Johan Sebastiaan Ploem - Work On Fluorescence Microscopy

Work On Fluorescence Microscopy

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" ((illumination and detection from one side of the sample)). At the time of his first communication 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 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 Feulgenpararosaniline 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 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 worldwide 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 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. 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 490 nm. Ploem combined this SP filter with a 1 mm 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 520 nm 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 520 nm (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).