Johan Sebastiaan Ploem
This chapter describes the invention and development of multi wavelengths epi illumination fluorescence microscopy ed fluorescence 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, Harvard univ ersity,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.
Ploem's prototype fluorescence epi-illuminators and microscopes form a part of the permanent exposition of the Dutch National Museum of Science and Medicine (Boerhaave Museum, Leiden, the Netherlands) A comprehensive report has been made for this museum presenting published reviews of Ploem's contributions to fluorescence microscopy, citations and web links to his inventions and developments. From this report a website has been made:
Ploem-fluorescence-microscopy.net or Ploem-fluorescence-microscopy.com
Ploem described a new sub-category of digital art. Transforming digital algorithms were used to create novel pictorial handwritings for digital painting. See website:
Review from the WEB
J.S. PLOEM AND E. LEITZ GERMANY 50 YEARS OF EPIFLUORESCENCE'
James Averill FRAEN COMPANY: Published on May 8, 2014
It has been just about 50 years since Dr. Ploem and E. LEITZ Wetzlar revolutionized fluorescence microscopy.
Prior to this fantastic innovation, all fluorescence microscopy was primarily performed in transmitted light using glass exciter filters and oiled darkfield condensers!
Over the decades, all microscope makers now offer their own proprietary Ploem illuminators.
We also offer the FRAEN LED epi fluorescence attachments for modern infinity corrected microscopes. Several makers offer LED and metal halide fluorescence illumination modules but these require the microscope maker's proprietary Ploem illuminator and filter cubes.
Biography J.S. Ploem
Bas Ploem - Purdue University Cytometry Laboratories www.cyto.purdue.edu/cdroms/cyto10a/cytometryhistory/ind... Bas Ploem. Professor Ploem is renowned for his research in fundamental light microscopy, especially his leadership in fluorescence microscopy. He developed This curriculum vitae was published at the occasion of Johan Sebastian Ploem receiving the Ernst Abbe Medal and Award from the New York Microscopical Society in the USA, November 17, 1998. Professor Ploem is renowned for his research in fundamental light microscopy, especially his leadership in fluorescence microscopy. He developed the first four-wavelength vertical fluorescence illuminator for excitation with a choice of narrow-band ultraviolet, violet, blue or green illumination. This epi-illumination instrument was first marketed for general fluorescence microscopy by Leica under the trade name “Ploem-opak.” The Ploem four wavelength epi-fluorescence illumination systems quickly became a standard for all major optical microscope manufacturers. Today, it is the dominant fluorescence microscopy technology. It is widely used in medicine, biology, and industry. This development significantly contributed to progress in cellular immunology and chromosome genetics. Later he developed, together with Leica, an improved optical design for reflected light microscopy for use in the biological and medical sciences. He proposed the name “Reflection-Contrast Microscopy” for this optical system. This microscope system produces images with very high definition and is successfully applied to thin sections in Immuno-cytochemistry applications. Professor Ploem’s collaboration with Leica also lead to a new instrument for correlative microscopy of the same specimen with fluorescence microscopy and scanning electron microscopy (1977). In this instrument, a fluorescence microscope system was built into the vacuum chamber of a scanning electron microscope, permitting simultaneous observation of the same specimen With LM and SEM. The early development of computer-operated microscopes was also advanced by Professor Ploem’s significant contributions. For automated image analysis of cervical specimens, a new fully automated computer-operated microscope, the AUTOPLAN, was developed by Leica in collaboration with Professor Ploem. By combining automated microscopes with image analysis software developments, Ploem and his team catalyzed development of several commercial systems for automated cytology. With his coworkers at Leiden University, one of the first systems for automated cervical cytology screening (LEYTAS) was developed in collaboration with Leica and the Institute for Mathematical Morphology (Fontainebleau, France). In a collaboration with Zeiss, a laser scanning fluorescence microscope was tested as early as 1980. Johan S. Ploem is Professor Emeritus at Leiden University, the Netherlands. He is a graduate of Utrecht University, the Netherlands, receiving an MD in 1972. He worked as Intern in the Broussais Hospital (Paris, France) with Professor Pasteur Valery-Radot. In 1963, Dr. Ploem was elected a Fulbright Fellow for Study at the Harvard University School of Public Health, receiving a Master of Public Health degree Cum Laude in 1954. He obtained a Ph.D. degree in 1967 from the University of Amsterdam, the Netherlands. Professor Ploem has served as visiting lecturer or professor at various universities: Dundee. Scotland; University of Florida, USA; Monash University, Melbourne, Australia; University of Beijing, China; and at the Free University of Brussels, Belgium. In 1980, he was appointed to a professorship in the Department of Cytochemistry and Cytometry at Leiden University. He retired from that position in 1992.Numerous honors and awards have been bestowed on Professor Ploem. In 1976 he was elected to the honorary Fellowship of the Royal Microscopical Society, Oxford England. In 1977 he received a Fellowship to the Papanicolaou Cancer Research Institute in Miami, Florida, and in 1979 a Fellowship to the Institute for Cell Analysis at the University of Miami, Florida. In 1982, he was the co-recipient of the C. E. Alken Foundation Award, Switzerland. In 1993, he was elected as the first Honorary Member of the International Society for Analytical Cytology. In 1993, Professor Ploem held the Erica Wachtel Medal Lecture at the British Society for Clinical Cytology meeting. In 1994, the European Society for Analytical Cellular Pathology established a Conference Keynote “Ploem” Lecture for invited scientists at its future general meetings. The International Society of Analytical Cytology invited Professor Ploem to present its inaugural “Robert Hooke” lecture. In 1995, he was invited by the Royal Microscopical Society to give the inaugural CYTO lecture. Professor Ploem has presented more than 200 invited lectures at Symposia and conferences outside of the Netherlands. He authored or co-authored more than 250 scientific publications. Professor Ploem’s memberships include the Council of the Dutch Society for Clinical Cytology; the Royal Microscopical Society; the Council of the International Society for Analytical Cytology; the Board of the National Foundation for Scientific Research (Belgium); the Royal Society of Medicine, England; the International Council on Automated and Quantitative Cytology; the Research Section “Krebsfrueherkennung” in Cytology and Hematology of the Bundesministerium fuer Forschung und Technology, Germany; the “Cell Board Subcommittee” of the Medical Research Council; and the Standing Steering Committee on Biomedical Image Processing of the IEEE Computer Society, USA. He is Emeritus Editor of the Journal of Analytical Cellular Pathology.
FLUORESCENCE MICROSCOPY  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. 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 good 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 separation of fluorescence emission from excitation with visible light (blue, green), using a dichroic beam splitter, is much easier than with transmitted light fluorescence microscopy, The rapidly growing interest in molecular biology, however, led to the development of new fluorescence markers like FITC and TRITC, that must be optimally excited with long wave blue light and green visible light. To study the detailed morphological location of several macromolecules in the cellular organelles, multiple 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 beam splitters for reflection of blue and green light for fluorescence microscopy using epi illumination. At the time of his first publication  on fluorescence microscopy using epi illumination with narrow-band blue and green light, he was not aware of the development of a dichroic beam splitter for UV excitation with incident light by Brumberg and Krylova . Neither was the Leitz company, 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 tetramethyl rhodamine 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 16 nm), close to the excitation maximum at 490 nm (long wavelength blue), with clear observation of the green fluorescence peak emission at 520 nm. 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 Feulgen pararosaniline with the strong mercury emission line at 546 nm (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 and Zeiss 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. 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 [3,6.7]. 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. The increasing worldwide use of routine immunofluorescence microscopy in medical diagnosis and molecular biology research could, however, profit from the new possibility @9 of epi-illumination permitting using narrow band excitation with blue and green light  . An important step for studies with live cells, was the design by Ploem of the first inverted fluorescence microscope with a multi wavelength epi illuminator under the microscope stage.. This provides free working stage above the microscope stage for eventual manipulation of the cells. . Following these suggestions by Ploem, Leitz also manufactured an inverted microscope with epi-illumination.
Fig. 3a: Liver tissue. Nuclei stained with Feulgen-pararosanilin for DNA, and visualized with transmitted green light. This stain was known as absorbing stain and not known to be fluorescent. One on the nuclei is illuminated with incident narrow-band green light (546 nm) resulting in a red fluorescence emission.
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 35 mm 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 . Fluorescence microscopy of formaldehyde induced neurotransmitter CA fluorophores can be observed for the first time as blue, by using a special dichromatic mirror . The independent detection of FITC (using blue excitation light) and of TRITC (using green excitation light) in the same cell uis possible with two dichromatic mirrors 
Fig. 6b: Same tissue and staining as Fig. 6a: Epi-illumination with narrow-band violet excitation light (LP 3mm GG 400 and SP(KP)425 interference filter), a dichroic beam splitter 495 nm, reflecting violet light and a barrier filter LP460nm. The filter cube permitted for the first time the observation of blue fluorescent adrenergic nerve fibers, distinctly different from yellow fluorescent mast cells (Ploem, 1971).
 Brumberg EM, Krylova TN: O fluoreschentnykh mikroskopopak. Zh. obshch. biol. 14: 461 (1953).
 Ploem JS: 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).
 The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incident light JS Ploem Z Wiss Mikrosk 68 (3), 129-142 246 1967
 Cormane RH, Szabo E, Hague LS: 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).
 JS Ploem A study of filters and light sources in immunofluorescence microscopy, Annals of the New York Academy of Sciences 177 (1), 414-429
 Walter F: Eine Auflicht-Fluorescenz-Einrichtung für die Routinediagnose. Leitz Mitt. Wiss. u. Techn. IV/6: 186–187 (1968).
 Pluta M: Handbook: Advanced Light Microscopy. Specialized Methods Vol. 2. Elsevier, Amsterdam (1989).m,
 Kraft W: Die Technologie des Fluoreszenzopak. Leitz Mitt. Wiss. u. Techn. IV/6: 239–242 (1969).
 Nairn RC, Ploem JS: Modern microscopy for immunofluorescence in clinical immunology. Leitz\ Leitz-Mitt. Wiss. u. Techn 4, 225-238 51 1969
 Multiple fluorescence in situ hybridization PM Nederlof, S Van der Flier, J Wiegant, AK Raap, HJ Tanke, JS Ploem, ...Cytometry 11 (1), 126-131 281 1990
 Ploem JS: The microscopic differentiation of the colour of formaldehyde-induced fluorescence. Prog. Brain Res. 34: 27–37 (1971).
 Hijmans W, Schuit HRE, Hulsing-Hesselink E: An immunofluorescence study on intracellular immunoglobulins in human bone marrow cells. Ann. N.Y. Acad. Sci. 177: 290–305 (1971).
Bas Ploem started painting as a small boy., While still at secondary school he attended an evening course in drawing and painting at the “Kunstnijverheidsschool Maastricht”.'
Meeting with the painters Frits and Yves Klein in Paris'
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.
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 an analogue painter, Ploem saw the possibility of also applying the methods of mathematical morphology to the creation of digital art .
DIGITAL TRANSFORMATIONAL ART
In his final contribution to digital art Ploem described and developed methodology for the creation of transformational digital art. The creative tool in classical digital art is often the use of the computer mouse. With digital transformational art, however, mathematical algorithms are the main creative tools and not the drawing by a computer mouse. The term transformational art has already been used earlier for some interesting categories of art. But in this website this term is again proposed for the special discipline of digital image transformation, following the earlier use of this term for image transformation, as described by Leonardo da Vinci in 1505. Transformational art, as described in this chapter, requires always a source image that has to be changed (transformed) into a target image. Algorithmic, mathematical and fractal art do not need source images, since they can create an entirely new image on their own. Ploem used for image rtransformation the QWIN program from LEICA, based on the French Mathematical Morphology program. The QWIN program can perform transformations on binary images and on gray scale images. A binary image is obtained by thresholding the gray scale image. A range of intensity (gray) levels in the image can be chosen for detection. It can be transformed with the various “amend” instructions from QWIN such as : erode, dilate skeleton, ultimate skeleton, opening, closing, prune . “skiz“ etc. The watershed transformation is performed on grey scale images. It is a rather aggressive algorithm that can damage some morphology in a picture. It is very effective in creating totally new pictorial handwritings. For technical details one could consult a website on transformational art (www.ploem-digital-transformational-art.com)
Mountain flowers as the first topic for digital image analysis
His first digital graphics of nature scenes were shown in his exposition at a 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
He was invited 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).
Portraits and architecture