SPECIAL ISSUE: NEW/EMERGING TECHNIQUES IN BIOLOGICAL MICROSCOPY

Editorial for PROTOPLASMA — SpecialIssue on Microscopy

J. W. Borst & David Robinson

Received: 14 December 2013 /Accepted: 14 December 2013# Springer-Verlag Wien 2014

Traditionally, two conceptual approaches underlying cell bi-ology were structure and function, whereby microscopy wasconsidered more or less synonymous with structure. Thesedays, however, the advent of confocal microscopy and relatedfluorescence techniques has led to a new discipline: “func-tional imaging”. Advances in our understanding of the mor-phology of cells and their constituent organelles have always,and continue to do so, gone hand-in-hand with developmentsin microscope construction and sample preparation tech-niques. The introduction of molecular biological methods intocell biology beginning in the 1980s has been accompanied bya renaissance in light microscopy. Today, virtually every newissue of a cell biological journal has at least several articlescontaining data obtained using a confocal microscope for thedetection of fluorescently labeled protein. This developmentwas given a kick-start with the discovery of green fluorescentprotein (GFP) and related fluorescent proteins in the 1990sand continues unabated. Unfortunately, the downside of this“get rich quick with GFP” trend was that after a few yearsinterest in electron microscopy temporarily decreased, andfewer people were prepared to invest the necessary timerequired to get proficient in this method. As a consequence,trained electron microscopists were achieving the status ofrare orchids at the beginning of this millennium. Not surpris-ingly, for a number of years, the quality of published electronmicrographs, due to poorly trained users and the lack ofqualified reviewers, was at a lower standard than in 1970.However, the gradual appreciation of the limits of GFP tech-nology for subcellular protein localization has led to an

increased demand for high resolution immunogold localiza-tions, a procedure which itself has profited enormously fromthe introduction of various cryotechniques since about 1985.GFP technology has also enabled the two disciplines of lightand electron microscopy to be combined in the form ofcorrelative light electron microscopy (CLEM). Further devel-opment and implementation of modern methods in light andelectron microscopy now enable us to go beyond the im-proved morphological description of an organelle and allownow the detection and visualization of individual molecules.

Examples of such developments are the so-calledsuperresolution and single molecule techniques, which arenowadays based on the available plethora of geneticallyencoded visible proteins, ultra-fast excitation light sources aswell as detectors with highly improved photon sensitivity. Theimpact of advanced fluorescence-based imaging approacheshas allowed for many new insights into biological processes.Fluorescence imaging in general offers a great opportunity toreveal dynamic processes in living cells, tissues, or wholeorganisms such as zebrafish, mice, or plants. Confocal mi-croscopy has opened doors for biologists to unravel proteinlocalization and, in combination with GFP technology, hasenabled the visualization of multiple fluorescently labeledproteins simultaneously.

Although colocalization studies can reveal whether bio-molecules coincide at the subcellular level, information aboutthe molecular composition of different fluorescently labeledbiomolecular complexes remains unknown. Since the early1970s, many in vitro studies have been conducted which haverevealed the molecular assembly of biomolecules. In thesestudies, Förster resonance energy transfer (FRET) methodol-ogy has been exploited to investigate, e.g., binding kinetics,protein complex formation, or lipid protein interactions.FRET is a dynamic spectroscopic ruler for unraveling dis-tances between molecules. It took quite some years, startingfrom the end of the 1990s, to be able to visualize proteininteractions in living cells. This was achieved by combingFRET with other fluorescence imaging techniques of which

Handling Editor: J.W. Borst

J. W. Borst (*)Wageningen University, Wageningen, The Netherlandse-mail: JanWillem.Borst@wur.nl

D. RobinsonUniversity of Heidelberg, Heidelberg, Germany

Protoplasma (2014) 251:273–275DOI 10.1007/s00709-013-0603-y

fluorescence lifetime imaging (FLIM) is one of the most robustmethods for quantifying FRET. Since FLIM set-ups have beencommercialized during the last decade, many biologists nowhave the possibility to access this technique via specializedimaging centers. This development has made FLIM a “standard”tool for the in vivo identification of protein interactions.

In this Special Issue, two reviews discuss FLIM applications;one is devoted to the medical sciences and discusses recenttechnical developments (Ebrecht et al.) and the second focuseson the potential of FLIM in plant systems (Bücherl et al.). FRET–FLIMmeasurements result in images with pixel information thatis beyond the diffraction limit of visible light as FRET takes placeat the nanometer scale. Alternative imaging approaches solvingthe diffraction-limited constraints associated with confocal mi-croscopy has led to the development of superresolution imagingtechniques such as stimulated emission depletion (STED) mi-croscopy, structured illuminationmicroscopy (SIM), and stochas-tic optical reconstruction microscopy (STORM)/ photoactivatedlocalization microscopy (PALM). The latter two techniques arefounded on the discovery of photoswitchable probes enabling thelocalization of fluorescent molecules with a precision far belowthe physical limit of conventional light microscopy. These recentcutting edge technologies are extensively discussed in the reviewof Hedde and Nienhaus.

Genetically encoded fluorescent proteins can also be used forthe characterization of biological processes such as monitoringcalcium fluxes by utilizing FRET-based biosensors. In the articleof Hamers and co-workers, a detailed overview of several engi-neering strategies for generating and optimizing biosensor per-formance is given along with a discussion of biosensor applica-tions in mammalian systems as well as in plants. FRETspectros-copy is further exploited by the application of single-moleculeFRET (smFRET), a powerful tool for elucidating biologicalstructures and mechanisms on the molecular level. The articleof Farooq and co-workers illustrates how smFRET can be ap-plied to precisely characterize dynamics and rarely occurringevents ofDNA/RNApolymerase activities at the singlemoleculelevel. In this Special Issue, there are twomore reviews discussingsingle molecule applications, but with a focus on in vivo appli-cations. The work of Langhans and Meckel elaborates on theapplication of single molecule detection and tracking in plantsystems. A procedure, inwhich all the necessary steps required toperform a single molecule experiment, is described from dataacquisition to analysis. Another single molecule-based techniqueis fluorescence fluctuation spectroscopy (FFS), which is exten-sively discussed by Mark Hink. This methodology can providequantitative molecular information on fluorescently labeled bio-molecules, e.g., diffusion rates, binding kinetics, and averagenumber of molecules. Though mainly developed and used forin vitro systems, this technique has in the meanwhile beensuccessfully applied in vivo. As FFS relies on monitoring differ-ences of fluorescence fluctuations, this approach can only beperformed at low nanomolar concentrations.

Since expression in living systems is sometimes difficult totune, there are alternative strategies available for monitoringdynamics of molecules at higher expression levels. This meth-od is known as fluorescence recovery after photobleaching(FRAP), which is discussed in detail by Malte Wachsmuth.An advantage of this methodology is that it can be performedon a regular confocal microscope. It allows revealing themobility of biomolecules as well as the binding kinetics inliving cells. All previous fluorescence-related techniques arealso referred to as the “F” techniques. However, since theborders of different disciplines in science have becomeblurred over the last decades, a better term for describing“F” techniques is “functional imaging”. Finally, Gualda andco-workers present developments of new fluorescence imag-ing platforms such as light sheet imaging. This new imagingconfiguration has led to high-resolution images that can cap-ture cell differentiation and proliferation of whole organismssuch as zebrafish, plants in vivo, and real time. The greatadvantage of this approach is performing live cell/tissue im-aging with reduced photodamage and fast acquisition rates.This new imaging concept is very promising, and initiativessuch as OpenSPIM and OpenSpinMicroscopy have emergedas open access platforms for light sheet and optical projectionimaging.

As already mentioned, the most significant advances inelectron microscopy over the last 30 years have involvedcryotechniques. Although not displacing classical chemicalfixation procedures, especially when large numbers of spec-imens are to be screened, e.g., in pathology, they have be-come the gold standard for today’s ultrastructural investiga-tions. In terms of the validity of the observations made,freeze-fixed specimens cannot be challenged. This does notmean that everything published before the cryo era is incor-rect; indeed, from the structural point of view, many organ-elles can be well-identified and monitored in chemically fixedspecimens. With cryo-methods not only is antigen preserva-tion enormously increased leading to better immunogoldlabeling, but these have also provided higher quality imageswith improved fine structural details. Moreover, in the formof cryo-electron microscopy (Cryo-EM), macromolecularcomplexes can now be studied at near atomic detail. ThisSpecial Issue on Microscopy contains two articles devoted tocryo-methods in electron microscopy. In one, KentMcDonald not only gives a detailed account of the latestfreezing techniques together with a hands-on account ofhow to prepare specimens and work with commerciallyavailable equipment, but he has also added a much appreci-ated historical perspective. The other article delivered byAndy Hönger is a comprehensive update on what couldconveniently be termed macromolecular electron microscopy.Cryo-electron tomography figures prominently in this re-search area and is fueled by new developments in computingpower and software as well as in the construction of super-

274 J.W. Borst, D. Robinson

stable microscopy stages and electron detectors. The readerwill soon realize that cryo-electron microscopy is very ex-pensive to establish and requires highly trained personnel.

Correlative light electron microscopy (CLEM) is whereGFP-technology meets light and electron microscopy, and,in terms of subcellular protein localization, is a goal that manygroups are striving for. To our knowledge, until now, nobodyhas succeeded in getting this technique to work on plants, butit has been achieved with varying degrees of success onanimal cells.

The article of Hodgson and co-workers presents a new wayof performing CLEM by including the Tokoyasu method ofcryosectioning for the immunogold labeling step in the pro-cedure. In this regard, it should be noted that while theTokoyasu method is widely used for conventionalimmunogold localizations on mammalian cells, it has rarelybeen performed on plant material.

Twomethods which have only very recently come onto thescene are described in the articles of Hughes et al. andCarziniga et al. The technique known as serial block facescanning electron microscopy (SBEM) is a new 3D imagingset-up for which a special type of scanning electron micro-scope is required. This is realized by incorporating an ultra-microtome within the specimen chamber, which is again anexpensive piece of equipment. It essentially involves imagingthe cut surface of the specimen block rather than looking at thesection and provides similar information that of serial sectiontransmission electron microscopy. This technique has great

potential and may challenge electron tomography data obtain-ed from plastic sections cut from high-pressure frozen-freezesubstituted specimens. Unfortunately, such samples tend notto have sufficient contrast, so there is certainly room forimprovement. A completely new method which does notinvolve electron microscopy, but can provide nanoscale 3Ddata from cryo-fixed, unstained whole cells is cryo-soft X-raytomography (Cryo-SXT). Required is a source of soft X-rays—usually from a synchrotron and is therefore not univer-sally available. In addition, the microscope needs a specialcryogenically cooled device with tilting and specimen rotationcapabilities. SXT data sets from rapidly frozen samples arethen analyzed with software packages similar to those in usefor electron tomography. The current best resolution with thisnew technique is around 20 nm. Immunogold labeling ispossible but at its infancy and it will therefore be intriguingto see how Cryo-SXT develops.

The guest editors of this Special Issue of PROTOPLASMAare delighted to have had the opportunity of assembling amixture of high quality scientific reviews, which highlightrecent developments in the fields of electron and light micros-copy.We hope that our readers appreciate this as well, and thatthese contributions will be inspiring and useful for their ownfuture research.

Conflict of interest The authors declare that they have no conflict ofinterest.

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