both and and lasers are now available as ultraviolet the built at were computer computers were for most flow cytometers until the early by which time could the work of an of The was with a computer system built by a few computer were integrated into commercial flow with to for its and and and and later of the of flow cytometers become with the incorporating integrated and signal processing The development of and video in large in the and in the of which are in data for any of flow cytometers The used with flow cytometers had only or it to use to process with a large This the use of for While this approach is when or are it is to for modern in which measurements of or more be The is which is to data to a of at least the early et al. in the which data, the for and have developed their own to high-resolution data and that, as been the case for and techniques in flow will their to of in this measurement in the near which were for cell in flow require light and lasers are too but and lasers light the the of the in late the cell and the other will be at the Most are but this be an as as cell are flow cytometers to continue to in size and and hope that will in It is known that flow it possible to and over cells second, to cells that only one of 10 cells in to measure light at two or and in or more to measure with a precision than one and to and a few of fluorescent antibody to a cell It is less that it be difficult or to two or of these at but it is that will be new flow with than have already been used to single fluorescent and, are made available to the right for analysis of and The incorporated in some of these it possible to stop and of cells to be on an to and over at the cells over time is a for a flow but is using down in and cost from microscopes through scanning as those developed by Corporation MA) to microscope cytometers using and even less light The Society for Analytical was in and became the Society for Analytical in the provided flow with the computer people a in both and its it is to at least some of its working cytometric apparatus to in the and other problems in both developed and developing thus light the even it is only at the