MICROSCOPY RESEARCH AND TECHNIQUE 77:479–482 (2014)
Fluorescence Microscopy in the Spotlight Identifying a specific biomolecule within its cellular environment and without perturbation, is like looking for a specific needle in a haystack of needles without destroying the haystack. The number of biomolecules that constitutes a cell is immense and each of these biomolecules is optically indistinguishable from each other. The complexity increases when the scouting location switches from a single cell to a whole organism. The introduction of fluorescence-based assays in general, and fluorescence microscopy in particular, represented a significant jump toward the solution of this problem. In a fluorescent light microscope only the biomolecule of interest appears “bright” and it is highlighted from all the other molecules, thus allowing very sensitive and specific analysis. As the image formation process is mainly based on the use of light, the invasivity remains low. The theory and phenomenon of fluorescence (incident light of short wavelength pushes a fluorophore to emit light at a longer wavelength) was extensively studied in the 1800s and early 1900s. Certainly, the fluorescence phenomenon was well known to August K€ohler, who in 1904, during early experiments in ultraviolet (UV) microscopy, observed that some objects emitted visible light upon UV illumination. Although, in the context of the UV microscope the fluorescence light was a problem, since it reduced the specimen’s contrast, K€ohler realized immediately the potential of fluorescence within the microscopy scenario. A few years later, K€ohler together with Henry Wilhelm Siedentopf, demonstrated the first fluorescence microscope (1908). Unfortunately, they used a spark gap as illumination light to induce fluorescence which was rather inefficient. Fluorescence microscopy remained impractical until steady state UV light sources were developed. In 1911, Heinrich Lehmann of the Carl Zeiss factory used an arc lamp and the UV filtering technique proposed by Robert Williams Wood, to make an efficient prototype of a fluorescence microscope. Independently, Otto Heimst€ adt of the Carl Reichert firm worked along a similar approach. As a result, both firms released similar commercial fluorescence microscopes. The first applications of fluorescence microscopy were limited to autofluorescent specimens so only biomolecules that naturally emit fluorescence light were observed. In others words, only some particular needles in the haystack could be identified. Right away this limitation was solved thanks to the advent of optimized fluorescent compounds. The development of an increasing range of fluorophores and selective staining methods boosted the success of fluorescence microscopy. Since the word “fluorochrome” was coined by Haintinger, several staining protocols based on secondary fluorescence were introduced. However, it was not until the early 1940s that Albert Coons developed
DOI 10.1002/jemt.22393 Published online in Wiley Online Library (wileyonlinelibrary.com).
C 2014 WILEY PERIODICALS, INC. V
immunofluorescence, thus increasing the labeling specificity using fluorophore-tagged antibodies able to localize specific antigens in tissues. Later in the early 1960s, Osamu Shimomura isolated the green fluorescent protein (GFP) from Aequorea jellyfish, setting the basis of a new era in cell biology. As a result in the 1990s fluorescent proteins started to be used as a fusion tag for a wide variety of protein to monitor cellular processes in living systems. The importance of GFP was recognized by the award of the 2008 Nobel Prize in chemistry (Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien). In parallel, the advances in instrumentation were crucial in the development of the fluorescence microscope. Of significance are two distinguished inventions that lead to Nobel Prize recognition (obliviously, we do not pretend to be exhaustive). The invention of the laser in the late 50s (Charles Hard Townes, Nicolay Gennadiyevich Basov, and Aleksandr Mikhailovich Prokhorov, 1964 Nobel Prize in Physics) and of the charge-coupled device in the late 60s (George Smith and Willard Boyle, 2009 Nobel Prize in Physics). At the beginning of 90s, the fluorescence microscope was ready to become one of the most important tools for investigating biological processes at the subcellular level and under real physiological conditions. Thanks also to the simplicity of the technique, there were many new applications and new scientists found the fluorescence microscope the perfect tool for addressing their questions. To meet this increasing demand, attention has been progressively focused toward the development of new fluorescence-based techniques. As a result, the last two decades have seen an exponential increase of the number of scientific reports based on the use of fluorescence microscopy (Fig. 1). This special issue concerns works strictly linked to some of the most important advances in fluorescence microscopy of the last 25 years (Fig. 1). Until the beginning of 90s, diffraction had been thought to be an insurmountable limit to the spatial resolution of any far-field light microscope, including the fluorescence microscope. In 1893, Ernest Abbe demonstrated that a far-field light microscope cannot discern objects any closer together that distance d 5 k/(2n sin a), with k the wavelength of the light used and n sin a < 1 the numerical aperture of the microscope’s objective lens. Based on Abbe’s results, K€ohler built the UV microscope to improve the spatial resolution using shorter wavelength light and he unexpectedly realized the first fluorescence microscope. Thereby, the fluorescence microscope can be considered as the first outcome of the obsession to improve the spatial resolution of light microscopy. The very same obsession that drove scientists, at the beginning of the 21st century, to develop effective fluorescence microscopy methods, the so-called super-resolution techniques, able to overcome the limit, imposed by diffraction, toward an unlimited spatial resolution. In general all these methods acquire subdiffraction images by exploring the photophysical properties of the fluorochromes and
480
CELLA ZANACCHI ET AL.
Fig. 1. Number of peer-reviewed papers published annually relating to fluorescence microscopy. Data was generated using Scopus, search term “fluorescence microscopy.” Lines indicate the years that the techniques used in this special issue were invented.
causing the fluorochromes of nearby features (
Fluorescence Microscopy in the Spotlight Identifying a specific biomolecule within its cellular environment and without perturbation, is like looking for a specific needle in a haystack of needles without destroying the haystack. The number of biomolecules that constitutes a cell is immense and each of these biomolecules is optically indistinguishable from each other. The complexity increases when the scouting location switches from a single cell to a whole organism. The introduction of fluorescence-based assays in general, and fluorescence microscopy in particular, represented a significant jump toward the solution of this problem. In a fluorescent light microscope only the biomolecule of interest appears “bright” and it is highlighted from all the other molecules, thus allowing very sensitive and specific analysis. As the image formation process is mainly based on the use of light, the invasivity remains low. The theory and phenomenon of fluorescence (incident light of short wavelength pushes a fluorophore to emit light at a longer wavelength) was extensively studied in the 1800s and early 1900s. Certainly, the fluorescence phenomenon was well known to August K€ohler, who in 1904, during early experiments in ultraviolet (UV) microscopy, observed that some objects emitted visible light upon UV illumination. Although, in the context of the UV microscope the fluorescence light was a problem, since it reduced the specimen’s contrast, K€ohler realized immediately the potential of fluorescence within the microscopy scenario. A few years later, K€ohler together with Henry Wilhelm Siedentopf, demonstrated the first fluorescence microscope (1908). Unfortunately, they used a spark gap as illumination light to induce fluorescence which was rather inefficient. Fluorescence microscopy remained impractical until steady state UV light sources were developed. In 1911, Heinrich Lehmann of the Carl Zeiss factory used an arc lamp and the UV filtering technique proposed by Robert Williams Wood, to make an efficient prototype of a fluorescence microscope. Independently, Otto Heimst€ adt of the Carl Reichert firm worked along a similar approach. As a result, both firms released similar commercial fluorescence microscopes. The first applications of fluorescence microscopy were limited to autofluorescent specimens so only biomolecules that naturally emit fluorescence light were observed. In others words, only some particular needles in the haystack could be identified. Right away this limitation was solved thanks to the advent of optimized fluorescent compounds. The development of an increasing range of fluorophores and selective staining methods boosted the success of fluorescence microscopy. Since the word “fluorochrome” was coined by Haintinger, several staining protocols based on secondary fluorescence were introduced. However, it was not until the early 1940s that Albert Coons developed
DOI 10.1002/jemt.22393 Published online in Wiley Online Library (wileyonlinelibrary.com).
C 2014 WILEY PERIODICALS, INC. V
immunofluorescence, thus increasing the labeling specificity using fluorophore-tagged antibodies able to localize specific antigens in tissues. Later in the early 1960s, Osamu Shimomura isolated the green fluorescent protein (GFP) from Aequorea jellyfish, setting the basis of a new era in cell biology. As a result in the 1990s fluorescent proteins started to be used as a fusion tag for a wide variety of protein to monitor cellular processes in living systems. The importance of GFP was recognized by the award of the 2008 Nobel Prize in chemistry (Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien). In parallel, the advances in instrumentation were crucial in the development of the fluorescence microscope. Of significance are two distinguished inventions that lead to Nobel Prize recognition (obliviously, we do not pretend to be exhaustive). The invention of the laser in the late 50s (Charles Hard Townes, Nicolay Gennadiyevich Basov, and Aleksandr Mikhailovich Prokhorov, 1964 Nobel Prize in Physics) and of the charge-coupled device in the late 60s (George Smith and Willard Boyle, 2009 Nobel Prize in Physics). At the beginning of 90s, the fluorescence microscope was ready to become one of the most important tools for investigating biological processes at the subcellular level and under real physiological conditions. Thanks also to the simplicity of the technique, there were many new applications and new scientists found the fluorescence microscope the perfect tool for addressing their questions. To meet this increasing demand, attention has been progressively focused toward the development of new fluorescence-based techniques. As a result, the last two decades have seen an exponential increase of the number of scientific reports based on the use of fluorescence microscopy (Fig. 1). This special issue concerns works strictly linked to some of the most important advances in fluorescence microscopy of the last 25 years (Fig. 1). Until the beginning of 90s, diffraction had been thought to be an insurmountable limit to the spatial resolution of any far-field light microscope, including the fluorescence microscope. In 1893, Ernest Abbe demonstrated that a far-field light microscope cannot discern objects any closer together that distance d 5 k/(2n sin a), with k the wavelength of the light used and n sin a < 1 the numerical aperture of the microscope’s objective lens. Based on Abbe’s results, K€ohler built the UV microscope to improve the spatial resolution using shorter wavelength light and he unexpectedly realized the first fluorescence microscope. Thereby, the fluorescence microscope can be considered as the first outcome of the obsession to improve the spatial resolution of light microscopy. The very same obsession that drove scientists, at the beginning of the 21st century, to develop effective fluorescence microscopy methods, the so-called super-resolution techniques, able to overcome the limit, imposed by diffraction, toward an unlimited spatial resolution. In general all these methods acquire subdiffraction images by exploring the photophysical properties of the fluorochromes and
480
CELLA ZANACCHI ET AL.
Fig. 1. Number of peer-reviewed papers published annually relating to fluorescence microscopy. Data was generated using Scopus, search term “fluorescence microscopy.” Lines indicate the years that the techniques used in this special issue were invented.
causing the fluorochromes of nearby features (
