Microscopy Milestones (Nature)

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mILESTonES TImELInE

1595 Invention of the microscope (Milestone 1)

1858 First histological stain (Milestone 2)

1871 Synthesis of fluorescein (Milestone 2)

1873 Diffraction limit theory (Milestone 3)

1911 First fluorescence microscopy (Milestone 4)

1929 First epifluorescence microscope (Milestone 4)

1935 Phase contrast microscopy (Milestone 5)

1939 Polarization microscopy (Milestone 6)

1942 Immunofluorescence (Milestone 7)

1955 Differential interference contrast (Milestone 8)

1961 Concept of confocal microscopy (Milestone 9)

1967 The dichroic mirror (Milestone 4)

1972 Fluorescence correlation spectroscopy (Milestone 10)

1976 FRAP (Milestone 10)

FRET (Milestone 11)

1980 Calcium probes (Milestone 12)

1981 Video-enhancement differential interference contrast (Milestone 8)

TIRF microscopy (Milestone 13)

1983 Deconvolution microscopy (Milestone 14)

1987 Realization of confocal microscopy (Milestone 9) 1990 Two-photon microscopy (Milestone 15)

1993 Light sheet microscopy (Milestone 16)

Single molecule microscopy (Milestone 17) 1994 GFP (Milestone 18) 1997 Fluorescent protein-based biosensors (Milestone 19) 1999 Red fluorescent proteins (Milestone 20) 2000 Breaking the diffraction limit: STED (Milestone 21) 2002 Photoactivatable fluorescent proteins (Milestone 20)

2006 Breaking the diffraction limit: PALM/STORM (Milestone 21)

was enormously influential and milestone 1 demonstrated for the first time what microscopy could do for science. Van Leeuwenhoek (1632–1723) The beginning was a Dutch draper with no formal scientific training, who became fascinated by the magnifying glasses used to count the threads in cloth. Letters by William Borel, the He learned how to grind and polish Dutch envoy to the court of France, lenses and developed such techniques mentions the Dutch spectacle-maker further. Van Leeuwenhoek made Hans Jansen and his son Zacharias more than 500 simple microscopes, as inventors of the first compound each containing a single, tiny convex microscope in 1595. Their micro- lens that could resolve detail as scope had two lenses combined in small as 1 micrometre. He became a tube in which the magnification extremely skilled in dissecting and was varied by altering the distance mounting specimens and, although between the lenses. However, the his microscopes were laborious Jansens are not known to have pub- to use, he was the first to describe lished any observations through their sperm cells and life in droplets of microscope; instead, the two names water in the form of bacteria and associated with the origin of micros- protozoa. From 1673 onwards, he copy are Robert Hooke and Antonie communicated his observations van Leeuwenhoek. to the Royal Society in a series of Hooke’s masterpiece, known letters, eventually being recognized widely as simply Micrographia, was for his careful observations when he published in 1665 and is the first was elected a member of the Royal book on microscopy by a scientist. At Society in 1680. the time of Micrographia’s publica- The introduction of multiple tion, Robert Hooke (1638–1703) lenses increased problems with was a Fellow of the Royal Society spherical and chromatic aberra- in London, holding the position of tion. Van Leeuwenhoek’s simple Curator of Experiments. His refined instruments were, in fact, superior compound microscope, described to compound microscopes such as and illustrated in the preface to his those of Hooke, and many micro- Drawings of the instruments used by Robert Hooke. Image is reproduced from R. Hooke (1665). book, was a forerunner of modern scopists continued to use single lens microscopes — equipped with a microscopes until achromatic lenses stage, a light source and three optical (corrected for chromatic aberration) lenses. Micrographia puts forward became widely available in the early If a microscope is defined as an scientific theories on many subjects, nineteenth century. … and by instrument that enables the visualiza- as well as containing descriptions Christina Karlsson Rosenthal, Associate the help of tion of objects or structures that are of biological and other specimens Editor, Nature Cell Biology Microscopes, usually invisible to the naked eye, that are accompanied by beautiful there is then microscopes appeared at the drawings made by Hooke himself. PRimARY ReFeRenCes Hooke, R. Micrographia: end of the sixteenth century. The Among these are drawings of a louse or Some Physiological Descriptions of Minute Bodies, nothing so Made by Magnifying Glasses with Observations and properties of concave and convex and a flea, the compound eye of Inquiries Thereupon (James Allestry, 1665) | small as to lenses were first described around the a fly, seeds and plant sections. He Van Leeuwenhoek, A. The Selected Works of Antony year 1000, although magnifiers in the observed the porous structure of cork van Leeuwenhoek Containing His Microscopical escape our Discoveries in Many of the Works of Nature form of lens-shaped crystals or glass and famously described the pores inquiry … (Arno Press, 1977) may have been used much earlier. as “cells”. Although these were not FURtHeR ReADinG Clay, R. S. & Court, T. H. The Robert Hooke Spectacles were invented around cells in the biological meaning of the History of the Microscope: Compiled From Original Instruments and Documents, Up To the Introduction 1300 and became widely used across word, the modern term derives of the Achromatic Microscope (Holland Press, 1975) Europe. from Hooke’s usage. The book

milestone 2 Kircher described similar observations nearly a century later). John Herschel’s description of the fluorescent properties of quinine sulphate in Stains and fluorescent dyes 1845 is considered the ‘modern’ milestone for observing fluorescence and realizing what it was, together with Robert Hooke may have been the previous staining solutions had George Stokes’s 100-page monograph first to describe the light microscopic been too concentrated, and also published in 1852 that describes appearance of a stained object, in noted that the dye was absorbed by a vast collection of fluorescent the form of dyed wool and hair (see specific cellular elements and could substances, from quinine sulphate Milestone 1). And although others not be washed out. Von Gerlach to oporto wine. Although David subsequently reported the use of therefore deserves credit for recog- Brewster first used the term ‘internal staining solutions, any differential nizing the importance of staining dispersion’ to describe fluorescence staining that was observed is thought and for carefully describing his phenomena in 1838, it was Stokes to have been mostly accidental. staining method. who coined the term ‘fluorescence’ Hartig and osborne, who independ- The late 1800s and early 1900s to describe light emission induced ently observed the colouring of the marked the discovery of many during excitation. cell contents in plants, are sometimes cytological phenomena and the With the development of the cited as the discoverers of staining in development of new stains and synthetic dye industry, it was not microscopy, but neither contributed synthetic dyes. The use of silver long before Adolf von Bayer syn- significantly to the development of staining in cytology was pioneered thesized the first fluorescent dye, the technique. Hartig even casually by Camillo Golgi in 1873 and made fluorescein, in 1871. Paul Ehrlich mentioned the use of carmine, a dye famous by Santiago Ramón y Cajal’s used the fluorescent dye uranin derived from female scale insects, in detailed neuroanatomical observa- (a sodium salt of fluorescein) in … it was an 1854 paper. tions. The notion that basic and 1882 to determine the pathway of not long It was Joseph von Gerlach who, acidic dyes are histologically distinct secretion of aqueous humour in the before Adolf in 1858, while experimenting was important for the development eye — representing the first use of with solutions of carmine and of the haematoxylin and eosin a fluorescent dye in animal physiol- von Bayer leaving a section of brain tissue in a stain (Paul Mayer, 1896), which ogy. In 1914, not long after the first synthesized dilute carmine solution overnight, became a key diagnostic stain. An fluorescence microscope was devel- the first reported good differential staining acidic dye (eosin) and a basic dye oped (see Milestone 4), Stanislav fluorescent of the nucleus and nuclear granules (methylene blue) also form the basis von Provazek used fluorescent dyes compared with little or no staining of the Giemsa stain (Gustav Giemsa, at a microscopic level as a means to dye … of the cytoplasm and intercellular 1904), which is still used to diag- enhance the autofluorescence of cells substance. He concluded that nose malaria and other parasites. and tissues — representing the first Robert Feulgen’s discovery in 1924 use of a fluorescent dye as a stain in that chromosomal material can be cell biology. stained by a chemical reaction based Arianne Heinrichs, Chief Editor, on acid hydrolysis of DNA became Nature Reviews Molecular Cell Biology a cornerstone of cytochemistry. The cytochemical staining of peroxidase PRimARY ReFeRenCes Von Gerlach, J. activity, as reported by Graham et Mikroskopische Studien aus dem Gebiet der menschlichen Morphologie (Enke, 1858) | al., was subsequently developed as Bayer, A. Ueber eine neue Klasse von an immunohistochemical approach Farbstoffen. Ber. Dtsch. Chem. Ges. 4, 555–558 by Sternberger et al. in 1970. (1871) FURtHeR ReADinG Herschel, J. F. W. On a case Whereas stains provide finite of superficial colour presented by a contrast by changing the light homogeneous liquid internally colourless. absorption properties of different Phil. Trans. R. Soc. Lond. 135, 143–145 (1845) | Stokes, G. G. On the change of refrangibility of cellular structures, fluorescence light. Phil. Trans. R. Soc. Lond. 142, 463–562 provides infinite contrast with the (1852) | Graham, R. C. Jr, Lundholm, U. & Karnovsky, M. J. Cytochemical demonstration of right equipment — although such peroxidase activity with 3-amino-9- equipment obviously did not exist ethylcarbazole. J. Histochem. Cytochem. 13, when fluorescence was discovered, 150–152 (1965) | Sternberger, L. A., Hardy, P. H. Jr, Cuculis, J. J. & Meyer, H. G. The unlabeled and is still being improved today. antibody enzyme method of The earliest description of fluores- immunohistochemistry: preparation and cence is thought to date from the properties of soluble antigen-antibody complex Fluorescein powder dropped into a solution of tap water under ultraviolet light, (horseradish peroxidase-antihorseradish after approximately 15 seconds have elapsed. Image is reproduced with sixteenth century, when Nicolás peroxidase) and its use in identification of permission from Bricksnite and licensed under the Creative Commons Monardes reported the fluorescent spirochetes. J. Histochem. Cytochem. 18, 315–333 Attribution 3.0 Unported License (1970) | Clark, G. & Kasten, F. H. History of (http://creativecommons.org/licences/by/3.0/) properties of wood extract from Staining 3rd edn (Williams & Wilkins, 1983) Lignum nephriticum (Athanasius

milestone 3 improve tissue penetration at the expense of point separation. Abbe’s mathematical foundations of … Abbe’s image formation and lens aberrations Knowing the limit provided for the proper design of study Without light microscopy, our affecting the spatial resolution or, in microscope lenses, accomplished influenced the knowledge of ‘little biology’ would be other words, the smallest separation in collaboration with Carl Zeiss and field so greatly Otto Schott. Abbe’s quantitative severely impaired. The ability to observe that two objects can have and still be that very few migrating cells, examine the distribution discerned. When this opening is a lens, insights greatly enhanced the quality of organelle populations or predict the diffraction pattern created by light of microscope optics, contributing attempts putative protein interactions (based on passage through the illuminated circular enormously to improved data collection were made and an enhanced user experience proximity) drive modern cell-biological aperture appears as an ‘Airy disc’, as to overcome research. However, these observations described by George Airy in 1835. for the microscopist. Of course, the have physical limits, governed by the Later, the mathematical foundations success of Abbe, Zeiss and Schott in the diffraction properties of light, which consequently for quantifying diffraction-limited designing lenses also had an enormous limit… restrict our view of this cellular world. microscopy were noticed by Emile Verdet impact on the eventual success of In light microscopy, when light passes but fully described and formalized by the microscopy manufacturer Carl through an opening it is diffracted, Ernst Abbe. Zeiss itself. But besides these tangible In his landmark paper of 1873, Abbe legacies, another long-lasting legacy brought about by Abbe’s work reported that the smallest resolvable PRimARY ReFeRenCe λ included the establishment of physical d = distance between two points using a Abbe, E. Beiträge zur Theorie 2n sin a conventional microscope may never boundaries in imaging for quite some des Mikroskops und der mikroskopischen be smaller than half the wavelength time. In fact, Abbe’s study influenced the field so greatly that very few Wahrnehmung. Archiv für of the imaging light. Although no Mikroskopische Anatomie 9, mathematical equations actually appear attempts were made to overcome the 413–418 (1873) FURtHeR ReADinG y in the paper, Abbe stated in this and diffraction limit, despite the increasing necessity to enhance resolution and Airy, G. B. On the diffraction following papers that the resolution of an object-glass with was limited by diffraction to half the to improve the visualization of cellular circular aperture. Trans. Cambridge Phil. Soc. 5, wavelength modified by the refractive structure. Rather, other techniques were developed to provide these 283–291 (1835) | Airy, G. B. index of the medium and the angle of On the diffraction of an the cone of focused light. Based on the data, such as replacing photons annular aperture. London, resulting equation, one can improve with electrons in the development Edinburgh, and Dublin of electron microscopy. Only more Philosophical Magazine Series spatial resolution by using light with 3 18, 132–133 (1841) | recently have scientists revisited Abbe’s x a shorter wavelength (for example, Verdet, E. Lecons d’optique ultraviolet), but in biological samples, limit and successfully increased the physique (Victor Masson et Blurred by diffraction, the image of two point resolution of light microscopy through fils, Paris, 1869) | Rayleigh, this is undesirable because of the Lord. On the theory of objects (red) can just about be resolved at distance a variety of innovative strategies (see d. Line profile (bottom) quantifies the brightness greater potential for sample damage optical images, with special along the direction of separation. Diagram and increased light scattering within a Milestone 21). reference to the microscope. courtesy of S. Hell, MPI Biophysical Chemistry, Phil. Mag. 42, 167–195 (1896) Göttingen, Germany. tissue. Reciprocally, longer wavelengths Noah Gray, Senior Editor, Nature

milestone 4 excitation light would enter the objective lens and provided high contrast for the fluorescence signal. The fluorescence microscope Although Heimstädt successfully imaged bacteria, he wasn’t convinced Inspired by Ernst Abbe’s finding this observation as the basis for the that fluorescence microscopy would that shorter wavelength light leads construction of the first successful have a lasting impact. He concludes to higher resolution, August Köhler fluorescence microscope. his paper with the words: “If and to constructed the first ultraviolet Heimstädt noted two main what degree fluorescence micro- (uV) microscope at Zeiss optical challenges: one was to concentrate scopy will widen the possibilities Works in Jena, Germany, in 1904. enough uV light on the sample to of microscopic imaging only the The instrument used uV illumina- make it fluoresce; the other was to future will show.” In fact, the reliance tion from a cadmium arc lamp and capture the emitted fluorescent light on autofluorescence of the imaged allowed photographic reproduction free of any noise. To address the object and the need for transmitted of objects at twice the resolution of former, he used cuvettes constructed illumination and darkfield condensers visible light microscopes. Köhler also by Hans Moritz Lehmann to eliminate limited the initial applications of the noted that some objects emitted light all but the uV light from the white microscope. of longer wavelength on illumination light generated by an arc lamp; to Both hurdles were overcome dur- with uV, but it was the physicist address the latter, he employed ing the next two decades, when the oskar Heimstädt who, in 1911, used darkfield illumination that ensured no Austrian investigator Max Haitinger ▶

▶ together with other scientists devel- excitation light from interfering which also functions as a condenser, oped the technique of secondary with observation. Ellinger and Hirt ensuring even excitation fluorescence, which involved applying imaged kidney and liver tissue in of the sample. The emitted longer exogenous fluorescent chemicals to rodents injected with the fluoro- wavelength fluorescent light is samples. Haitinger also coined the chromes fluorescein and trypaflavin. collected by the objective, and passes term ‘fluorochrome’. Another major advance came back through the dichromatic mirror Fluorochromes were essential to almost 40 years later, with the inven- and a barrier filter to the eyepiece. the use of fluorescence microscopy on tion of dichromatic beamsplitters, Dichromatic mirrors converted the If and to living tissue, as was the development or dichromatic mirrors. Instead of epi-fluorescence microscope from a what degree of the incident light (epi)-fluorescence absorbing certain wavelengths, as tool that could be used only by trained fluorescence microscope. Here, the light source lies traditional filters did, dichromatic specialists to a universal and indispen- on the same side of the sample as the filters reflect a narrow width of sable instrument for modern biology. microscopy objective, and excitation and emission wavelengths while transmitting all Nicole Rusk, Senior Editor, will widen the light pass through the objective. This others, allowing illumination of the Nature Methods

possibilities configuration allows more efficient sample with a precise wavelength PRimARY ReFeRenCes Heimstädt, O. Das of microscopic sample excitation and the imaging of and ensuring that no excitation light Fluoreszenzmikroskop. Z. Wiss. Mikrosk. 28, opaque objects. is transmitted to the ocular. 330–337 (1911) | Ellinger, Ph. & Hirt, A. imaging only Mikroskopische Beobachtungen an lebenden The prototype of the epi- The Dutch scientist Johan Organen mit Demonstrationen the future fluorescence microscope was designed Sebastiaan Ploem is closely associ- (Intravitalmikroskopie). Arch. Exp. Pathol. Phar. 147, in 1929 by German pharmacologist ated with the development of these 63 (1929) | Ploem, J. S. The use of a vertical will show. illuminator with interchangeable dichroic mirrors Philipp Ellinger and anatomist August mirrors and described incident verti- for fluorescence microscopy with incident light. Oskar Hirt. In what they called an ‘intravital cal illumination with dichromatic Z. Wiss. Mikrosk. 68, 129–142 (1967) ° FURtHeR ReADinG Brumberg, E. M. & Heimstädt microscope’ the excitation light passed mirrors in 1967. Fixed at a 45 angle Krylova, T. N. Primenenie interferentsionnykh through a series of filters before the to the incoming excitation light, delitel’nykh zerkal v fluorestsentnoi mikroskopii. right wavelength hit the objective lens the mirror reflects up to 95% of the Zh. Obshch. Biol. 14, 461–464 (1953) | Ploem, J. S. Die Möglichkeit der Auflichtfluoreszenzmethoden and triggered emission of fluorescent shorter wavelength excitation light bei Untersuchungen von Zellen in light in the observed tissue. A yellow but transmits longer wavelength Durchströmungskammern und Leightonröhren. barrier filter between the objective light. The reflected beam of excita- Xth Symposium d. Gesellschaft f. Histochemie, 1965. Acta Histochem. 7 (Suppl.), 339–343 (1967) and ocular prevented most reflected tion light then enters the objective,

milestone 5 strip to his telescope, the striped pattern on the grating reappeared. Zernike immediately saw the potential of translating his discovery to the world of Finding phase microscopy, where transparent objects found in cells had previously been compared to gratings. In 1930, physicist Frits Zernike was studying By placing his phase strip in the focal plane of the optics of diffraction gratings when his lab a microscope, similarly to the way in which he obtained a large concave grating. Owing to its had used it with the telescope, Zernike was size, Zernike had to position a small telescope able to visualize otherwise transparent objects 6 metres from its surface to visualize the light without the need for a stain. Zernike published patterns created by the gratings. Normally, this a description of his method, which he coined resulted in a striped pattern, the effect of seeing Negative phase-contrast micrograph of epithelial ‘phase contrast’, in 1935. a principal spectral line of direct light and weaker cells from the mouth. Image is reproduced, with Shortly after his discovery, Zernike excitedly permission, from L. C. Martin Nature 159, 827–830 spurious lines of diffracted light to the left and © (1947) Macmillan Publishers Ltd. All rights demonstrated the method to microscope maker right caused by slight imperfections associated reserved Carl Zeiss, who were less than enthusiastic about with the gratings. But when Zernike focused his its potential at first glance. It took another 10 telescope on the gratings themselves, something contained information about the object of years before companies started manufacturing surprising happened — the striped patterns interest, and realized that he would need an microscopes with the ability to perform phase disappeared. Determining why this occurred adequate reference surface if he were to make contrast, but today most high-end microscope would change how microscopists look at cells use of it. systems have this capability, enabling and earn Zernike the 1953 Nobel Prize in Physics. Zernike then recalled the 1900 work of Lord researchers to see cellular structures in real time Through a series of experiments, Zernike Rayleigh, who described an operation to make without the need for specialized stains or dyes. came to understand that the direct light and shallow etchings in glass with very dilute Nathan Blow, Technology Editor, hydrofluoric acid. Using this approach, Zernike the diffracted light were actually in different Nature and Nature Methods phases. When the telescope was focused on the created ‘phase strips’, in which the direct gratings, the resulting image, created through light hit a thin etching on a glass surface while PRimARY ReFeRenCes Zernike, F. Das interference, was rendered invisible because, the diffracted light passed through the glass. Phasenkontrastverfahren bei der mikroskopischen unlike changes in amplitude or brightness, The result was to shift the direct light by 90o, Beobachtung. Z. technische Physik 16, 454–457 (1935) | the eye cannot pick up phase differences. making it darker than a uniform background of Zernike, F. Phase contrast, a new method for the microscopic observation of transparent objects. Physica 9, 974–986 (1942) But Zernike knew that the phase differences coherent light. When Zernike added a phase

milestone 6 Seeing is believing: subcellular structures brought to life

The development of fluorescence microscopy caused by aligned protein units. However, First maturation division spindle in a living oocyte of the marine annelid Chaetopterus pergamentaceous imaged with a technologies has made live-cell imaging although football-shaped spindles were polarization microscope (left) and traced the projected routine for the current generation of cell visible in Schmidt’s pictures, his microscope image negative (right). Image is reproduced, with permission, from S. Inoué © 1953. biologists. However, the ability to visualize the did not provide adequate resolution to see cell’s architecture was not taken for granted discrete fibres. until the 1950s. Although images of cells and The definitive visual demonstration of the microscopy offers certain advantages over subcellular organelles had been produced existence of spindle fibres in untreated living fluorescence microscopy in that it is label-free as early as the mid-nineteenth century, they cells was given by Shinya Inoué more than a and therefore a non-invasive mode of imaging originated from fixed material. As a result, the decade later. Inoué built his first polarizing that can be used, for example, to monitor existence of three-dimensional macromolecular microscope by hand in post-war Japan and the viability of in vitro fertilized oocytes. structures within cells had remained a source of managed to repeat Schmidt’s observations. Furthermore, the development of the ‘pol-scope’ controversy. However, it was only later at Princeton University in 1995 overcomes the limitation of previous The view of the cell’s internal organization in New Jersey, USA, that he built an improved microscopes in that the polarization contrast is was revolutionized in the 1940s and 1950s polarizing microscope that could image weakly independent of the orientation of the sample. with techniques that were initially developed birefringent structures. In 1953 this led to the Kim Baumann, Associate Editor, to image the mitotic spindle. Until then, the publication of the structure of the metaphase Cell Migration Gateway observed assembly of polymers into fibres was and anaphase spindles in oocytes of the marine believed to be an artefact originating from annelid Chaetopterus pergamentaceous and in fixation and staining treatments that induced Lilium longiflorum pollen mother cells: “With the PRimARY ReFeRenCes Schmidt, W. J. Doppelbrechung der Kernspindel und Zugfasertheorie der protein coagulation and gave the spindle a increased resolution and sensitivity achieved by Chromosomenbewegung. Chromosoma 1, 253–264 (1939) | fibrous appearance. The first evidence of fibrous the new instrument, I have been able to observe Inoué, S. Polarization optical studies of the mitotic spindle. I. organization in the living spindle came from the detailed structure of the spindle in living The demonstration of spindle fibers in living cells. Chromosoma 5, 487–500 (1953). W. J. Schmidt’s observations of developing cells and to follow its change during mitosis,” he FURtHeR ReADinG Inoué, S. A method for measuring sea-urchin eggs made in 1937, and reinterpreted wrote. Further observations led Inoué to suggest small retardations of structures in living cells. Exp. Cell Res. 2, in 1939, with a polarizing microscope. This how microtubule dynamics generate the force 513–517 (1951) | Inoué, S. & Sato, H. Arrangement of DNA in living sperm: a biophysical analysis. Science 136, apparatus exploits the optical anisotropy for chromosome movement and also to deduce 1122–1124 (1962) | Inoué, S. & Sato, H. Deoxyribonucleic of transparent materials to reveal detailed the arrangement of DNA and chromatin folding acid arrangement in living sperm. In Molecular Architecture information about the internal structure of in living sperm. in Cell Physiology (eds Hayashi, T. and Szent-Gyorgi, A. G.) 209–248 (Prentice-Hall, New Jersey, 1964) | Oldenbourg, R. & samples — information that was not available Schmidt’s and Inoué’s findings were the first Mei, G. New polarized light microscope with precision with any other optical microscopy technique at demonstrations of the structural complexity universal compensator. J. Microsc. 180, 140–147 (1995) | the time. Schmidt reported that spindles have of the cell interior and showed the great Inoué, S. Microtubule dynamics in cell division: exploring living cells with polarized light microscopy. Annu. Rev. Cell positive birefringence — a measure of optical potential of live-cell imaging for understanding Dev. Biol. 24, 1–28 (2008). anisotropy — a feature that he concluded to be complex biological processes. Polarization

in dilute solutions, so Coons et al. milestone 7 turned to a fluorescent compound, anthracence isothiocyanate. They obtained brightly fluorescent anti- Recipe for fluorescent antibodies bodies that could agglutinate bacteria, indicating that the conjugation During a seminar in 1961, Albert group A haemolytic streptococci in had not affected the antibodies’ Coons humbly mentioned that “the the Aschoff nodules that character- binding properties. However, when … the basic hour of the fluorescent antibody” ize rheumatic fever. Initially, Coons they tested the fluorescence in tissue idea remains had started. Nearly 70 years since and his colleagues repeated the sections, they discovered that Coons and colleagues published work of John Marrack, who had human and mouse tissues autofluo- unchanged… the first report describing the use successfully modified antibodies resce at the same wavelength as of fluorescent antibodies, immun- with tetrazotized benzidine to pro- anthracence. ofluorescence staining remains a duce coloured antibodies without Coons and his co-workers then cornerstone of cell biology. disturbing their antigen-binding turned to fluorescein, which was The idea to make antibodies properties. These coloured antibod- a known fluorophore. However, a visible was conceived to visualize ies were too faint to be detected form of fluorescein that could be ▶

▶ conjugated to protein, fluorescein- fluorescent antibodies. When Coons iso-cyanate, had to be made from and colleagues were able to see bright scratch. In collaboration with fluorescent patches in the organs of oRiGinAl ReseARCH colleagues at Harvard university the infected, but not uninfected, ani- PAPeRs Coons, A. H., in Boston, Massachusetts, Coons mals, the era of immunofluorescence Creech, H. J. & Jones, R. N. obtained a crude preparation of microscopy was born. Immunological properties of an antibody containing a this compound and used it to label Many improvements have fluorescent group. Proc. Soc. an anti-Pneumococcus strain 3 followed, primarily in the lenses, Expt. Biol. Med. 47, 200–202 (1941) | Coons, A. H., serum. The labelling was performed microscopes and cameras that the Creech, H. J., Jones, R. N. & by mixing the antiserum and field relies on, as well as through Berliner, E. The demonstration fluorescein-iso-cyanate, dissolved the introduction of fluorophores of pneumococcal antigen in tissues by the use of in dioxane, in a buffer of saline and with different, non-overlapping fluorescent antibody. sodium carbonate at pH 9.0 — a excitation and emission spectra. J. Immunol. 45, 159–170 (1942) protocol that is almost unchanged The resolution has moved from FURtHeR ReADinG Coons, A. H. & Kaplan, M. H. today. The antibodies remained the tissue to the intracellular level, The location of the extracellular domain (green) and Localization of antigen in specific for Pneumococcus strain 3, the intracellular domain (red) of the epithelial and the technique is now used for tissue cells: improvements and Pneumococcus strain 2 did not cell-adhesion molecule (EpCam) was revealed by subcellular localization in even the in a method for the double-label immunofluorescence. FaDu cells were detection of antigen by become fluorescent when incubated stained with primary antibodies against the smallest cells. However, the basic means of fluorescent with the antibodies. To test whether different parts of EpCam. The primary antibodies idea remains unchanged, almost antibody. J. Exp. Med. 91, were visualized with fluorescently labelled 1–13 (1950) | the antibodies could detect bacteria secondary antibodies that specifically recognize one seven decades after its first Coons, A. H. The beginnings in tissues, organs of mice infected of the two primary antibodies. Scale bar, 18.83 mm. description. of immunofluorescence. with Pneumococcus strain 3 were Image is reproduced, with permission, from J. Immunol. 87, 499–503 D. Maetzel et al. Nature Cell Biol. 11, 162–171 Christiaan van Ooij, Chief Editor, (1961) harvested and stained with the © (2009) Macmillan Publishers Ltd. All rights reserved. Nature Reviews Microbiology

milestone 8 Light detector Contrast by interference Polarizing filter

In the 1950s, differential-interference ‘halo’ artefact in DIC images, unlike Wollaston contrast (DIC) systems were that in phase-contrast images. (Nomarski) prism designed as an alternative technique In the first DIC systems such as to phase contrast (see Milestone 5). those of Smith in 1955, one of the Objective lens In DIC, polarized light is separated Wollaston prisms that were used as into two beams, which take slightly beam splitters was located at or near Sample different paths through a sample the focal plane of the objective, where depending on its optical density. it was often inaccessible. The French When the beams are recombined, physicist Georges Nomarski, who their interference reveals interfaces described the theoretical basis for between regions of different thick- DIC, modified the Wollaston prisms ness and/or refractive index and gives so that they could be physically Condensing lens the illusion of a three-dimensional located away from the focal planes, image. DIC allows for high- making the system more practical to resolution imaging of unstained and use. This design was then developed Wollaston living cells and organisms, and for commercially by microscopy manu- (Nomarski) prism ‘optical sectioning’ of thick samples. facturer Carl Zeiss and its theory and Another advantage is that there is no potential applications were described Polarizing filter by Allen, David and Nomarski in 1969. The full potential of DIC was The full potential of DIC realized only when video cameras was realized only when entered the stage. In 1981, two papers demonstrated that changing video cameras entered camera gain and offset could increase Light source the stage. resolution and contrast in the image. The components of the basic differential interference contrast microscope set-up. Adapted with permission from Richard Wheeler and licensed under the GNU Free of course, the video camera also Documentation License (http://commons.wikimedia.org/wiki/Commons:GNU_ enabled researchers to follow events ▶ Free_Documentation_License).

▶ over time. Allen, Allen and Travis the cell at high resolution. For exam- PRimARY ReFeRenCes Smith, F. H. Microscopic interferometry. Research (London) exemplified the possibilities of the ple, the technique was used to dis- 8, 385–395 (1955) | Nomarski, G. Microinterféromètre différentiel à ondes polarisées. J. Phys. Radium 16, 9S–11S (1955) | Allen, R. D., David, G. B. & Nomarski, G. The technique by visualizing the dynamic cover the fast transport of membrane Zeiss-Nomarski differential interference equipment for transmitted-light microscopy. movement of microtubules in the vesicles, some 50–100 nanometres in Z. Wiss. Mikrosk. 69, 193–221 (1969) | Allen, R. D., Allen, N. S. & Travis, J. L. Video- marine protist Allogromia. Inoué diameter, along microtubules within enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial demonstrated the dramatic improve- neuronal axons and, subsequently, in network of Allogromia laticollaris. Cell Motil. Cytoskel. 1, 291–302 (1981) | Inoué, S. ment of both polarized light and DIC in vitro motility assays that revealed Video image processing greatly enhances contrast, quality, and speed in imaging by video enhancement. the microtubule motors that pull this polarization-based microscopy. J. Cell Biol. 89, 346–356 (1981) FURtHeR ReADinG Allen, R. D., Metuzals, J., Tasaki, I., Brady, S. T. & Video-enhanced DIC (VE-DIC) vesicle cargo (see Further Reading). Gilbert, S. P. Fast axonal transport in squid giant axon. Science 218, 1127–1129 was subsequently used in numerous Christina Karlsson Rosenthal, Associate (1982) | Salmon, E. D. VE-DIC light microscopy and the discovery of kinesin. Trends studies of dynamic behaviour within Editor, Nature Cell Biology Cell Biol. 5, 154–158 (1995)

CitinG tHe milestones The Nature Milestones in Light Microscopy supplement has been published using material from Nature Cell Biology, Nature Methods and Nature Reviews Molecular Cell Biology. However, most referencing formats and software do not allow the inclusion of more than one journal name or volume in an article reference. Therefore, should you wish to cite any of the Milestones, please reference the page number (Sxx–Sxx) as a supplement to Nature Cell Biology. For example, Nature Cell Biol. 11, Sxx–Sxx (2009). To cite articles from the Collection, please use the original citation, which can be found at the start of each article.

milestone 9 Seeing the wood for the trees

ConventionalConfocal During the 1960s, researchers worked of the Charles university in Prague, Detector to overcome a debilitating feature of Czechoslovakia, began a collabora- epifluorescence microscopy — back- tion with David Egger and Robert ground signals. Efforts began in both Galambos at Yale university. Back America and Czechoslovakia to do home, Petrán went on to build away with these unwanted signals, and a pocket-size pinhole scanning the result was the advent of confocal microscope; he then returned to microscopy. Yale where, in 1967, he and Egger The resolution achievable with reported unstained images of brain conventional microscopy can be and ganglion cells using reflected negated by out-of-focus information. light. In 1981, the design of Petrán’s Objective Confocal microscopy avoids this microscope was discussed in a issue by restricting both the field of theoretical analysis of conventional Specimen illumination and the light reaching the versus confocal microscopes by objective to a single point in the same Colin Sheppard and Tony Wilson, focal plane. An image of the region who proposed a theory on how to Condenser of interest is then built up by raster combine “the resolution and depth scanning through the sample — either discrimination improvements of by moving the sample, or by moving a confocal microscopy with the ease laser beam or pinhole disk. By exclud- of operation of the conventional ing the out-of-focus light, the image microscope”. contrast is increased and finer detail Several follow-up studies made a Light source can be seen. key adjustment to the confocal design The initial patent for a confocal — the use of laser beams. This allowed In a conventional microscope (left), a large region of the specimen is illuminated by microscope was granted to Marvin faster scanning and higher resolution, the light source and condenser. The detector forms an image from both the in-focus Minsky — better known for his but, most importantly, provided light (black lines) and the out-of-focus light (red and blue lines). Conversely, a confocal microscope (right) restricts both the illumination and the light that can pioneering work in Artificial the illumination needed to obtain reach the detector by introducing two pinholes, so only the in-focus light is Intelligence — who was working fluorescence images. Two papers pub- detected (black lines). Adapted, with permission, from Spector D. L. & Goldman, R. D. Basic Methods in Microscopy (Cold Spring Harbor Laboratory at Harvard university in 1961. lished in 1987 used such instruments Press, Cold Spring Harbor, 2006). Shortly thereafter, Mojmir Petrán in the first key applications of confocal ▶

— produced by epifluorescence versus conditions. Together with the discov- confocal techniques. The second ery of green fluorescent protein (see If I could have only one study came from Gerrit van Meer and MILESToNE 18), this ushered in the era single apparatus in the colleagues, working in Kai Simon’s of in vivo imaging. laboratory at the European Molecular Alison Schuldt, Senior Editor, lab, it would be a point Biology Laboratory in Heidelberg, Nature Cell Biology scanner. Kees Jalink Germany, who used a laser scanning PRimARY ReFeRenCes Minsky, M. Microscopy Apparatus. US Patent 3,013,467 (1961) | confocal microscope developed Egger, M. D. & Petrán, M. New reflected-light microscope for viewing unstained brain by Wijnaendts-van Resandt et al. and ganglion cells. Science 157, 305–307 (1967) | Sheppard, C. J. & Wilson, T. The together with fluorescent ceramide theory of the direct-view confocal microscope. J. Microsc. 124, 107–117 (1981) | White, J. G., Amos, W. B. & Fordham, M. An evaluation of confocal versus conventional ▶ microscopy to cell biology. In the first labels to follow the transport of newly imaging of biological structures by fluorescence light microscopy. J. Cell Biol. 105, study, John White, William Bradshaw synthesized sphingolipids in epithe- 41–48 (1987) | Van Meer, G., Stelzer, E. H. K., Wijnaendts-van Resandt, R. W. & Simons, Amos and Michael Fordham, working lial cells. In this way, the advantages K. Sorting of sphingolipids in epithelial (Madin–Darby canine kidney) cells. J. Cell Biol. 105, 1623–1635 (1987) at the Medical Research Council labo- of confocal microscopy for analysing FURtHeR ReADinG Petrán, M., Hadravsky, M., Egger, M. D. & Galambos, R. Tandem- ratory in Cambridge, uK, used their subcellular processes became clear. scanning reflected-light microscope. J. Opt. Soc. Am. 58, 661–664 (1968) | Brakenhoff, G. J., Bloem, P. & Barends, P. J. Confocal scanning light microscopy with high aperture laser scanning confocal microscope By revealing clear images in thick lenses. J. Microsc. 117, 219–232 (1979) | Brakenhoff, G. J. New imaging modes in to compare images of various cells tissue samples, confocal microscopes confocal scanning light microscopy (CSLM). J. Microsc. 117, 233–242 (1979) | Cox, I. J. and tissues — from the endoplasmic obviated the need for tissue Scanning optical fluorescence microscopy. J. Microsc. 133, 149–154 (1984) | Wijnaendts-van Resandt, R. W. et al. Optical fluo rescence microscopy in three reticulum of plasmacytoma cells to sectioning, and allowed tissues to be dimensions: microtomos copy. J. Microsc. 138, 29–34 (1985) the mitotic spindles of worm embryos imaged under more physiological

milestone 10 FCS and FRAP: illuminating cellular processes

By the late 1960s, fluorescence 1990). The widespread adoption of imaging was routinely used in a FCS was initially constrained by the number of applications. But in requirement for extensive calibration …the Webb 1972, the Webb laboratory put measurements and specialized equip- fluorescence microscopy to a novel ment, but FCS modules for confocal laboratory put use: measuring reaction kinetics. microscopes are now available. fluorescence Neither ethidium bromide (EtBr) Twenty-five years after the initial microscopy to Laser nor DNA fluoresces independently, studies using FCS, the development a novel use: but in complex the two emit an of fluorescence cross-correlation Lase orange glow under ultraviolet light. spectroscopy (FCCS) permitted measuring r By measuring the fluctuations in the measurement of intermolecular reaction fluorescence of EtBr binding to interactions. In 1997, Schwille et al. kinetics. DNA, Magde et al. calculated the labelled oligonucleotides with two thermodynamics of complex discrete fluorophores — red and formation and diffusion at green — and measured the kinetics equilibrium, and derived precise of red–green complex formation APD mathematical formulae explaining to deduce average annealing time. their interdependencies. This technique has since been used Between 1972 and now, this to measure protein–protein interac-

APD technique, termed fluorescence tions, including dynamic correlation spectroscopy (FCS), has co-localization, trafficking and been applied to measure the kinetics caspase-mediated cleavage. Principle of dual-colour fluorescence of chemical and biological reactions, FCS could be used to measure cross-correlation spectroscopy (dcFCCS). Intensity fluctuations from two spectrally distinct diffusion and flow. The rates of dif- the diffusion of membrane proteins, species of labelled molecule (red and green) in the fusion and flow were measured by but a technique called fluorescence focal volume are recorded by two different avalanche photodiode (APD) detectors. detecting the stochastic movement of recovery after photobleaching Co-diffusion of molecules that are tightly linked to fluorophores into and out of a focused (FRAP) enabled researchers to each other induces coincident fluctuations in both channels and, consequently, non-negative cross laser beam. Detection techniques have elucidate the movement of proteins correlation. The cross-correlation amplitudes now improved, making it possible within and between cellular mem- directly reflect on the degree of interactions. Image courtesy of P. Schwille, Technical University to quantify the dynamics of single branes at micrometre-scale resolu- of Dresden, Dresden, Germany. molecules (Rigler and Widengren, tion. In 1974, Bähr and colleagues ▶

▶ photobleached half of an erythrocyte and to quantitate the binding and supported additional applications of plasma membrane labelled with disassociation of membrane proteins fluorescent probes. fluorescein isothiocyanate (FITC) in vivo. Emily J. Chenette, Associate Editor, and found that membrane proteins In 1976, Koppel et al. undertook UCSD-Nature Signaling Gateway did not diffuse rapidly from the a side-by-side comparison of FCS unbleached area into the bleached and FRAP, and noted that each PRimARY ReFeRenCes Magde, D., Elson, E. & area. The robustness of FRAP, and system has distinct advantages and Webb, W. W. Thermodynamic fluctuations in a its utility in a range of applications, disadvantages. Whereas FCS can reacting system — measurement by fluorescence correlation spectroscopy. Phys. Rev. permitted Axelrod et al. to measure provide information about single Lett. 29, 705–708 (1972) | Axelrod, D., intracellular membrane trafficking molecules, its potential to pho- Koppel, D. E., Schlessinger, J., Elson, E. & Webb, W. W. Mobility measurement by analysis just 2 years later. Examining the tobleach samples limits the length of fluorescence photobleaching recovery recovery curves of photobleached and scope of experiments, and the kinetics. Biophys. J. 16, 1055–1069 (1976) | Koppel, E., Axelrod, D., Schlessinger, J., samples allowed the authors to infer experimental set-up is sensitive to Elson, E. L. & Webb, W. W. Dynamics of whether recovery was accomplished perturbation. unlike FCS, FRAP fluorescence marker concentration as a probe of by lateral diffusion, flow from intra- can detect immobile elements in cell mobility. Biophys. J. 16, 1315–1329 (1976) FURtHeR ReADinG Peters, R., Peters, J., cellular compartments or a combina- membranes and can also be applied Tews, K. H. & Bähr, W. A microfluorimetric study of tion of the two. The development of across populations of living cells, but translational diffusion in erythrocyte membranes. Biochim. Biophys. Acta 367, 282–294 (1974) | precise mobility-rate formulae also the required photobleaching step can Rigler, R. & Widengren, J. Ultrasensitive detection permitted the comparison of diffu- irrevocably damage delicate samples. of single molecules by fluorescence correlation spectroscopy. Bioscience 3, 180–183 (1990) | sion and flow between heterogeneous Nonetheless, the development and Schwille, P., Meyer-Almes, F.-J. & Rigler, R. Dual- samples. Today, FRAP is widely refinement of these complementary color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in used to study trafficking within and techniques ushered in a new era solution. Biophys. J. 72, 1878–1886 (1997) between subcellular compartments of fluorescence microscopy and

milestone 11 on biology, and on microscopy in particular. Förster’s proposal was confirmed FRET is uniquely suited Time to start FRETting experimentally in 1967 by Lubert Stryer and Richard Haugland, who to imaging protein– demonstrated that the phenomenon protein interactions of resonance energy transfer could in cells… be used as a ‘spectroscopic ruler’ to determine the distance between two chromophores. These findings paved the way for using FRET to observe interactions in cells because for a interactions and conformational change to be seen in the FRET signal dynamics in purified proteins the two fluorophores must be in very in vitro. close proximity. The first papers But Förster’s theory also led to demonstrating this application of the another application: mapping pro- technique were published in the mid- tein distributions and interactions 1990s, after fluorescence microscope in living cells with high resolution technology had improved and the using a fluorescence microscope. confocal fluorescence microscope Fernandez and Berlin first showed had become available. In 1993, in 1976 that the concept of FRET Brian Bacskai, Roger Tsien and their Förster (or fluorescence) The theory behind Förster resonance could be used to examine the colleagues used FRET and confocal resonance energy transfer microscopy has been used to energy transfer (FRET) — or fluores- dynamic distribution of receptor fluorescence microscopy to follow address questions about the cence resonance energy transfer, as complexes on the cell surface. This the fate of the catalytic and regulatory existence of lipid rafts. Image is reproduced, with it is also known — was first formally was a substantial advance for the subunits of cyclic AMP-dependent permission, from R. Varma & proposed in the 1940s by Theodor field because it facilitated the protein kinase, as well as free cAMP, S. Mayor © (1998) Macmillan Publishers Ltd. All rights Förster, who showed that electronic determination of receptor distribu- in response to stimulation of sensory reserved. excitation energy can be transferred tion with much higher spatial neurons from a marine snail with from a donor fluorophore to an resolution than with standard serotonin. acceptor chromophore in close prox- fluorescence tagging. However, Thomas Jovin’s group soon imity, with an efficiency dependent unlike the authors of work to come, followed with the pioneering of on the inverse of the sixth power of Fernandez and Berlin did not show FRET microscopy techniques that the distance separating the two. This any microscopy images. improved the sensitivity and accuracy relatively simple equation would turn FRET is uniquely suited of FRET so as to allow quantitative out to have a considerable impact to imaging protein–protein measurements of protein interactions ▶

▶ in cells. In 1995, Jovin and colleagues as a microscopy technique has PRimARY ReFeRenCes Fernandez, S. M. & Berlin, R. D. Cell surface distribution of introduced donor photobleaching been borne out in its application to lectin receptors determined by resonance energy transfer. Nature 264, 411–415 (1976) | Bacskai, B. J. et al. Spatially resolved dynamics of cAMP and protein kinase A subunits and fluorescence lifetime imaging challenging biological questions, such in Aplysia sensory neurons. Science 260, 222–226 (1993) | Gadella, T. W. J. Jr & Jovin, T. M. microscopy (FLIM), which they used as the existence of lipid rafts. A wide Oligomerization of epidermal growth factor receptors on A431 cells studied by time- to study epidermal-growth-factor variety of FRET-based biosensors resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. J. Cell Biol. 129, 1543–1558 (1995) | Bastiaens, P. I. H. et al. Imaging

receptor oligomerization on single are now available for detecting small the intracellular trafficking and state of the AB5 quarternary structure of cholera toxin. cells. They introduced acceptor molecules and enzyme activities in EMBO J. 15, 4246–4253 (1996) FURtHeR ReADinG Förster, T. Energiewanderung und Fluoreszenz. photobleaching in 1996, which they cells. And the technology continues to Naturwissenschaften 6, 166–175 (1946) | Stryer, L. & Haugland, R. P. Energy transfer: a used to follow the subcellular locali- evolve, as novel donor–acceptor pairs spectroscopic ruler. Proc. Natl Acad. Sci. USA 58, 719–726 (1967) | Kenworthy, A. K. & Edidin, M. Distribution of a glycosylphosphatidylinositol-anchored protein at the zation and oligomerization state of are developed, new methods are intro- apical surface of MDCK cells examined at a resolution of <100 Å using imaging the cholera toxin in cells. duced and instrumentation improves. fluorescence resonance energy transfer. J. Cell Biol. 142, 69–84 (1998) | Varma, R. & Mayor, S. GPI-anchored proteins are organized in submicron domains at the cell Since these landmark develop- Allison Doerr, Associate Editor, surface. Nature 394, 798–801 (1998). ments, the importance of FRET Nature Methods

milestone 12 calcium, a messenger under the spotlight

“It is scarcely necessary any longer to successful. Compared with Quin2, stress the importance of intracellular the new dyes showed much stronger free Ca2+ as a second messenger” fluorescence, higher wavelength shift reads the opening line of Roger on Ca2+ binding, weaker affinity for Ca2+ Tsien’s 1980 paper, reporting for the and better selectivity against other first time the synthesis of rationally ions. Soon after, high-resolution digital designed fluorescent Ca2+ probes imaging of single smooth muscle cells for intracellular use. Indeed, studies labelled with Fura-2 allowed Tsien, on calcium date back to the late Fredric Fay and their co-workers to nineteenth century, underscoring perform unprecedented spatiotemporal its significance. Reliable methods measurements of Ca2+ gradients in advances such as fast cameras and Carbachol-evoked Ca2+ to measure intracellular Ca2+ subcellular compartments, revealing automated filter wheels, and their uses signals in human embryonic concentrations first appeared in the the differential regulation of Ca2+ kidney cells recorded using have been further expanded by the the fluorescent indicator 1960s, but it was not until the 1980s concentrations in the cytoplasm, concomitant development of imaging Fluo-4. Warmer colours that the rapid expansion of new nucleus and sarcoplasmic reticulum of denote a higher Ca2+ technologies such as fluorescence Ca2+-measurement techniques opened these contracting cells. concentration. Image lifetime imaging and multiphoton courtesy of Z. Ding and the way to spatial and temporal Critical to the widespread microscopy. Today, fluorescent calcium C. W. Taylor, University of analysis of the complex dynamics of popularity of the polycarboxylate Cambridge, UK. dyes remain key to studies that combine Ca2+ concentrations in living cells. Ca2+ indicators was the synthesis of imaging with electrophysiological In his original paper, Tsien introduced their acetoxymethyl esters, which analyses. a new generation of fluorescent are membrane permeable and allow Silvia Grisendi, Associate Editor, polycarboxylate dyes derived from non-disruptive loading and efficient Nature Cell Biology the selective Ca2+ chelator ethylene- trapping of the dyes in intact cells once glycoltetraacetic acid (EGTA). Despite the esterifying groups are hydrolysed their ground-breaking potential, the (Tsien, 1981). One disadvantage of PRimARY ReFeRenCes Tsien, R. Y. New calcium indicators and buffers with high selectivity prototype BAPTA and its derivative the Fura-2 family of dyes was that against magnesium and protons: design, 2+ Quin2 had relatively limited application they required excitation at ultraviolet synthesis, and properties of prototype structures. … Ca owing to restrictions imposed by their wavelengths, which limited their range Biochemistry 19, 2396–2404 (1980) | Tsien, R. Y. measurement A non-disruptive technique for loading calcium absorption spectrum, low selectivity of potential applications. In 1989, Tsien buffers and indicators into cells. Nature 290, techniques and fluorescence intensity. However, all and colleagues further developed the 527–528 (1981) | Grynkiewicz, G., Poenie, M. & 2+ opened the of the polycarboxylate dyes visible-wavelength indicators Rhod-2, Tsien, R. Y. A new generation of Ca indicators with greatly improved fluorescence properties. way to spatial currently available are derived from Fluo-2 and Fluo-3, which are among the J. Biol. Chem. 260, 3440–3450 (1985) and temporal this original design, the flexibility of most widely used non-ratiometric Ca2+ FURtHeR ReADinG Williams, D. A., Fogarty, K. E., which enabled the synthesis of many indicators so far, thanks in part to their Tsien, R. Y. & Fay, F. S. Calcium gradients in single analysis … smooth muscle cells revealed by the digital improved dyes within a few years of the general applicability to many cell types, imaging microscope using Fura-2. Nature 318, original report. including neurons. 558–561 (1985) | Minta, A., Kao, J. P. & Tsien, R. Y. In 1985, Tsien and his colleagues Besides their crucial role in Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. introduced six new indicators, among dogma-changing discoveries, synthetic J. Biol. Chem. 264, 8171–8178 (1989) which Fura-2 and Indo-1 were the most Ca2+ indicators have driven technological

milestone 13 TIRF: imaging at the cellular edge

If you want to visualize what happens at a reflected back into the slide. At the same time, a exocytosis of synaptic vesicles, and visualized biological interface or on the surface of a cell, tiny part of the beam called an evanescent wave the vesicles approaching the plasma membrane it is vital to eliminate the background that manages to penetrate a few hundred nanometres and unloading their contents. In the same year, comprises all the stuff that sits beyond that first into the sample and can stimulate fluorescence Axelrod and colleagues similarly imaged the layer. This is exactly the approach used in total in a biological medium that is tagged with fusion of post-Golgi carriers with the plasma internal reflection fluorescence microscopy chromophores. membrane. These studies are also early examples (TIRF). Building on studies from the late 1800s The evanescent wave penetrates the sample of through-the-objective TIRF, which, through the on the detection of scattered photons from TIR only for some 200 nm and its intensity decreases use of high numerical aperture objectives, further excitation, Daniel Axelrod built a microscope in exponentially, thereby producing exquisite simplifies the setup and sample handling. Given which a laser beam hits, at an angle, a glass slide z-axis sensitivity. Restricting illumination to the number of important events that take place — on the other side of which the sample is placed. a thin section at the interface of the sample at the cellular membrane, including endocytosis, The incident angle is greater than the critical and the glass slide eliminates background exocytosis, receptor activation and ion transport, angle, and therefore the beam is completely fluorescence from deeper in the sample and having a technique that can access this layer allows longer imaging periods by limiting has set the stage for what can be done with TIRF bleaching of chromophores that can move into at present. or out of the illuminated layer during imaging. Stefano Tonzani, Associate Editor, Nature In 1981, Axelrod demonstrated the imaging capabilities of the technique by visualizing PRimARY ReFeRenCes Axelrod, D. Cell-substrate cellular focal adhesions on a glass slide. contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89, 141–145 (1981) | Funatsu, T., In the mid-1990s, Yanagida and colleagues Harada, Y., Tokunaga, M., Saito, K. & Yanagida, T. Imaging of took the thin illumination layer provided by single fluorescent molecules and individual ATP turnovers by TIRF to its logical consequence and performed single myosin molecules in aqueous solution. Nature 374, 555–559 (1995) | Zenisek, D., Steyer, J. A. & Almers, W. single-molecule detection of biological events in Transport, capture and exocytosis of single synaptic vesicles aqueous solutions and in real time. They reported at active zones. Nature 406, 849–854 (2000) | Schmoranzer, J., individual ATP turnover events by a single Goulian, M., Axelrod, D. & Simon, S. M. Imaging constitutive exocytosis with total internal reflection fluorescence myosin molecule in 1995. This setup predated microscopy. J. Cell Biol. 149, 23–32 (2000) confocal microscopy by several years, and to a FURtHeR ReADinG Thompson, N. L., Burghardt, T. P. & large extent it is much simpler and less expensive Axelrod, D. Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching because it can be easily fitted on to conventional recovery or correlation spectroscopy. Biophys. J. 33, 435–454 Clathrin-coated pits (red), actin stress fibres (parallel green inverted fluorescence microscopes. (1981) | Merrifield, C. J., Feldman, M. E., Wan, L. & Almers, W. lines) and local foci of actin polymerization (green dots) near In 2000, Almers and colleagues used the high Imaging actin and dynamin recruitment during invagination of the plasma membrane of a fibroblast. Image courtesy of single clathrin-coated pits. Nature Cell Biol. 4, 691–698 (2002) W. Almers, Vollum Institute, Portland, Oregon, USA. z-sensitivity of TIRF to image the process of

milestone 14 Imaging without the blur

The distribution and structure of hoped to reconstruct 3D images from knowledge of the microscope’s molecules within the cell are central stacks of ‘optical sections’ — images optical properties to model the to their functions. By enabling captured at consecutive focal planes process by which imaging distorts high-resolution three-dimensional through a thick object. However, the ‘real’ object to produce the raw (3D) imaging of the cell’s interior, interpreting the information in any image. In several rounds of iterative the application of deconvolution given focal plane was complicated by computation, estimates of the object algorithms to microscopy has made a the presence of out-of-focus fluores- are entered into the equation and a vital contribution to our understand- cence from neighbouring planes. comparison of the calculated outcome ing of cell biology. In 1983, Agard and Sedat brought to the raw image is used to improve By the 1980s, fluorescent probes deconvolution — a method to the estimate, until the object’s true for many specific cellular molecules eliminate out-of-focus fluorescence properties are determined. In this were available. However, visualizing or restore it to its original source way, Agard and Sedat obtained their distribution in relatively thick — to the attention of cell biologists. the first high-resolution images of specimens was tricky. Researchers Deconvolution algorithms use the structure and organization of ▶

calculations. However, as desktop computers became more powerful, cell biologists quickly realized the advantages of deconvolution, partic- ularly for long-term, live-cell imaging in which reduced light intensities are important and confocal microscopy techniques (see Milestone 9) are limited. The contribution of deconvolution to the visualization of organelles and cellular macromolecules has thus been central to our current understanding of many cellular processes. A HeLa cell in metaphase, before (left) or after (right) deconvolution. Staining shows DNA (blue), microtubules (white), centrosomes (green) and the centrosomal protein ASPM (red). Images courtesy of A. Ferrand, Katherine Whalley, Senior Editor, University of Dundee, Dundee, UK. Nature Reviews Neuroscience These papers … PRimARY ReFeRenCes Agard, D. A. & demonstrated Sedat, J. W. Three-dimensional architecture of a ▶ fluorescently labelled chromatin in circumvent this problem, the authors polytene nucleus. Nature 302, 676–681 (1983) | the high the nuclei of Drosophila melanogaster introduced constraints to the object Fay, F. S., Carrington, W. & Fogarty, K. E. Three- dimensional molecular distribution in single cells level of 3D salivary glands. estimates: they eliminated estimates analysed using the digital imaging microscope. macromolecular Numerous deconvolution algo- in which the object was not ‘regular’ J. Microsc. 153, 133–149 (1989) Richardson, W. H. structure rithms exist (see Further Reading); (or smooth) or in which fluorescence FURtHeR ReADinG Bayesian-based iterative method of image present in however, another key advance levels were less than zero, a physically restoration. J. Opt. Soc. Am. 62, 55–59 (1972) | was made in 1989, when Fay and impossible situation. This yielded Lucy, L. B. An iterative technique for the cells and the rectification of observed distributions. colleagues were examining the high-resolution images that enabled Astron. J. 79, 745–754 (1974) | Shepp, L. A. & structural molecular basis of force generation in the quantitative analysis of α-actinin Vardi, Y. Maximum likelihood reconstruction complexity of smooth muscles. Basic deconvolution distribution in smooth muscle cells. for emission tomography. IEEE Trans. Med. Imaging 1, 113–122 (1982) | Carrington, W. A. the cell interior. algorithms can amplify noise in the Initially, deconvolution was et al. Superresolution three-dimensional images, making it difficult to nar- limited to those researchers who had images of fluorescence in cells with minimal Jason Swedlow row the range of possible estimates access to the large computing facili- light exposure. Science 268, 1483–1487 (1995) to determine the real object. To ties required to perform the intensive

m i l e s to n e 1 5 It takes two photons to tango

A microscopist’s dream is to be dissertation in 1931. Thirty years able to visualize phenomena later, Kaiser and Garrett observed occurring in cells deep inside … provides clear the phenomenon in crystals. tissues with minimum photo- imaging at a ten times Two-photon excitation results damage to the live samples. from the almost simultaneous The achievement of this goal greater depth than absorption of two photons by a is hampered by the presence of a normal confocal molecule. The wavelength of the out-of-focus flare, which blurs microscope. excitation light needs to be about images obtained with conventional twice that required for single- epifluorescence microscopes. photon excitation because photon In confocal laser scanning energy is inversely proportional ▶ microscopy (see MILESToNE 9), out-of-focus background is to the focal volume, so fluores- eliminated by a pinhole, but the cence detection is less sensitive to Maximum-intensity side projection of a large excitation volume means light-scattering issues. two-photon image stack obtained in a transgenic mouse expressing Clomeleon, a genetically that light scattering limits the Two-photon absorption is an encoded chloride indicator, preferentially in deep extent to which this background old concept in quantum optics layer 5 pyramidal cells. Data were taken with a 40x, NA 0.8 water immersion objective. Scale bar, 100 can be reduced. In two-photon and was first described by Maria mm. Image courtesy of F. Helmchen and T. Kuner, microscopy, excitation is confined Göppert-Mayer in her doctoral University of Zurich, Switzerland.

▶ to the wavelength. In two-photon milestone 16 microscopy, a laser beam is focused on a sample so as to increase the probability of the fluorophore Seeing the light, perpendicularly becoming excited, and the laser focus is scanned across a field Despite tremendous advances in light microscopy, of view to create an image. This visualizing large biological structures in three only became feasible in 1990, when dimensions (3D) has been problematic. Images mode-locked lasers became available from thin histological sections do not convey that could deliver photon densities enough spatial information, and sectioning a million times higher than those cannot be used with live specimens. Furthermore, of conventional lasers, in ultrashort non-destructive, optical sectioning (for example, pulses with typical durations in the by confocal microscopy) offers a limited imaging femtosecond range. depth for large samples, and the axial (‘z’) Denk, Strickler and Webb resolution is inherently much lower than the were the first to use this method lateral resolution. A simple solution might to image chromosomes in live overcome these issues: illuminating the specimen cultured cells. But Denk et al. at a 90° angle with a light sheet. soon adapted it to image neurons The idea of illuminating samples from the side Images of embryo development obtained with digital scanned in situ, and the full potential of the with a light sheet was first described in 1903, laser light sheet fluorescence microscopy (DSLM), an improved version of selective plane illumination microscopy (SPIM), both of technique became apparent when but was not explored until Voie et al. pioneered which were developed by the Stelzer laboratory. The digital Svoboda et al. used it in 1997 to the same principle in 1993 to develop embryo (left half) shows patterns of cell migration in different orthogonal-plane fluorescence optical sectioning directions: dorsal migration (cyan), ventral migration (green), measure sensory-stimulus-induced migration towards the body axis (red) or away from it (yellow), and dendritic calcium dynamics in (ORFOS). In this approach, the specimen is migration towards the yolk (pink). Image is reproduced, with optically sectioned by a thin light sheet that is permission, from P. J. Keller et al. © (2008) American Association pyramidal neurons in intact for the Advancement of Science. rat brain. produced by a cylindrical macroscopic lens. The By confining excitation to the light sheet is projected on to one plane of a focal volume, two-photon laser transparent, fluorescently labelled specimen, only one plane of the embryo. This helped to scanning fluorescence microscopy perpendicularly to the detection axis. Because only overcome a major challenge: visualizing allows the correct assignment of one plane of the specimen is illuminated, no light is embryogenesis over long periods of time. Indeed, in photons to the spatial position emitted from regions outside the focal plane and, 2008 Stelzer and co-workers reported an improved from which they originated. The therefore, there are no out-of-focus components in version of SPIM that could image the development use of long excitation wavelengths the image. By rotating their hamster cochlea of zebrafish embryos over 24 hours and track every and the reduced sensitivity to scat- specimen, Voie et al. collected serial images at cell nucleus, thereby obtaining a complete map of tering associated with excitation different angles and reconstructed a 3D cell division and cell migration patterns. confinement provides clear imag- representation of the cochlea, showing the fine With the Stelzer team now collaborating with ing at a ten times greater depth internal structures in great detail. microscope manufacturer Carl Zeiss to improve than a normal confocal micro- Although this method offered enormous this method and develop a commercially scope. In addition, two-photon potential for visualizing large specimens in detail, it available product, light sheet microscopy will no excitation greatly diminishes was not developed commercially and was used only doubt further our understanding of biological photobleaching and photodamage, by a few research groups — most notably Fuchs processes in 3D. making it ideal for imaging living et al., who in 2002 developed a light sheet system Rachel David, Assistant Editor, Nature Reviews cells buried deep inside intact using a microscope lens to visualize aquatic bacteria Molecular Cell Biology and Nature embryos or organs. in their natural setting. In 2004, however, Stelzer and Reviews Microbiology colleagues developed a light sheet microscopy Nathalie Le Bot, Senior Editor, application with even broader appeal, which could Nature Cell Biology PRimARY ReFeRenCes Voie, A. H., Burns, D. H. & Spelman, F. A. be used to image both fixed and live embryos. Orthogonal-plane fluorescence optical sectioning: three- Termed selective plane illumination microscopy dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229–236 (1993) | Huisken, J., Swoger, J., Del Bene, F., PRimARY ReFeRenCe Denk, W., Strickler, J. H. & (SPIM), the technique offers 3D images at high Wittbrodt, J. & Stelzer, E. H. K. Optical sectioning deep inside live Webb, W. W. Two-photon laser scanning resolution with impressive penetration depth of live embryos by selective plane illumination microscopy. Science 305, fluorescence microscopy. Science 248, 73–76 embryos. Furthermore, phototoxicity over time is 1007–1009 (2004) (1990) FURtHeR ReADinG Siedentopf, H. & Zsigmondy, R. Über FURtHeR ReADinG Göppert-Mayer, M. reduced because the thin sheet of light illuminates Sichtbarmachung und Grössenbestimmung ultramikroskopischer Über Elementarakte mit zwei Quantensprüngen. Teilchen, mit besonderer Anwendung auf Goldrubingläser. Ann. Phys. 9, 273–294 (1931) | Kaiser, W. & Annalen der Physik 10, 1–39 (1903) | Stelzer, E. H. K. & Lindek, S. Garrett, C. G. B. Two-photon excitation in Fundamental reduction of the observation volume in far-field CaF : Eu2+. Phys. Rev. Lett. 7, 229–231 (1961) | 2 light microscopy by detection orthogonal to the illumination axis: Denk, W. et al. Anatomical and functional … the technique offers 3D confocal theta microscopy. Opt. Comm. 111, 536–547 (1994) | imaging of neurons using 2-photon laser Fuchs, E., Jaffe, J. S., Long, R. A. & Azam, F. Thin laser light sheet scanning microscopy. Neurosci. Methods 54, images at high resolution with microscope for microbial oceanography. Opt. Express 10, 151–162 (1994) | Svoboda, K., Denk, W., Kleinfeld, 145–154 (2002) | Keller, P. J., Schmidt, A. D., Wittbrodt, J. & D. & Tank, D. W. In vivo dendritic calcium impressive penetration depth Stelzer, E. H. K. Reconstruction of zebrafish early embryonic dynamics in neocortical pyramidal neurons. development by scanned light sheet microscopy. Science 322, Nature 385, 161–165 (1997) of live embryos. 1065–1069 (2008)

m i l e s to n e 1 7 single molecules in the dark

The potential to visualize bright — including its use for Förster (or 0 min 24 min 48 min 72 min fluorescent molecules on a dark back- fluorescence) resonance energy trans- ground using fluorescence microscopy fer (FRET) as a molecular ruler for lit up the field by providing molecular single molecules — others turned to specificity and image contrast unparal- alternatives. In 1995, Toshio Yanagida lelled by other dyes. Theory suggested and colleagues used an optimized that these properties should permit the implementation of total internal reflec- 96 min 120 min 144 min 168 min detection and imaging of single mole- tion fluorescence microscopy (TIRF) cules. A first glimpse of the possibilities to image single myosin molecules came in 1976, when Thomas Hirshfeld labelled with one fluorophore and to used a fluorescence microscope to detect the turnover of ATP labelled detect single molecules bound by with a different fluorophore. unlike dozens of fluorophores as they passed previous methods using immobilized through a thin layer of illumination. molecules, this work showed that seemingly well-characterized systems. Time-lapse images of Escherichia coli cells showing But technical limitations hindered TIRF could image many molecules in Similar enzymatic-turnover assays single-molecule imaging of progress. Although the next two dec- aqueous solution for periods of several developed by other groups eventually the expression of single fluorescent fusion proteins. ades witnessed the detection of single seconds before bleaching occurred, led to the sequencing of individual The fluorescent molecules fluorophores at low temperatures, and demonstrated the suitability DNA molecules. are photobleached in the intervals between each or while transiting a highly focused of this method for true biological Methods continued to be devel- image. Image is reproduced, laser beam (see MILESToNE 10), such applications. oped and refined as highlighted by a with permission, from J. Yu et al. © (2006) American methods were incapable of repeatedly Although TIRF would come to number of groundbreaking studies Association for the imaging identified molecules in dominate most single-molecule on ribozymes, molecular motors and Advancement of Science. ambient environments. imaging applications, it was not the gene expression in single living cells In 1993, this situation changed first technique to image the move- as scientists pushed the limits of markedly when Eric Betzig and Robert ment of single fluorophores. This technology and imagination. Chichester reported the first repetitive first was claimed by conventional Daniel Evanko, Editor, Nature Methods imaging of single fluorophores at room epifluorescence microscopy (see temperature with a new technique MILESToNE 4) with a highly sensi- PRimARY ReFeRenCes Betzig, E. & Chichester, R. J. called near-field scanning optical tive charge-coupled device (CCD) Single molecules observed by near-field scanning optical microscopy. Science 262, 1422–1425 (1993) | microscopy (NSoM) that repeatedly camera. Hansgeorg Schindler and Funatsu, T., Harada, Y., Tokunaga, M., Saito, K. & scans an extremely small optical his co-workers adapted the method Yanagida, T. Imaging of single fluorescent molecules probe over a sample. This provided of Jeff Gelles et al. for single-particle and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, molecule-scale spatial localization and tracking based on Gaussian-peak 555–559 (1995) | Schmidt, T., Schütz, G. J., information on molecular orientation. fitting to determine the positions of Baumgartner, W., Gruber, H. J. & Schindler, H. The potential biological applications individual fluorophore-labelled lipids Imaging of single molecule diffusion. Proc. Natl Acad. Sci. USA 93, 2926–2929 (1996) | Lu, H. P., of single-molecule imaging captured in a membrane with an accuracy of Xun, L. & Xie, X. S. Single-molecule enzymatic the imaginations of microscopists about 15 nanometres and to track dynamics. Science 282, 1877–1882 (1998) FURtHeR ReADinG Hirschfeld, T. Optical and biologists alike, but because of its their movements over time. microscopic observation of single small molecules. invasiveness and complexity NSoM up until this point, single-molecule Appl. Opt. 15, 2965–2966 (1976) | Gelles, J., proved largely unsuitable for complex fluorescence microscopy applications Schnapp, B. J. & Sheetz, M. P. Tracking kinesin-driven movements with nanometre-scale precision. Nature biological samples. had an almost proof-of-principle feel 331, 450–453 (1988) | Shera, E. B., Seitzinger, N. K., Although a number of people to them, and for the most part novel Davis, L. M., Keller, R. A. & Soper, S. A. Detection of continued to try to improve NSoM biological insights were limited. But single fluorescence molecules. Chem. Phys. Lett. 174, 553–557 (1990) | Macklin, J. J., Trautman, J. K., in 1998, Sunney Xie and colleagues Harris, T. D. & Brus, L. E. Imaging and time-resolved used the intrinsic on–off fluorescence spectroscopy of single molecules at an interface. Science 272, 255–258 (1996) | Ha, T. et al. Probing the switching of flavin adenine dinucle- interaction between two single molecules: … Eric Betzig and otide in the active site of cholesterol fluorescence resonance energy transfer between a Robert Chichester oxidase to reveal that enzyme activity single donor and a single acceptor. Proc. Natl Acad. Sci. USA 93, 6264–6268 (1996) | Zhuang, X. et al. reported the first is influenced by a form of molecular A single-molecule study of RNA catalysis and memory residing in conformational folding. Science 288, 2048–2051 (2000) | Yildiz, A. repetitive imaging of changes in the protein. This behaviour et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. single fluorophores at was completely unanticipated from Science 300, 2061–2065 (2003) | Yu, J., Xiao, J., Ren, X., room temperature … ensemble experiments and showed Lao, K. & Xie, X.S. Probing gene expression in live cells, one protein molecule at a time. Science 311, unequivocally that single-molecule 1600–1603 (2006) microscopy could shed new light on

milestone 18 GFP: the green revolution

In 1994, Chalfie et al. published a report for experiments to assess its utility as an Cell bodies of two touch-receptor neurons in Science showing that the green in vivo tag for proteins. Two years later, from the worm fluorescent protein (GFP) from the Chalfie et al. showed that GFP could Caenorhabditis elegans jellyfish Aequorea victoria could be used fluoresce when expressed in bacteria are labelled with green fluorescent protein as a marker for protein localization and worm cells. In the worm, GFP was expressed from the and expression in living bacteria and expressed from the promoter of a gene gene encoding β-tubulin. Image is worm cells, in the absence of any that encoded β-tubulin. Its spatial and reproduced, with auxiliary factors from A. victoria. This temporal expression in specific neurons permission, from demonstration of GFP as a tool to study of the worm mimicked that of the M. Chalfie et al. © (1994) American proteins in vivo fundamentally altered endogenous β-tubulin gene, thus proving Association for the the nature and scope of the issues that that GFP could be a faithful marker for Advancement of Science. could be addressed by cell biologists. monitoring gene-expression GFP was first discovered fortuitously patterns. Soon thereafter, Roger Tsien’s in 1962 by Shimomura and colleagues laboratory engineered native GFP to during the purification of the become brighter, more photostable and bioluminescent protein aequorin from excitable at a wavelength that matched of the green fluorescent protein, GFP”, A. victoria. Subsequent purification, that of conventional microscope filter acknowledging the seminal nature of crystallization and reconstitution of sets, increasing its practical usability this discovery. energy transfer in vitro from aequorin to (as summarized in his 1998 review). The Sowmya Swaminathan, Senior Editor, GFP by Morise and colleagues in 1974 next breakthrough in GFP technology Nature Cell Biology provided insight into the fluorescent came with the development of GFP properties of GFP, which was shown to variants to produce blue, cyan and yellow emit green light on energy transfer from fluorescent proteins (also described in PRimARY ReFeRenCe Chalfie, M., Tu, Y., Euskirchen. G., Ward, W. W. & aequorin. Tsien’s review), thus enabling imaging Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994) Whether GFP would need aequorin experiments that could follow multiple FURtHeR ReADinG Shimomura, O., Johnson, F. H. & Saiga, Y. Extraction, and possibly other factors from the tagged proteins in cells and organisms. purification and properties of aequorin, a bioluminescent protein from the luminous jellyfish to fluoresce in heterologous Almost half a century after GFP was hydromedusan Aequorea. J. Cell. Comp. Physiol. 59, 223–239 (1962) | Morise, H., systems remained an open question discovered, the 2008 Nobel Prize in Shimomura, O., Johnson, F. H. & Winant, J. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 13, 2656–2662 (1974) | Prasher, D. C., for many years. In 1992, 30 years after Chemistry was awarded to Osamu Eckenrode, V. K., Ward, W. W., Prendergast, F. G. & Cormier, M. J. Primary structure of the its discovery, Prasher et al. cloned the Shimomura, Martin Chalfie and Roger Aequorea victoria green-fluorescent protein. Gene 111, 229–233 (1992) | gene encoding GFP, paving the way Tsien “for the discovery and development Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998)

milestone 19 2.70 2.36 0s 40s 60s 80s100s 2.03 1.69 Sensing second messengers 1.35 1.01 0.68 0.34 The discovery of green fluorescent 0.00 protein (GFP) as a probe for These visualizing proteins in vivo (see Ratio 460/545 2+ MILESToNE 18) laid the founda- Förster (or fluorescence) resonance energy transfer (FRET) between subunits of protein kinase A two Ca (PKA) tagged with green fluorescent protein is disrupted after cyclic AMP stimulation (time in tion for even more sophisticated biosensors seconds), which causes PKA subunits to dissociate. Image is reproduced, with permission, from microscopy techniques. GFP M. Zaccolo et al. © (2000) Macmillan Publishers Ltd. All rights reserved. were mutants with shifted excitation or the first emission wavelengths that emit in genetically blue, cyan, green and yellow can used FRET to engineer biosensors myosin light-chain kinase (M13) serve as donors and acceptors for that measure changes in Ca2+ and green GFP. on binding by encoded Förster (or fluorescence) resonance concentrations in living cells. Ca2+, CaM wraps around M13, molecular energy transfer (FRET) — energy Ca2+ binds to calmodulin decreasing the distance between sensors. transfer between two fluorophores (CaM), changing its conformation the GFP proteins and allowing that depends on their proximity and and allowing it to bind to target FRET. In bacteria, the presence of orientation, and allows the accep- proteins. Miyawaki et al. made Ca2+ was visualized as a change in tor fluorophore to emit at a longer a construct encoding blue GFP, emission on violet excitation wavelength. In 1997, two groups CaM, the CaM-binding peptide of from 445 nm to 510 nm, and ▶

▶ this biosensor reported Ca2+ so that Ca2+–CaM binding induced changes in cAMP level and vali- concentrations from 10–7 to 10–4 a conformational change in GFP dates FRET as a tool for studying M. Independently, and in parallel, that increased the intensity of its protein–protein interactions. Today, Romoser et al. linked blue- and fluorescence. Fluorescence output fluorescent biosensors allow protein green-emitting GFPs through M13 from this high-affinity Ca2+ probe activity, protein interactions and to detect Ca2+. In the absence of was enhanced when Ca2+ concentra- small-molecule signals to be sensed Ca2+, excitation of blue GFP causes tions were increased in myotubes. and quantified with high spatial and the green acceptor to fluoresce Another advance was made temporal resolution. at 505 nm as a result of FRET. in 2000 when, using a biosensor Katharine H. Wrighton, Associate Editor, 2+ Binding of Ca and CaM to M13 for cyclic AMP, Zaccolo et al. Nature Reviews Molecular Cell Biology increases the inter-fluorophore showed that FRET can measure distance, eliminates FRET and protein–protein interactions. The PRimARY ReFeRenCes Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997) | Romoser, V. A. causes donor emission at 448 nm. authors linked green GFP and blue et al. Detection in living cells of Ca2+-dependent changes in the fluorescence emission Fluorescence responses to Ca2+ GFP to catalytic and regulatory of an indicator composed of two green fluorescent protein variants linked by a in cells were enhanced by co- subunits of protein kinase A (PKA), calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13270–13274 (1997) | Zaccolo, M. et al. A genetically encoded, fluorescent indicator for expressing CaM with the biosensor. respectively, and co-expressed these cyclic AMP in living cells. Nature Cell Biol. 2, 25–29 (2000) | Nakai, J. et al. A high signal- These two Ca2+ biosensors were the constructs in cells. Excitation at to-noise Ca2+ probe composed of a single green fluorescent protein. Nature Biotechnol. 19, 137–141 (2001) first genetically encoded molecular 380 nm resulted in FRET, which FURtHeR ReADinG Adams, S. R. et al. Fluorescence ratio imaging of cyclic AMP in sensors. suggested that these subunits single cells. Nature 349, 694–697 (1991) | Miesenböck, G. Visualizing secretion and In 2001, Nakai et al. developed interacted in vivo. Physiological synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 2+ 192–195 (1998) | Baird, G. S. Circular permutation and receptor insertion within green a Ca biosensor with a higher stimulation of cAMP induced a loss fluorescent proteins. Proc. Natl Acad. Sci. USA 96, 11241–11246 (1999) | Ng, T. et al. signal-to-noise ratio that used just of FRET consistent with its ability Imaging protein kinase Cα activation in cells. Science 283, 2085–2089 (1999) | Ghosh, I. one GFP protein. They positioned a to dissociate PKA subunits. Thus, et al. Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658–5659 (2000) GFP variant between M13 and CaM this biosensor detects physiological

milestone 20 …the next day the Beyond gFp: Dsred and pA-gFp sample had turned red.

Following the isolation of green fluorescent with individual proteins (red or green) or that it moved between lysosomes in a protein (GFP) from the luminescent both proteins (yellow) at the tadpole stage. microtubule-dependent manner. jellyfish Aequorea victoria in 1962 and its This represented the first double-labelling Later that year, Ando et al. cloned a first application as a fluorescent probe in experiment. gene encoding a bright GFP from the coral 1994 (see Milestone 18), the race was on Three years later, Patterson et al. Trachyphylla geoffroy and accidentally to expand the colour palette of fluorescent generated the first photoactivatable GFP left an aliquot of the expressed protein on proteins and to produce new probes suitable (PA-GFP), which turned out to be a crucial the bench: the next day the sample had for studying dynamic cellular processes. advance for super-resolution fluorescence turned red. The fluorescent protein, named Blue, cyan and yellow fluorescent pro- microscopy approaches. The PA-GFP vari- Kaede (Japanese for maple leaf), changed teins were quickly obtained by modifying ant contains a Thr-to-His mutation that to red when irradiated with ultraviolet GFP, but variants emitting in the orange and makes it devoid of green fluorescence until (350–400 nm) or violet light. using a red spectra (which would allow multicolour it is activated by violet light (390–415 nm). focused ultraviolet pulse, the authors imaging experiments and be less phototoxic This leads to a 100-fold increase in green highlighted a single neuron in red among than other colours owing to their longer fluorescence (with an emission peak at many green ones expressing Kaede, thereby excitation wavelengths) remained elusive. A 504 nm), which is ideal for rapidly marking showing the protein’s potential use as an breakthrough occurred in 1999, when Matz a selected population of molecules within optical marker. et al. proposed that the fluorescent property cells and following their kinetics over time. Despite these advances, the available of GPF-like proteins might not necessarily Indeed, Patterson et al. demonstrated the fluorescent proteins emitting in the orange be linked to bioluminescence and searched value of PA-GFP by tagging a lysosomal and red regions were not optimal for for them in non-bioluminescent Anthozoa membrane protein with it and showing multicolour live imaging (in terms of ▶ species — reef corals that exhibit bright fluorescent colours. This led to the cloning of the first red fluorescent protein (known Purified fluorescent proteins are shown in fluorescence (from left to right, mHoneydew, mBanana, mOrange, as DsRed, for Discosoma sp. red). Injection tdTomato, mTangerine, mStrawberry and mCherry). of mRNAs encoding DsRed or a green Image is reproduced, with permission, from N. C. Shaner variant into blastomeres of Xenopus laevis et al. © (2004) Macmillan Publishers Ltd. All rights reserved. embryos revealed regions that were marked

▶ brightness and photostability, for exam- fusions, have faster maturation rates and PRimARY ReFeRenCes Matz, M. V. et al. Fluorescent ple). However, the use of directed protein are brighter than previously identified red- proteins from nonbioluminescent Anthozoa species. Nature evolution by Shaner et al. generated red, shifted monomeric fluorescent proteins. Biotechnol. 17, 969–973 (1999) | Patterson, G. H. & Lippincott- Schwartz, J. A photoactivatable GFP for selective orange and yellow fluorescent proteins with The availability of fluorescent proteins in photolabeling of proteins and cells. Science 297, 1873–1877 improved properties. These proteins, which many colours, and the ability of some to be (2002) | Ando, R. et al. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent were named after fruits that bear colours photoactivated (PA-GFP) or photoconverted protein. Proc. Natl Acad. Sci. USA 99, 12651–12656 (2002) | similar to the emission profiles (for example, (Kaede), has revolutionized live-cell imaging. Shaner, N. C. et al. Improved monomeric red, orange and mStrawberry, mCherry and mBanana), are Francesca Cesari, Associate Editor, Nature yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004). monomeric, tolerant to amino-terminal Reviews Molecular Cell Biology

and high-accuracy single-molecule milestone 21 localization to break the diffraction barrier. Although the details differ, all three methods are based on a single Light microscopy at the limit principle: a sparse subset of fluorophores is switched on at any one time, and each fluorophores at its rim through stimulated molecule is localized with high accuracy. emission with a doughnut-shaped STED These fluorophores are then switched off beam of red-shifted light. The tiny spot is and the process is repeated with another then scanned over the sample to generate subset until enough information has been a sub-diffraction image. In the first collected to generate a sub-diffraction experimental implementation of STED, image. which included imaging of live yeast and The hope for super-resolution bacteria, resolution was improved both microscopy is that visualizing cells at axially and laterally and reached about this unprecedented scale will yield 100 nm. The resolution has since been unprecedented insights. Although improved further, allowing the technique the application of these methods to to be used to show that the synaptic biological questions has begun, the full vesicle-associated protein synaptotagmin realization of their potential is still to remains clustered on the neuronal plasma come. Their extension to imaging in all membrane after exocytosis. three dimensions and within living cells — Stimulated-emission- For most of the twentieth century, it While STED was being demonstrated, capabilities that are developing rapidly — depletion image recorded from a living neuron of an was held that far-field light microscopy Mats Gustafsson was developing an will be a crucial part of this process. organotypical hippocampal could not resolve objects closer than approach called structured illumination Natalie de Souza, Associate Editor, slice, showing dendritic 150–200 nanometres (see Milestone 3). microscopy (SIM). In SIM, the excitation Nature Methods spines in unprecedented detail. The neuron is This is because light diffracts as it passes light is structured in a controlled pattern, expressing yellow through different media, so that the which renders normally unresolvable PRimARY ReFeRenCes Klar, T. A., Jakobs, S., fluorescent protein. Image Dyba, M., Egner, A. & Hell, S. W. Fluorescence courtesy of K. Willig, V. light emanating from a point is detected information accessible after image as emerging from a larger volume. Light microscopy with diffraction resolution barrier Nägerl, N. Urban, T. processing. Although still diffraction- broken by stimulated emission. Proc. Natl Acad. Sci. Bonhoeffer & microscopy was therefore said to be limited, SIM improved lateral resolution USA 97, 8206–8210 (2000) | Betzig, E. et al. Imaging S. W. Hell, MPI Biophysical intracellular fluorescent proteins at nanometer Chemistry, Göttingen, ‘diffraction-limited’. by about twofold and produced clearer Germany Yet many cellular processes take resolution. Science 313, 1642–1645 (2006) | images of biological structures. The Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction- place within these diffraction-limited technique has since been extended to limit imaging by stochastic optical reconstruction distances, at length scales of tens three dimensions using 3D structured microscopy (STORM). Nature Methods 3, 793–796 (2006) to hundreds of nanometres (nm). To light; moreover, it enables the diffraction FURtHeR ReADinG Hell, S. & Stelzer, E. H. K. be able to visualize these processes, barrier to be broken, using nonlinear Fundamental improvement of resolution with a therefore, microscopy with substantially processes. 4Pi-confocal fluorescence microscope using two- improved resolution was needed. Finally, by exploiting the known photon excitation. Opt. Commun. 93, 277–282 (1992) | Hell, S. W. & Wichmann, J. Breaking It became necessary to ‘break’ the It became ability to localize single molecules the diffraction resolution limit by stimulated diffraction barrier. with nanometre precision, several emission: stimulated-emission-depletion necessary After early work that achieved the single-molecule approaches to fluorescence microscopy. Opt. Lett. 19, 780–782 (1994) | Gustafsson, M. G. L. Surpassing the lateral to ‘break’ the maximum theoretical resolution — achieving sub-diffraction resolution resolution limit by a factor of two using structured diffraction including the standing-wave illumination were developed. The pioneers of these illumination microscopy. J. Microsc. 198, 82–87 reported by Fred Lanni and Jans Taylor methods — Eric Betzig and Harald (2000) | Gordon, M. P., Ha, T. & Selvin, P. R. Single- barrier. and the two-photon 4Pi microscope molecule high-resolution imaging with Hess for photoactivated localization photobleaching. Proc. Natl Acad. Sci. USA 101, of Stefan Hell and Ernst Stelzer — Hell microscopy (PALM), Xiaowei Zhuang 6462–6465 (2004) | Willig, K. I., Rizzoli, S. O., showed, first theoretically and then for stochastic optical reconstruction Westphal, V., Jahn, R. & Hell, S. W. STED microscopy experimentally, that a stimulated- reveals that synaptotagmin remains clustered after microscopy (STORM) and Samuel synaptic vesicle exocytosis. Nature 440, 935–939 emission-depletion (STED) microscope Hess for fluorescence photoactivation (2006) | Hess, S. T., Girirajan, T. P. K. & Mason, M. D. can break the diffraction barrier. To do localization microscopy (FPALM) — used Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. this, the excitation focal spot is shrunk the combination of photoactivatable 91, 4258–4272 (2006) to a very small size by depleting the or photoswitchable fluorophores