The Development of Fluorescence Microscopy:
The theory of fluorescence was extended by the physics professor Eugen Cornelius Joseph von Lommel in 1875; he posited that a substance must absorb light before it can emit the light as fluorescence. Others investigated the phenomenon of luminescence included Alexandre Edmond Becquerel who investigated the phenomena of fluorescence and posphorescence.
Advances in fluorescent dyes and instrumentation were crucial in the development of the fluorescence microscope.
In 1903 Henry Friedrich Wilhem Siedentopf (1872–1940), who worked at the optical works of Carl Zeiss, collaborated with Richard Adolf Zsigmondy (1865–1929) and they invented the ultramicroscope to observe colloids. Although their microscope could not image colloids, it could detect each colloid as a bright spot of light.
Siedentopf constructed a dark-field condenser that blocked the incident light from entering the microscope objective, thereby improving the specimen’s contrast. Siedentopf reported that the use of ultraviolet light produced specimen fluorescence, and that the fluorescence was a problem since it reduced the specimen’s contrast. Later in 1914, Siedentopf and Zsigmondy refined their original invention and produced a slit ultramicroscope and an immersion
ultramicroscope. The Carl Zeiss firm manufactured and sold their ultramicroscope which was used for the study of
colloids (Figure 1,Figure2).
It is of historical interest that modern microscopic techniques that are based on ‘illumination with a light sheet’ follow from the early works of Siedentopf and Zsigmondy on lateral illumination.
August Kohler (1866–1948) working in the Jena Zeiss factory developed in 1893 a new system of microscope illumination (later named Kohler illumination) for microscopic photographic purposes.
In 1904, August Kohler invented the ultraviolet absorption microscope that preceded the fluorescence microscope. A camera was required to detect the very weak image.
The motivation to use the ultraviolet light was based on the fact that shorter wavelengths of incident light results in increased resolution of optical microscopes (from the previous work of Abbe).
Together with Siedentopf, they made a fluorescence microscope with excitation at the wavelength of 275nm from a spark source. They made microscopic observations of autofluorescence (selffluorescence) of biological specimens excited with ultraviolet light. Siedentopf observed the ultraviolet image of unstained chromatin in the cell nucleus with incident light of wavelength of 275 nm. In spite of these successes, the spark gap was an unreliable source of ultraviolet light for microscopy.
The development of a steady source of ultraviolet light was a prerequisite to the first reliable fluorescence microscope.
In 1903, Robert W Wood (1868–1956) working at Johns Hopkins University demonstrated how to isolate a band of ultraviolet light (300–400 nm) from an arc lamp with a dye solution of nitrosodiumethylaniline. Then, in 1910,HLehmann of the Carl Zeiss factory in Jena used the modified Wood’s light source (the addition of gelatin to Wood’s solution plus another quartz chamber containing copper sulfate solution) to make a prototype bright-field fluorescence microscope (the ultraviolet exciting light entered the microscope objective to illuminate the specimen).
This modified Wood’s light source blocked all visible light.
In 1914, Stanislaus von Prowazek (1875–1915) was the first to introduce vital cellular staining into fluorescence
microscopy, that is his fluorescence staining of living protozoa. (analog this example, of course with the microscopes of that time!:)
At the same time the firm Carl Reichert of Vienna developed a competing fluorescence microscope with a new dark-field quartz condenser (the ultraviolet light did not enter the microscope objective).
In 1911, Hans Stubel, a physiologist at Jena University, observed autofluorescence (naturally occurring fluorescence without the application of a fluorescence dye to the specimen) during his microscopic studies of cells and tissues. Subsequent observations by Herwig Hamperl (University of Vienna) who initiated the use of the fluorescence microscope in pathology confirmed the autofluorescence of the ocular lens of the eye, plant tissues, chlorophyll, amyloid, collagen, fibrin and elastic fibres. In 1913, the Zeiss firm introduced its commercial luminescence bright-field microscope.
The fluorescence microscopes sold by the Zeiss firm and the Reichert firm used quartz lenses for the illumination with ultraviolet light, and glass lenses in the microscope. Both of these fluorescence microscopes worked in the transmission mode. They only differed in the type of condenser; the Zeiss microscope used a bright-field condenser, and the Reichert microscope used a dark-field condenser.
In England, the Beck microscope company manufactured an ultraviolet microscope that had higher resolution than that obtainable with visible light.
Philipp Ellinger (1887–1952), a pharmacology professor whoworked in Heidelberg in the 1920s, was instrumental in the development of the intravital fluorescence microscope. Between 1925 and 1932, Ellinger (the senior investigator) collaborated with August Hirt on the development of the intravital fluorescence microscope. This microscope was patented with the names of the inventors: Ellinger and Hirt.
In 1929, the firm Carl Zeiss in Jena produced a commercial intravital microscope with vertical illumination, a waterimmersion microscope objective and an ultraviolet light source for the investigations of the distribution of fluorescent dyes in the kidneys and the liver of frogs and mice.
In 1933, Ellinger who was Jewish was forced to leave his position and subsequently Hirt, who was a member of the Nazi SS, falsely claimed that he was the sole inventor of the intravital fluorescence microscope (Kasten, 1991)
A major advance in the incident-light fluorescence microscope was the 1948 invention by Evgenii Brumberg; he called his technique as ‘illumination from above’. It consisted of an interference mirror that directed the illumination light to the microscope objective, and separated the exciting light and the fluorescence light that was collected by the microscope objective (Brumberg, 1959). Brumberg’s invention was subsequently developed by Johan Sebastiaan Ploem (1967). It consisted of the dichromatic beamsplitting plate (dichroic mirror) that is used to separate the excitation light from the longer wavelength fluorescence.
The sensitivity and specificity of the binding of antibodies and antigens was applied to fluorescent labels and that resulted in the ability to image single proteins in cells and tissues.
Albert Coons (1912–1978) and Melvin Kaplan (1921–) are the inventors of immunofluorescence (1950) in which a fluorescent dye (fluorescein isocyanate) is chemically attached to an antibody; this technique resulted in an enormous increase in specificity (due to the antigen–antibody specificity) and resulted in many advances in cell biology. For example, in 1982, Mary Osborne and Klaus Weber used the fluorescence microscope together with fluorescent monoclonal antibodies to visualize the cytoskeleton protein tubulin in cells.
Max Haitinger (1868–1946) who is considered the founder of modern fluorescence microscopy and the fluorochroming technique introduced the term ‘fluorochroming’ or fluorescent staining of specimens, and he developed many of the staining techniques for the observation of specimens in the fluorescence microscope.
Max Haitinger (1959) made advances in both staining and instrumentation as described in his 1938 monograph: Fluorescenzmikroskopie Ihre Anwendung in der Histologie und Chemie.
Microscopy: Introduction to Fluorescence Microscopy (Nico Stuurman) Video: