Milestones of Light Microscopy

  • Fig. 1: Robert Hooke microscope. © TrondarneFig. 1: Robert Hooke microscope. © Trondarne
  • Fig. 1: Robert Hooke microscope. © Trondarne
  • Fig. 2: Zeiss microscope ca. 1901.  © Bryn Mawr College Special Collections
  • Fig. 3: Patent of the confocal microscope by Marvin Minsky.
  • Fig. 4: Image from the patent for the tandem scanning microscope
  • Fig. 5: 4Pi image © Andy Nestl
  • Fig. 6: Prototype of the Zeiss LSM  © ZEISS Microscopy
  • Fig. 7: Phalloidin-labeled actin filaments as a comparison between STED and confocal microscopy  © Howard Vindin

–1950: Who Invented it?

Historians are still arguing about the names of those who are said to have built the first optical microscope with assembled lenses. It is, inter alia, reported that Zacharias Janssen, a spectacle maker from Middelburg, was the first to invent it at the end of the 16th century. In 1609, the Italian astronomer Galileo Galilei developed a microscope composed of a convex and a concave lens. In 1625, the term "microscope" (from the Greek, the prefix “micros” stands for small and “skopein” for seeing) was introduced by the German physician and botanist Johannes Faber of Bamberg.
In his work "Micrographia", Robert Hooke published numerous microscopic pictures in 1665 (Fig. 1). In 1676, the Dutch naturalist Antonie van Leeuwenhoek described living organisms and bacteria (bacilli, cocci, and spirilla) for the first time. He also designed a new method of producing lenses that allowed 270x magnification. However, he took his art of lens-making to the grave so that after his death it lasted nearly 250 years until it was possible again to construct microscopes with comparable high resolution. In 1830, the physicist Joseph Jackson Lister described a way to eliminate spherical aberration by combining two achromatic cemented elements. Ernst Abbe then founded the theorem, which is still valid today, on the limits of resolution in microscopy in 1871-73. The companion of Carl Zeiss and co-owner of the optics company of the same name in Jena developed the theory of image formation in the microscope, thus putting microscope construction on a scientific basis. One of the essential merits of the German physicist was to replace the conventional and complex testing procedure for the composition of the optical systems, the so-called "pröbeln" (test by trial and error), with a calculated optical system.
In 1930-32, the Dutch chemist and physicist Frits Zernike developed the first phase contrast microscope, which allows transparent samples to be observed, e.g. cells, without previous staining. The significance of this development was initially underestimated, and thus the phase contrast microscope did not experience its industrial breakthrough until 1941.

Zernike was awarded the Nobel Prize for Physics for this achievement in 1953. As early as 1946, the German physicochemist Theodor Förster described the Förster / Fluorescence Resonance Energy Transfer (FRET), in which the energy of an excited fluorescent dye (donor) is transferred without radiation to the immediately adjacent fluorescent dye (acceptor), that then fluoresces. With this method, protein-protein interactions can be observed in real time with a temporal resolution in the millisecond range.

50s: Confocal

This decade marks the birth of the "confocal principle". Marvin Minsky built the first point-scanning confocal microscope (Fig. 3). His reasoning was based on avoiding stray light interference that greatly disturbs the observation of the tissue structures of brain sections.
The concepts for confocal raster scanning microscopes by Klaus Weber are not so well known. The employee at Ernst Leitz in Wetzlar saw three possibilities for scanning: firstly, synchronously rotating Nipkow disks: one in the plane of the luminous-field diaphragm and one in the intermediate image plane; secondly, synchronously moving pinhole diaphragms in the luminous-field plane and in the intermediate image plane, and thirdly, the displacement of the beam path by means of tilted mirrors.
Since there were no suitable digital or photographic documentation possibilities at this time, it took another 30 years for the concept of confocal microscopy to finally take hold.
The 50s were characterized by microphotography. The installation or adaptation of small-image or narrow-film cameras in or to the microscope opened up the possibility of documentation for wide-field imaging, and from then on, observations could be recorded on small-format and narrow film.

60s: Fluorescence

Stewart J. Strickler and Robert A. Berg paved the way for the development of fluorescence lifetime imaging microscopy (FLIM) with their description of a correlation between the absorption intensity and the fluorescence lifetime of molecules.
The two physicians Mojmír Petrá and Maurice David Egger published scientific data observed with the aid of the first imaging confocal microscope with a Nipkow disk (tandem scanning microscope, TSM) (Fig. 4). The birth of the first confocal laser raster- scanning microscope dates back to the end of the 1960s. Maurice Egger and Paul Davidovits published a work on the first confocal microscope with laser light as a point scanner. The authors were then already speculating on the use of fluorescent dyes for observations in vivo.

70s: Another World

The technical development of light microscopy also led to an expansion of the number of visualizable microstructures in the 1970s. Significant progress was made especially in cell physiology. For instance, Dieter Weiss and other employees provided a detailed description of microtubules and the systems associated with them. These important steps in the elucidation of tubulin structure have led to further scientific breakthroughs in medicine, including cancer research.
On the basis of the confocal microscope, Douglas Magde and other scientists developed fluorescence correlation spectroscopy (FCS). This technique, for example, plays an important role in molecular biophysics when determining the size and the folding state of proteins. Pharmaceutical research also benefited from new methods in fluorescence microscopy. In particular, FRAP (Fluorescence Recovery after Photobleaching) should be mentioned here. If fluorescence markers in a sample are locally deactivated by a laser pulse (this process is referred to as photobleaching), fluorescent molecules from the environment diffuse back into the deactivated region, and the diffusion rate can be determined by fluorescence intensity measurement. This method opened up the possibility of investigating the microscopic mobility and interaction of molecules with unprecedented accuracy.
Light microscopy was given a major innovative impetus in the 1970s through the work of Christoph Cremer and Thomas Cremer. The scientists proposed to focus laser light from all sides (space angle 4Pi) to a point with a diameter of less than 200 nm and then to scan the sample point by point (Fig. 5). Insights from this 4Pi microscopy also contributed to the concept of confocal laser scanning microscopy (CSLM). Here, too, a sample is scanned point by point by a focusing laser beam with specifically marked regions being excited to show fluorescence. The image is then recomposed electronically spot by spot, as in scanning electron microscopy.
Using a reflected-light confocal microscope, Colin Sheppard and Tony Wilson showed a valuable advancement in which the sample could be moved not only in the focal plane but also along the optical axis. Thus they obtained optical serial sections of, for example, integrated optical circuits.
Analog video cameras made it possible to record image sequences. This complement to the instrumentation showed its strength particularly in the visualization of the changes in cell components concerning position and shape as well as in the representation of concentrations of different substances in living cells.

80s: Revolutions

At the beginning of the 1980s, electronics increasingly moved into light microscopy. With advances in information technology, opportunities evolved to combine the classical methods of light microscopy with digital imaging and image processing. The performance of light microscopy was no longer linked to the properties of the human eye. Operations that could be visualized so far only with the electron microscope could now be presented in a time-resolved manner in vivo. In addition to video cameras, particularly light-sensitive charge-coupled-device (CCD) cameras were also used for image recording. Immediately after the recording, digital image data were processed in a fast processor for contrast enhancement, background subtraction and filtering, and finally used as a basis for in-depth analyses.
Technical improvements to existing concepts have also contributed to overcoming the hitherto existing limitations of image rate and control elements, sample loading, lateral and axial resolution: when performing a scan with a confocal laser scanning microscope using the point scanner principle, up to now the specimen was moved while the illumination point remained fixed. These instruments were often sensitive to shocks and also slow. In the mid-1980s, W. Amos and J. White developed the first confocal beam scanning microscope in which the sample was fixed and the illumination point was moved. Suitable software allowed for precise control of the scanning motors for the mirrors. Based on their design it was already possible to record four images per second with 512 lines each.
The technical innovations very quickly found their way into the commercial sector. The first laser scanning microscopes were offered in 1982 by Oxford Optoelectronics (SOM-25) and Carl Zeiss (LSM). The LSM prototype can now be viewed at the Deutsches Museum, Munich.
Driving factors in the instrumental and methodical development of light microscopy were, in particular, the increasing computing power of computers and graphics cards, increasing memory space, easier operation, further motorization and automation. The use of the green fluorescent protein (GFP) also had a lasting effect on the development of light microscopy, in particular the techniques FLIM, FRAP, and FRET. The provision of various variants of the GFP is perhaps the most well-known contribution of Roger Tsien who received the Nobel Prize for Chemistry in 2008.
Both the digitization of light microscopy and the further instrumental development have led us beyond the limits of "microscopy with the eye". Dieter Weiss speaks of a renaissance of light microscopy and a paradigm shift in cell biology: "It has replaced the static image of the cell, which has been dominated by electron microscopy since the 1950s, by a completely new view of the highly ordered intracellular movements." The dynamics of cell structures could be studied in more detail, individual protein molecules could be quantitatively displayed and tracked in the living cell, cellular processes could be resolved with high precision in relation to time, chemical-physical properties could be visualized.

90s: Sharper Than Light Allows

The pioneering methodological innovations of the past years reached market maturity. Renowned manufacturers such as Leica, Olympus, Carl Zeiss and Nikon introduced new confocal microscope systems and application-specific objective lenses. Particular progress was made in the development of fluorescence microscopy. In addition to many interesting innovations, especially the invention of light sheet fluorescence microscopy, LSFM, or single plane illumination microscopy, SPIM, 4Pi-confocal fluorescence microscopy (double-confocal scanning microscopy) and fluorescence quenching by stimulated emission (stimulated emission depletion, STED) are important milestones.
The Jena physicist Ernst Abbe recognized the central problem of light microscopy as early as in 1873, namely, that the resolution will never reach beyond half of the "wavelength of blue light by a significant value". It is the wave nature of light that is responsible for this. The new methods of far-field fluorescence microscopy have not been able to overcome the diffraction-limited resolution limit either. It is no wonder that in the past decades scientists have thought up other types of microscopy, such as electron, scanning tunneling or near-field microscopy. However, non-invasive imaging of the interior of cells was difficult to achieve with these high-resolution methods.
Stefan Hell and Thomas Klar succeeded in breaking the diffraction limit in fluorescence microscopy with the aid of the concept of fluorescence quenching by stimulated emission (stimulated emission depletion, STED). In this fundamental approach, a nearly spherical focal spot was generated, the resulting volume of 670 zeptoliters being about 18 times smaller than in the confocal microscope. The actual breakthrough idea was quenching the fluorescence of dye molecules at the rim of the originally excited region. For this purpose, the researchers worked with two sequences of ultrashort laser pulses. The intensity distribution of the STED beam took on ring-shape around the excitation spot. This way, the fluorescence at the focal rim was suppressed and the fluorescence in the center of the spot spared out. A first application of this method to E. coli bacteria yielded images with subdiffraction resolution and revealed details in the images that were hitherto concealed in conventional fluorescence microscopy. The neurosciences in particular profited from turbomicroscopy. Synapses are precisely at the resolution limit of the light microscopy in use so far. The transformation ability of the synapses influences neurological processes such as learning. With the help of STED, even details of the structure and the temporal changes of the tiny objects have been made visible.

2000s: Super-Resolution ­Microscopy

The news of the breaking of a longstanding physical limit had quickly spread. Other research teams also tried to develop methods of light microscopy to resolve the nanometer range. Photoactivated localization microscopy (PALM) was developed by Eric Betzig and colleagues. They were able to switch the fluorescence of individual molecules on and off by light-control. The switching process took place over a certain period of time, so that some pictures could be taken. Using the computer, the position of individual molecules beyond Abbe's resolution limit could be determined. The technique has also been developed in parallel by other groups, so that different names appear in the technical literature (S.T. Hess, FPALM, X. Zhuang, STORM).
STED and PALM appeared as important representatives of a new class of high-resolution techniques of light microscopy, commonly referred to as "super-resolution microscopy". In 2014 Eric Betzig, Stefan Hell and William Moerner received the Nobel Prize for Chemistry for the development of super-resolution fluorescence microscopy.
At the beginning of the 21st century the commercial sector also displayed extraordinary dynamism. The first mass-produced STED microscopes were launched by Leica Microsystems. With the development of high-resolution light microscopy, there was also a need for complementary laboratory technology, for example, incubators for live cell imaging and products for automation. The innovation upsurge among methods also led to a significant further development of components such as cameras, objective lenses with high numerical aperture, lenses with adaptive optics, apochromatically corrected lenses, etc.
As Ernst Abbe had already written in 1873, the resolution limit at half the wavelength used as he had formulated is valid only "as long as no arguments are made which go completely beyond the established theory." Obviously, this has been done.

 

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