Far‐field Light Microscopy


Novel developments in optical technology and photophysics made it possible to radically overcome the diffraction limit (ca. 200 nm laterally, 600 nm along the optical axis) of conventional far‐field fluorescence microscopy. Presently, three principal ‘nanoscopy’ families have been established: Nanoscopy based on highly focused laser beams; nanoscopy based on structured illumination excitation; and nanoscopy based on localisation microscopy approaches. With such ‘superresolution’ or ‘nanoscopy’ techniques, it has become possible to analyse biostructures with a substantially enhanced light optical resolution down to a few tens of nanometre in 3D, and a few nanometre in the object plane, corresponding to 1/100 of the exciting wavelength. These methods allow to study individual membrane complexes, cellular protein distributions, nuclear nanostructures, bacteria or individual viruses down to the molecular level; they open new perspectives to combine molecular and structural biology to unravel the basic mechanisms of life and their emergence from fundamental laws.

Key Concepts

  • Light microscopy of biostructures has reemerged as an essential tool of the life sciences.

  • Conventional light microscopy methods suffer from the limited resolution and thus prevent the direct study of nanostructural details.

  • Novel developments in optics and photophysics have made it possible to overcome the conventional resolution limits.

  • Present ‘superresolution’/‘nanoscopy’ techniques are based either on focused laser beam excitation; or on patterned/structured illumination schemes; or on homogeneous wide‐field illumination.

  • In addition to advanced optical techniques, nanoscopy requires appropriate photostable/photoswitchable fluorochromes with specific spectral characteristics.

  • Far‐field light microscopy based ‘nanoscopy’ techniques presently allow to discriminate (resolve) in a cellular environment individual fluorescent molecules down to a minimum distance of few nanometre.

  • The combination of the novel nanoscopy approaches with molecular biology methods is expected to open an avenue towards a substantially improved understanding of the basic mechanisms of life and its emergence from the basic laws of nature.

Keywords: superresolution; light microscopy; light optical nanoscopy; focused nanoscopy; structured illumination microscopy; localisation microscopy; biological nanostructures; 4Pi‐, STED, SIM, SMI microscopy; single molecule microscopy; GSDIM; PALM; SPDM; STORM; photoswitching

Figure 1.

Two‐colour 4Pi microscopy images of PML bodies. Immunostaining was conducted with a secondary antibody labelled with Alexa Fluor 568 (PML, red colour) and Atto 647 (green colour) against (a) a SUMO‐1 or (b) a SUMO‐2/3 primary antibody. 3D image reconstructions of the 4Pi stacks are also shown (right). Scale bars: 0.5 µm (the same in x,y object plane; and in z axial direction). The first three image columns (from left) show the PML, SUMO and corresponding merged image (merge 1) of two PML‐NBs. Then the merged PML‐SUMO image of two other PML‐NB is presented (merge 2). SUMO‐1 was distributed more sparsely and also more aggregated than PML. A partial colocalization of PML (red) and SUMO‐1 (green) in the same spherical shell was evident from the line profiles 1 and 2 shown in (c). By contrast, SUMO‐2/3 was located also in the interior of the PML‐NB (b, merge 1). A fraction of PML‐NBs showed only a very weak SUMO‐ 2/3 signal in the interior (b, merge 2). (c) Image‐intensity profiles taken along the broken lines indicated by white numbers in a (1,2) and in b (3); red: PML; green: SUMO. (d) The 3D‐structure of a PML‐Sp100 4Pi‐reconstruction is shown for comparison. Sp100 was distributed similarly to PML in the outer shell of spherical shape. No extrusions from the shell were apparent but the two proteins were present in distinct patches. Reproduced from Lang et al. (). © Company of Biologists Ltd.

Figure 2.

SMI imaging of replication foci. For SMI imaging (C2C12 mouse cells), a stack of data with a number of axial positions (z) is registered. (a) shows one of the frames obtained at conventional optical resolution. After acquisition of the raw data (a), a filtering and thresholding procedure was used to identify the positions of fluorescence labelled individual foci. Once the positions of the foci have been established, a model function is fitted at the location of each focus to extract the object size and a more accurate position estimate. The results of this fitting procedure were visualised by rendering a sphere at each object position, which was coloured according to the object size. This resulted in the representation shown in (b). Objects that could not be fitted, due to either insufficient signal level or a size outside the effective range (40–200 nm) for SMI measurements were coloured black. Scale bar=5 µm. Reproduced from Baddeley et al. (). © Oxford University Press.

Figure 3.

Structured Illumination Microscopy (SIM) of nuclear topography of nascent RNA, nascent DNA, and Ser‐2P‐RNA Pol II. Comparison between optical sections of a small part of a murine C127 cell nucleus obtained from deconvolved conventional wide‐field images (a,b) and 3D‐SIM images (a*,b*) of the same regions obtained with a commercial SIM system (OMX, Applied Precision Instruments). Ser‐2P RNA Pol II (red) and Pol 3/3 (green) primary antibodies were used to mark the CTD domain repeats phosphorylated at serine 2, and the RPB1 domain of the enzyme, respectively, together with secondary antibodies conjugated to Alexa 594 (red) and Alexa 488 (green). DNA was counterstained with DAPI (grey). Scale bar, 500 nm. Reproduced with permission from Markaki et al. (). © Cold Spring Harbor Laboratory Press.

Figure 4.

Principle of resolution enhancement by spectrally assigned localisation microscopy (SALM). Three point‐like objects are assumed to be located in the xy‐plane within mutual distances of 50 nm, i.e. substantially smaller than the conventional resolution. Furthermore, they are assumed to be labelled with the same spectral signature in (a) producing the diffraction pattern shown in (b) or with three different, unique spectral signatures B,G,R in (d), producing the diffraction patterns shown in (e and f). In (f), the different spectral components are imaged simultaneously whereas in (e) the same signals (B,G,R) are registered independently from each other, producing three independent diffraction patterns B,G,R. Linescans through the diffraction patterns in (b) and in (e) B,G,R are shown in (c) and (g) respectively. Reproduced from Kaufmann et al. (). © SPIE.

Figure 5.

Localisation microscopy (SPDM) of nanostructures in human cell nuclei. (a) Dual‐colour localisation microscopy (SPDM) of histones and chromatin remodelling proteins. Left: Conventional wide‐field fluorescence image of H2A proteins (red) and Snf2H proteins (green) in a human U2OS nucleus. Right: Localisation microscopy (SPDM) of the same nucleus; about 120 000 protein signals were localised and counted in an optical section of approximately 600‐nm axial thickness (b) Localisation microscopy (SPDM) of histones H2B labelled with GFP in HeLa cell nuclei. Left: Conventional wide‐field fluorescence image of a nucleus under live cell conditions. Middle: Localisation microscopy (SPDM) of the same cell under live cell conditions (insert from image left). Approximately 11 000 individual GFP molecules were localised in this region. Scale bar 500 nm. Right: SPDM image of histones H2B in a nucleus of a fixed cell. Approximately 16 000 GFP molecules were detected in this section. (c) Statistical analyses of the distribution of GFP‐labelled histone H2B in human fibroblast cells and two different strains I, II of HeLa cells. Visualised local densities of H2B from SPDM measurements for a fibroblast cell (a) and a HeLa cell of strain I (b). Graphs middle/right: Shown are the densities of blocks of linear dimension b=500 nm. They display strong deviations from a random distribution, indicating that structure in the histone distribution exists on that scale. (c) Block density distributions, for different values of block size b=200 nm (squares), b=500 nm (circles), and b=1000 nm (triangles). Data are scaled with the average density, to make the distribution independent of labelling efficiency. (d) The cumulants Ub of the density distribution. For a thermodynamically equilibrated system, cumulants should display a value of zero (dashed line) above a block size b larger than the scale of density fluctuations. (e) Nanostructure of nuclear histone distribution. The radial density g(r) calculated from the single molecule positions revealed distinct differences from a random distribution (i.e. g(r)=1), highlighting the existence of distinct structures on the scale below 100 nm. Structural hallmarks are found for HeLa as well as fibroblasts independent of labelling methods, but quantitative differences exist. The grey horizontal line corresponds to the (normalised) average density, that is, a random spatial distribution of histones. (d) Localisation microscopy (SPDM) of double‐strand break‐repair complex proteins Rad 52 labelled with GFP in a retina pigment epithelium cell (RPE1). Left: Conventional wide‐field fluorescence image. The insert shows a magnification. Middle: SPDM image. Right: Line scans through the conventional wide‐field image (blue, FWHM approximately 310 nm) and through the SPDM localisation microscopy image (red, FWHM approximately 100 nm). Reproduced with permission from Cremer et al. (). © WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 6.

Virtual multicolour ‘nanoscopy’ of nuclear genome structure. (a) 3D visualisation and projections to the lateral (x, y) plane and the vertical (x, z) and (y, z) planes of a computer simulation of a 1 Mbp chromatin domain of total diameter 500 nm (Cremer and Cremer, ) assumed to comprise 10 condensed 100 kbp domains formed by nucleosome chains, having an individual size in the 100 nm range. The small ‘beads’ represent the individual nucleosomes. Here, a labelling of all 10 domains with the same spectral signature was assumed. (b–d) Virtual microscopy representations (3D visualisation, projections) of the monospectral computer simulated 1Mbp domain shown in (a). For this, the simulated 3D data set of (a) was convoluted with the effective point spread function for the optical resolution assumed. (b) Virtual microscopy assuming a theoretical 3D resolution of 25 nm. The positions and sizes of the ten 100 kbp regions are all resolved individually. (c) Virtual microscopy assuming an effective optical resolution of 100 nm. Some structural resolution of the subdomains is still maintained. (d) Virtual microscopy assuming an effective optical 3D resolution of 250 nm (achieved, for example, by merging confocal images obtained from different angles. All structural information has been lost, except the total size of the 1Mbp domain. (e) Virtual microscopy of (a) assuming a multicolour 3D resolution of 10 nm, realised, for example, by combinatorial localisation microscopy with four detectable differences in spectral excitation/emission. The bar indicates 100 nm. (Figure kindly provided by Dr G. Kreth, Heidelberg). (f) Multicolour localisation microscopy of FISH labelled sequence specific oligonucleotide probes would allow to unravel the folding of an individual 100 kbp subdomain. Modified from P. Diesinger, Ph.D. Thesis Physics, University of Heidelberg 2009). © P. Diesinger Scale bar 30 nm.



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Further Reading

Best G, Amberger R and Cremer C (2013) Super‐resolution microscopy: interference and pattern techniques. In: Kubitscheck U (ed.) Fluorescence Microscopy: From Principles to Biological Applications, 2nd edn, pp. 345–374. Boston, Oxford, Berlin, Singapore, Tokyo: Wiley‐Blackwell.

Cremer C (2012) Optics far beyond the diffraction limit: from focused nanoscopy to spectrally assigned localization microscopy. In: Träger F (ed.) Springer Handbook of Lasers and Optics, 2nd edn, pp. 1351–1389. Berlin, Heidelberg, New York: Springer.

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Pawley JB (ed.) (2006) Handbook of Biological Confocal Microscopy, 3rd edn. New York: Springer Science+Business Media, LLC.

Rouquette J, Cremer C, Cremer T and Fakan S (2010) Functional nuclear architecture studied by microscopy. International Review of Cell and Molecular Biology 282: 1–90.

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Cremer, Christoph(Aug 2014) Far‐field Light Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005922.pub2]