Johan Sebastiaan Ploem

Johan Sebastiaan Ploem

name = Johan Sebastiaan Ploem
image_width = 200px
birth_date = birth date and age|1927|08|25
birth_place = Sumatra, Indonesia
nationality = Dutch
field = Advanced Microscopy; Molecular Diagnostics; Digital Painting
work_institutions = University of Miami; University of Amsterdam; University of Leiden
prizes = Fellow of the Papanicolaou Cancer Research Institute (1977) C. E. Alken Foundation award
footnotes = Member of the Society of Analytical Cytology, the Dutch Society of Cytology, the International Academy of Cytology and the Royal Microscopical Society, for which he served as president in 1986.

Personal life

Ploem was born on August 25, 1927 in Sawah Lunto, Sumatra, Indonesia, where his Dutch father was employed as a coal-mining engineer. At the age of two he returned with his parents to the Netherlands where he remained for the rest of his youth in Heerlen, a town in the south of the Netherlands. He started painting as a small boy and was educated in drawing and painting in Maastricht but after finishing high school opted to study medicine instead of art. During Ploem’s entire career painting has remained a factor in his life, even during extensive scientific work in medicine, medical research and advanced microscopy.


Ploem received his education at the University of Utrecht in the Netherlands, Harvard University and the University of Amsterdam. He has since then been employed by a number of academic institutions, including the University of Miami, the University of Amsterdam, and the University of Leiden, where he served as a professor at the Faculty of Medicine. He also cooperated with industry, in particular in the branch of optics and concentrated on research in image analysis, participating in a project aiming to automate cancer cell recognition.
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“Boil-in-the-bag rice”

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Fluorescence Microscopy

Fluorescence is a molecular phenomenon in which a substance absorbs light (excitation) and radiates light of longer wavelength (emission) for a very short period of time. When a fluorochrome absorbs light, energy in the form of photons is taken up, leading to excitation of electrons to higher energy states. The process of absorption of photons is extremely rapid and is immediately followed by a return to lower energy states, which is then accompanied by emission of photons, which can be observed if light is emitted in the visual range.This short duration (nanoseconds) distinguishes it from other forms of luminescence such as phosphorescence. The difference in wavelengths between the excitation and emission peaks is referred to as the Stokes shift. Fluorochromes with a large Stokes shift are easy to excite and to observe their emission in a fluorescence microscope. With transmitted light illumination, a small Stokes shift may make it impossible to illuminate a fluorochrome at its excitation peak and to observe the fluorescence colour at its emission peak.

Confocal laser scanning fluorescence microscopy enables two-photon excitation with photons of longer wave-length than the emitted light. The electron is then brought into its excited state with two photons of half the required energy, arriving simultaneously. In most cases the electron ends up in the same excited state as with normal single-photon excitation before it drops down to the ground state. Similarly, the fluorescence emitted is similar to that given off by normal single-photon excitation.

Many molecules, both non-organic and organic, show fluorescence emission, especially with excitation using high energy radiation. When plant or animal tissue is excited with UV-light very often a bluish fluorescence emission is observed which is called primary fluorescence, or autofluorescence. Naturally occurring substances often have very broad excitation and emission spectra. With the development of molecular biology, research has been focussed on special dyes with bright fluorescence to distinguish them from possible autofluorescence. The dyes used to selectively stain biologically important molecules are called fluorochromes. When conjugated to antibodies or nucleic acids, they are referred to as fluorescent markers or probes. Today fluorochromes are available with peak emissions in the violet, blue, green, orange, red and near-infrared regions of the spectrum. The possibility to excite fluorochromes with orange and red light and detect the red and infrared fluorescence with a photomultiplier or a CCD-camera has extended fluorescence microscopy beyond the visual range. The excitation and emission of a fluorochrome may shift with changes in cellular environment. Some dyes are especially chosen because their excitation or emission spectra are shifted, following changes in concentration of some ions in the intracellular medium such as calcium, natrium, and of the pH.

Early developments in epi-illumination fluorescence microscopy
For a review of the history of fluorescence microscopy the reader is referred to Kasten. Epi-illumination (vertically incident exciting light) was used already by Policard and Paillot. Several instruments were made by Leitz (Leica), Bausch & Lomb, Reichert and Zeiss, which were partially based on suggestions by Ellinger and Hirt, Singer and Mehler and Pick. An early Leitz (Leica) fluorescence epi-illumination system was described by Haitinger. For more details on the evolution of epi-illumination fluorescence microscopy the reader is referred to Rost and for early applications of incident-light fluorescence microscopy to Hauser.
A major contribution in epi-illumination fluorescence microscopy was the introduction of a dichromatic mirror for incident illumination with UV light by Brumberg and Krylova. Epi-illumination has definite optical advantages because, unlike transmitted illumination where the condenser and the objective have independent optical axes which must be perfectly aligned. The objective functions both as a condenser and as a light-collecting objective, avoiding all alignment problems. The separation of fluorescence emission from excitation light, using a dichroic beamspitter, is much easier than with transmitted light fluorescence microscopy, These possibilities did, however, not lead to a general acceptance by industrial microscope manufacturers of epi-illumination for routine fluorescence microscopy. The main reason for this could have been that transmitted-light darkfield UV excitation gave already excellent results in most applications of fluorescence microscopy. Its replacement by UV epi-illumination would not have had significant advantages. The use of transmitted light using a darkfield condenser remained the industry standard until the late sixties. The rapidly growing interest in molecular biology, however, led to the development of many (monoclonal) antibodies for the detection of important macromolecules in the cell. To study the detailed morphological location of several macromolecules in the cellular organelles, fluorescent markers with different colours were increasingly used. UV excitation – as used traditionally for fluorescence microscopy – was not optimally suited for detecting multiple fluorochromes simultaneously in a cell.

Around 1962 Ploem started work in collaboration with Schott on the development of dichroic beamspitters for reflection of blue and green light for fluorescence microscopy using epi-illumination. At the time of his first communication [1965] and publication on epi-illumination with narrow band blue and green light, he was not aware of the development of a dichroic beamsplitter for UV excitation with incident light by Brumberg and Krylova. Neither was the Leitz company, from which he obtained an "Opak" epi-illuminator with a neutral beamsplitter. This illuminator had to be modified to contain a slider in the incident light path containing four dichroic beamsplitters, for respectively UV, violet , blue and green excitation light. This device, developed at the University of Amsterdam, permitted the easy exchange of different dichroic beamsplitters in the incident light path. The wavelength of the excitation light could thus be easily and rapidly changed.
Soon it became clear that excitation with narrow-band blue and green light opened optimal possibilities for the detection of the widely used immunofluorescence labels fluorescein-isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). The use of blue and green excitation also minimized autofluorescence of tissue components, an undesired effect encountered with conventional transmitted illumination with UV light. FITC could now be excited with narrow band blue light (using a band interference filter with a half width of 16nm), close to the excitation maximum at 490nm (long wavelength blue), with clear observation of the green fluorescence peak emission at 520nm. Autofluorescence of tissue components was minimized (Fig. 2a, b) resulting in a high image contrast. Excitation of FITC near its excitation maximum enabled such an efficient excitation that even a mercury high-pressure arc lamp, having no strong emission peak in the blue wavelength range, could be used. Furthermore epi-illumination with a green reflecting dichroic mirror enabled for the first time the excitation of Feulgenpararosaniline with the strong mercury emission line at 546nm (Fig. 3a, b).
In his second publication on the multi-wavelengths epi-illuminator, describing a Leitz prototype with four dichroic beam-splitters, Ploem could acknowledge the contribution of Brumberg and Krylova. The inaccessibility of Russian research in that time period, and the absence of any major industrial development of epi-fluorescence microscopy in Russia or East Germany was the reason that Leitz had not been aware earlier of such a development. The possibility to introduce epi-illumination with UV light, although useful for several applications, had not been a motive for a new technological development at Leitz, since they had already excellent transmitted dark field UV excitation available. The increasing world-wide use of routine immunofluorescence microscopy in medical diagnosis and molecular biology research could, however, profit from the new possibility of epi-illumination using narrow band excitation with blue and green light. Since standard high-pressure mercury arc lamps could be used, this seemed a practical proposition. Subsequently Leitz developed a novel multi-wavelength fluorescence epi-illuminator (Leitz PLOEMOPAK) with four rotating dichroic beamsplitters for respectively UV, violet, blue and green light. In successive generations of Leitz illuminators (containing four dichroic beamsplitters) barrier filters and a rotating turret for excitation filters were added. Finally an elegant epi-illuminator was constructed by Kraft containing multiple sets of a combination of an excitation filter, a dichroic beamsplitter and a barrier or emission filter, mounted together in a filter cube, also called filter block (Fig. 4). Since this illuminator permitted the filter cubes to be rapidly turned into the optical light path, multi-wavelength illumination of the same section of tissue became a practical proposition. Moreover, the four filter cubes in the illuminator could be exchanged by the user (Fig 1). Different sets of four filter cubes could be assembled, chosen from many filter cubes, containing combinations of excitation, barrier filters and dichroic beamsplitters, developed for different applications. Following suggestions by Ploem, Leitz also produced an inverted microscope with epi-illumination. For a review of the Leitz PLOEMOPAK illuminator for multi-wavelength fluorescence microscopy, the reader is referred to a review by Pluta.
The Leitz (Leica) filter cube system was so efficient that now, >45 years later, similar types of filter cubes are still used by most microscope manufacturers for multi-wavelength fluorescence microscopy. This development finally led within Leica to the development of automated multi-wavelength fluorescence epi-illuminators accommodating eight filter cubes for various wavelength ranges. When switching between filter cubes, pixel shift on the computer monitor is avoided or stays below the resolution power of a 35mm film due to a 0-pixel shift technology. This illuminator is now used for fluorescence in situ hybridisation methods (FISH) in the study of chromosomes.Ploem, van der Ploeg and Ploem and Nairn and Ploem further explored the filter combinations that had to be developed for many biomedical applications. This was done in collaboration with Schott and Leitz. Rygaard and Olson developed a novel shortwave pass high transmission interference filter with a very high transmission for blue light and a sharp cut-off towards wavelengths longer than 490nm.Ploem combined this SP filter with a 1mm GG 455 filter from Schott, which blocked UV excitation, and suggested the development by Balzers of a similar filter ( SP 560 = KP560) for excitation with green light and a filter for excitation with violet light (LP 425 = KP 425). The latter filter was applied in the investigation of neurotransmitters. In Fig. 6a, b the resulting blue fluorescence can be observed. From the optical industry side, early contributions and reviews on these developments were written by Kraft, Walter,Trapp and Herzog.
The main classes of filters used in epi-illumination fluorescence microscopy were defined in (1) the primary excitation filters LP (long pass) and SP (short pass) – in the German literature known as KP filter – and (2) the secondary filters such as barrier filters and emission filters. The latter were also described as fluorescence selection filters; these are for instance used to limit the observation to the peak fluorescence at 520nm of FITC. A recent extensive review on filters for fluorescence microscopy has been given by Reichman. Cormane was the first to demonstrate that narrow band blue light epi-illumination of the fluorescent label FITC gave an optimal contrast in immunofluorescence studies of human skin disease. Transmitted-light excitation with UV light used to cause such a strong auto-fluorescence of elastic fibres in the skin, so that visualization of the fluorescent antibody was severely hindered. The pioneering work of Leitz in epi-illumination fluorescence microscopy coincided in the seventies with a worldwide increase in the application of immunofluorescence and other molecular biology methods like FISH in medical diagnosis and research. Hijmans et al were the first to demonstrate the usefulness of the new Leitz multi-wavelength excitation epi-illuminator for the selective detection of certain classes of immunoglobu lines in cells, using antibodies conjugated with green fluorescent FITC and red fluorescent TRITC. They applied the two-wavelengths excitation method using blue and green light and the selection of the peak fluorescence of FITC by an emission filter at 520nm (Fig. 7). Brandtzaeg and Klein et al. made similar discoveries in identifying immunologically important cell types, using two-wavelength excitation with the Leitz epi-illuminator. In a staining of blood with "rosette" formation, the two-wavelengths excitation method using UV and green light can demonstrate erythrocytes around a mononuclear cell (Fig. 8).

Cell Screening / Image Analysis

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Digital Painting

Early days
Bas Ploem started painting as a small boy, making copies of paintings in the house of his parents in Heerlen, a town in the south of the Netherlands. He lived there until he was eighteen. While still at secondary school he used to take the train to the nearby town of Maastricht to attend an evening course in drawing and painting at the “Kunstnijverheidsschool Maastricht”, which was later converted into the ‘Academie Beeldende Kunsten Maastricht’ (Academy of Art, Maastricht). After finishing high school he had the chance to opt for a further education in art, but decided to study medicine instead.

Meeting with the painters Frits and Yves Klein in Paris
During his medical studies at the University of Utrecht in the Netherlands, he had the opportunity to work as an intern in the Hospital Broussais in Paris under the supervision of professeur Pasteur Vallery-Radot, the grandson of Louis Pasteur. Ploem’s presence in Paris was important for his knowledge and interest in art since he could regularly visit his cousins in Paris, the painter Frits Klein and his son Yves. He visited the Kleins when Yves was making his first monochromes.

Analogue paintings
During Ploem’s entire career, painting remained a factor in his life, its intensity varying with the workload, first in medicine and later in medical research. On the basis of this lifelong activity as a ‘Sunday painter’ an exposition of his analogue paintings was organized in 1992 in Pulchri Studio in The Hague on the occasion of his retirement as a professor from Leiden University, The Netherlands.

Computer image analysis for the creation of digital graphics
In the last years of his activities at the faculty of medicine at Leiden University, he concentrated on research in image analysis. He was asked to participate in a European project with the aim of automating cancer cell recognition using computer analysis. It concerned a collaborative project with the German optical company Leitz/Leica Microsystems, and the Institute for Mathematical Morphology in Fontainebleau, France. Together with a team, Professor Jean Serra at this institute had developed an image analysis method, now internationally known as ‘Mathematical Morphology’ (MM). With his experience as a analogue painter, Ploem quickly saw the possibility of also applying the methods of mathematical morphology to human faces, landscapes, buildings and flowers. Instead of looking at cells, the computer programmed for image analysis, can also look at the optical information of an image scanned into its memory by e.g. a camera. Unfortunately not Mathematical Morphology program was then available for use on a PC. It was only years later, on a visit to the firm Leica Imaging Systems in Cambridge, England, that Ploem accidentally saw a CD with the Mathematical Morphology program that could run in Windows on a PC. He received a copy of this program on loan and immediately started to use it for digital painting experiments.

Mountain flowers as the first topic for digital image analysis
Since Ploem is a nature enthusiast, he started with the application of mathematical morphology programs to the image analysis of meadows covered with mountain flowers. To get inspiration for his art work he frequently made nature walks in the region of the Pyrenees known as the Cerdagne, and specifically in the Eyne valley also known as the ‘La Vallée des Fleurs’. These first digital graphics of nature scenes were shown in his exposition at an regional art centre in the Pyrenees (Ossega, June, 1997).

Scientific interest in computer graphics created with mathematical morphology
As he was probably the first person to systematically use mathematical morphology for the creation of digital art, Ploem’s work attracted international attention and he was invited as a plenary speaker at an international mathematical conference in Amsterdam in 1998 to explain his new type of digital art. He also received an invitation to show his art work in an exposition at this conference. The organisers of this meeting asked Ploem to write a chapter on his novel technique for digital art in a book (Kluwer, ISBN 0-7923-5133-9) that was published on the occasion of this meeting.

Exposition at universities
When scientists in France became more aware of Ploem as a digital artist, they invited him for a symposium on ‘Art et Science’ at the University of Caen, France (April, 2001). At the art exposition connected with this symposium, he presented 6 digital graphics that were dominated by chaotic transformations of rock art themes. A similar invitation was made by the University of Basel in Switzerland (April, 2002). His exposition of digital graphics in Basel also showed works which were created with the so called ‘watershed transformation’ of Mathematical Morphology, resulting in pictures resembling mountain ranges.

Acceptance of digital art
Generally spoken, digital art still suffers from a lack of acceptance by a wider public. One of the reasons may be that this type of art is sometimes difficult to understand or to explain. For Ploem as one of the early developers of digital graphics using image analysis, it was encouraging that the well known museum ‘Fondation Beyeler’ in Basel, Switzerland already had a separate curator for digital art in 2002. At the time of his exposition in Basel he was invited for a discussion with this curator about the future of digital art. They discussed the massive interest in digital art while still waiting for the emergence of more significant digital art by young artists who have been growing up with computers. The words ‘Digital Art’ typed in together in the advanced search made of Google now results in more than 300 million references!

Portraits and architecture
In recent years Ploem has spent time studying more conventional topics in art like portraits and scenes with architecture. The availability of special mathematical morphology software such as the ‘Top Hat’ transformation made his attempts at creating portraits rather interesting. This famous algorithm was developed by Fernand Meyer. As an assistant of Professor Jean Serra in the earlier mentioned Institute of Mathematical Morphology, he came to the University of Leiden, the Netherlands in order to write programs for the recognition of cancer cells visualized under the microscope. He is now director of this institute in Fontainebleau. The ‘Top Hat’ algorithm enhances the recognition of structures mainly based on contrast in the image rather than on edge detection. If applied to a human face it makes a novel type of drawing that is different from a drawing made by most artists. If followed by several other MM image transformations, it can produce (digital) portraits characterized by structures that were not directly evident in the original picture.
For the painting of scenes with architectural components Ploem used a program for 3D rendering, that permits structures drawn in 2 dimensions to be visualised in 3D. This enabled him to effectively use perspective in some of his paintings.Whatever the future judgment about the artistic value of Ploem’s art form will be, it is clear that he has created novel approaches for the creation of digital graphics on the basis of image analysis, and as such he can be considered as a real pioneer in this field.

Awards and Recognition

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Bibliography Fluorescence Microscopy

Brandtzaeg, P.: Two types of IgA Immunocytes in Man. Nature (New Biology) 243, 142–143,1973.
Brumberg, E. M., Krylova, T. N.: O fluoreschentnykh mikroskopopak. Zh. obshch. biol. 14, 461, 1953.
Cormane, R. H., Szabo, E., Hauge, L. S.: Immunofluorescence of the skin: the interpretation of the staining of blood vessels and connective tissue aided by new techniques. Br. J. Derm. 82, Supplement 5, 26–43, 1970.
Ellinger, P., Hirt, A.: Mikroskopische Untersuchungen an lebenden Organen. I. Mitteillung: Methodik: Intravitalmikroskopie. Zeitschrift für Anatomie und Entwicklungs- Geschichte 90, 791–802,1929.
Ellinger, P., Hirt, A.: Eine Methode zur Beobachtung lebender Organe mit starksten Vergrösserung im Lumineszenzlicht (Intravitalmikroskopie). Handbuch der Histologischen Arbeitsmethoden 15, 1753–1764. Urban & Schwarzenberg, Berlin & Vienna, 1930.
Haitinger, M.: Fluoreszenzmikroskopie. Akademische Verlagsgesellschaft, Geest & Portig K.G., Leipzig, 1959.Hauser, F.: Das Arbeiten mit auffallemdem Licht in der Mikroskopie, Academische Verlagsgesellschaft, Geest & Portig K.G., Leipzig, 1960.
Herzog, F., Albini, B., Wick, G.: Comparison of filters used in immunofluorescent staining procedures with fluoresceinisothiocyanate (FTIC) conjugates. Journal of Immunol.3, 211–220, 1973.
Hijmans, W., Schuit, H. R. E., Klein, F.: An immunofluorescence procedure for the detection of intracellular immunoglobulins. Clin. exp. Immunol. 4, 457–472, 1969.
Hijmans, W., Schuit, H. R. E., Hulsing-Hesselink, E.: An immunofluorescence study on intracellular immunoglobulins in human bone marrow cells. Ann. N. Y. Acad. Sci. 177: 290–305, 1971.
Kasten, F. H.: The origins of modern fluorescence microscopy and fluorescence probes. In: E. Kohen and J. G. Hirschberg (eds), Cell Structure and Function by Microspectrophotometry. Academic Press, San Diego, 1989.
Klein, G., Gergely, L., Goldstein, G.: Two-colour immunofluorescence studies on EBV determined antigens. Clin. exp. Immunol. 8, 593–602, 1971.
Kraft, W.: Die Technologie des Fluoreszenzopak, Leitz Mitt. Wiss. u. Techn. IV/6, 239–242, 1969.
Kraft, W.: Ein neues FITC-Erregerfilter für die Routinefluoreszenz. Leitz Mitt. Wiss. u. Techn. V/2, 41–44, 1970.
Kraft, W.: Fluorescence Microscopy and Instrument Requirements. Leitz Mitt. Wiss. u. Techn. V/7, 193–206, 1972.
Mehler, L., Pick, J.: Über ein Mikroskop zur Untersuchung lebenden Gewebes. Vorläufig Mitteilung. Anatomische Anzeiger 75, 234–240, 1932.
Nairn, R. C.: Fluorescent Protein Tracing. E. & S. Livingstone Ltd. Edinburgh and London, 1969.
Nairn, R. C., Ploem, J. S.: Modern microscopy for immunofluorescence in clinical immunology. Leitz Sci. a. Techn. Information VI/3: 91–95, 1974.
Pick, J.: Über ein Mikroskop zur Untersuchung lebenden Gewebes. II. Mitteillung. Zeitschr. f. wiss. Mikroskopie 51, 257–262, 1934.
Ploeg, M. van der, Ploem, J. S.: Filter combinations and light sources for fluorescence microscopy of quinacrine mustard or quinacrine stained chromosomes. Histochemie 33: 61–70, 1973.
Ploem, J. S.: Die Möglichkeit der Auflichtfluoreszenzmethoden bei Untersuchungen von Zellen in Durchströmungskammern und Leightonröhren. Xth Symposium d. Gesellschaft f. Histochemie, 1965. Acta Histochem. Suppl. 7, 339–343, 1967.
Ploem, J. S.: The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incident light. Zeitschr. f. wiss. Mikroskopie 68, 129–142, 1967.
Ploem, J. S.: A study of filters and light sources in immunofluorescence microscopy. Ann. N. Y. Acad. Sci. 177, 414–429, 1971.
Ploem, J. S.: Immunofluorescence microscopy. In: E. H. Beutner and R. J. Nisengard
(eds.), Defined Immunofluorescence in Clinical Immunopathology. pp 248–270. Manual for School of Medicine, State University of Buffalo, 1971.
Ploem, J. S.: The microscopic differentiation of the colour of formaldehyde-induced fluorescence. Prog. Brain Res., 34: 27–37, 1971.
Ploem, J. S.: Immunofluorescence microscopy. In: E. H. Beutner, T. P. Chorzelski, S. F.Bean and R. E. Jordon (eds.), Immunopathology of the skin, Labeled Antibody Studies. pp. 248–270. Dowden, Hutchinson and Ross Inc., Stroudsburg, Penn. USA, 1973.
Ploem, J. S.: Fluorescence Microscopy. In: A.J. Lacey (ed.), Light Microscopy in Biology: A Practical Approach, 2nd Edition. pp 163–186. Oxford University Press, 1998.
Ploem, J. S., Tanke, H. J., Al, I., Deelder, A. M.: Recent developments in immunofluorescence microscopy and microfluorometry. In: W. Knapp, K. Holubar and G. Wick (eds), Immunofluorescence and related staining techniques. pp. 3–10. Elsevier/North-Holl. Biomedical Press, Amsterdam, New York, 1978.
Ploem, J. S., Cornelese-ten Velde, I., Prins, F. A., Bonnet, J.: Reflection-Contrast Microscopy: An Overview. Proceedings RMS 30, 185–192, 1995.
Ploem, J. S., Cornelese-ten Velde, I., Prins, F. A., Bonnet, J., Heer, E. D.: Reflection-Contrast Microscopy. Leitz Sci. u. Techn. Inform. XI/4, 98–113, 1997.
Pluta, M.: Handbook: Advanced Light Microscopy. Specialized Methods Vol. 2. Elsevier. Amsterdam, 1989.
Policard, A., Paillot, A.: Etude de la sécrétion de la soie à I'aide des rayons ultraviolets filtrés (lumière de Wood). Comptes Rendus de l'Académie des Sciences Paris 181, 378–380, 1925.
Reichman, J.: Handbook of optical filters for fluorescence microscopy. Chroma Technology Corporation, 2000.
Rost, F. W. D.: Quantitative fluorescence microscopy. Cambridge University Press, 1991.
Rygaard, J., Olson W.: Interference filters for improved immunofluorescence microscopy. Acta Path. Microbiol. Scand. 76, 14, 1969.
Schönenborn, J.: Leica Scientific and Technical Information. CDR 1, 37–46, 1988.
Singer, E.: A microscope for observations of fluorescence in living tissues. Science 75, 289–291, 1932.
Trapp, L.: Über Lichtquellen und Filter für die Fluoreszenzmikroskopie und über die Auflichtfluoreszenzmethode bei Durchlichtpräparaten. Acta Histochemica suppl. VII, 327–338, 1967.
Walter, F.: Eine Auflicht-Fluoreszenz-Einrichtung für die Routinediagnose. Leitz Mitt. Wiss. u.Techn. IV/6, 186–187, 1968.
Walter, F.: Fluoreszenzmikroskopie in Biologie und Medizin. Leitz Mitt. Wiss. u. Techn. V/2, 33–40, 1970.

Bibliography Cell Screening / Image analysis

Ploem JS, Verwoerd N, Bonnet J, and Koper G, J.: An automated microscope for quantitative cytology combining television image analysis and stage scanning microphotometry, Histochem Cytochem 27 (1979) 136-143.
J.J. Ploem, et al, Clinical Cytometry and Histometry, “Leytas – A Cytology Screening System Using The New Modular Image Analysis Computer (MIAC) From Leitz”, pp. 25-35 (1987).
I Al and JS Ploem: Detection of suspicious cells and rejection of artefacts in cervical cytology using the Leyden Television Analysis System, Journal of Histochemistry & Cytochemistry, Volume 27, Issue 1, pp. 629-634, 01/01/1979
JS Ploem, N Verwoerd, J Bonnet and G Koper: An automated microscope for quantitative cytology combining television image analysis and stage scanning microphotometry, Journal of Histochemistry & Cytochemistry, Volume 27, Issue 1, pp. 136-143, 01/01/1979
R. Strohmeier, H. Naujoks, A.M.J. van Driel-Kulker, und J.S. Ploem: Labortest eines vollautomatischen Bildanalysesystems für die vaginalzytologische Diagnostik, Zeitschrift Archives of Gynecology and Obstetrics, Springer Berlin/Heidelberg, Volume 254, Numbers 1-4, Dezember 1993

"'Additional Publications

PLOEM, J. S.: Die Möglichkeiten der Auflicht-Fluoreszenzme thode bei Untersuchungen von Zellen in Durchstromungs.. kammern und Leightonrohren. 10. Symposium d. Ges. F. Histochem. Nijmegen 1965. Acta histochem., Supply WI (1967), 339—343.
PLOEM, J.S.: Ein neuer llluminator-Typ für die Auflicht-Fluoreszenzmikroskopie. Leitz-Mitt. Wiss. u. Techn. 4/8 (1969), 225.
PLOEM, J. S.: A new microscopic method for the visualization of blue formaldehydeinduced catecholamine fluorescence. Arch. mt. Pharmacodyn. 182/2 (1969), 421.
PLOEM,J.S.: Standards for fluorescence microscopy. In: E.J. Holborow (ed.), Standardization in Immunofluorescence Blackwell Scientific Publication, Oxford and Edinburgh (1970).
PLOEM, J.S.: Quantitative Immunofluorescence, In: Standardization in Immunofluorescence. E.J. Holborow ed. p. 63—73. (1970). Blackwell Sd. PubI. Oxford and Edinburgh.
PLOEM, J.S., J.A. STERKE, J. BONNET, H. WASMUND: A microspectrofluorometer with epi-illumination operated under computer control. J. Histochem. Cytochem. 22 (1974) 668—677.
PLOEM, J.S.: General introduction. In: Fifth international conference on immunofluorescence and related staining techniques (eds. W. Hijmans and M. Schaeffer). Annals of the New York Academy of Sciences 254 (1975) 4.
PLOEM, J.S.: Automation of Immunofluorescence. In: Rapid methods and automation in microbiology (eds. H. H. Johnston and S. W. B. Newsom). 2nd International Symposium, Cambridge, England, September 19—25, 1976, (Learned Information (Europe) Ltd.), Oxford, New York 1976, p. 193—196.
PLOEM, J. S.: Quantitative fluorescence microscopy In: “Analytical and quantitative methods in microscopy” (eds. G.A. Meek and H. Y. Elder) Cambridge Univ. Press. Cam bridge-London, New York-Melbourne (1977) p. 55—89.
PLOEM: Identification of monocytes in suspensions of mononuclear cells. Blood 48 (1976), 139—147.
Wouters CH, Van der Weij JP, Koper GJM, Daems WT, De Vries E, and Ploem JS: Measurement of single-cell DNA synthesis by pokeweed mitogen-stimulated mono-nuclear cells with combined light and scanning electron microscopy, Immunology 58 (1986) 79-85.

Tanke HJ, Rothbarth PH, Vossen JMJJ, Koper GJM, and Ploem JS: Flow cytometry of reticulocytes applied to clinical hematology, , Blood 61 (1983) 1091-1097.
Dresden MH, Rotmans JP, Deelder AM, Koper G, and Ploem JS: Automated measurement of proteinase activity with a fluorogenic substrate using an inverted fluorescence microscope, An Biochem 126 (1982), 170-173.
Koper GJM, Bonnet J, Christiaanse JGM, and Ploem JS: An epi-illuminator/detector unit permitting arc lamp illumination for fluorescence activated cell sorters, Cytometry 3 (1982) 10-14.
Ploem JS, Koper GJM, Bonnet J, and Christiaanse JGM: An epi-illumination system added to fluorescence activated cell sorters, Cytometry 2 (1981) 122.
Koper GJM, Christiaanse JGM, Blanken R, and Ploem JS: Additions to a FACS-IV cell sorter, Cytometry 2 (1981) 109.
Tanke HJ, Nieuwenhuis IAB, Koper GJM, Slats JCM, and Ploem JS: Flow cytometry of human reticulocytes based on RNA fluorescence, Cytometry 1 (1980) 313-320.
Deelder AM, Koper G, De Water R, Tanke HJ, Rotmans JP, and Ploem JS: Automated measurement of immunogalactosidase reactions with a fluorogenic substrate by the aperture defined microvolume measurement method and its potential application to schistosoma mansoni immunodiagnosis, J Immunol Meth 36 (1980) 269-283.
Johan S. Ploem: Laser Scanning fluorescence microscopy, Applied Optics, Vol. 26, Issue 16, pp. 3226 -, 1987
J.S. Ploem, H.J. Tanke: Fluorescence Microscopy, BIOS Scientific Publ; 1 edition (Nov 11 2004)
J.S. Ploem and H.J. Tanke: Introduction to fluorescence microscopy,. Oxford University Press, New York, 1987.

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