Elsevier

Methods

Volume 123, 1 July 2017, Pages 11-32
Methods

Super-resolution microscopy approaches to nuclear nanostructure imaging

https://doi.org/10.1016/j.ymeth.2017.03.019Get rights and content

Highlights

  • Review of the available light optical super-resolution approaches to study nuclear nanostructure.

  • Nuclear genome structure studied at the single cell/single molecule level by Spectral Precision Distance/Position Determination Microscopy (SPDM), a variant of localization microscopy.

  • Single Molecule Localization Microscopy (SMLM) using conventional fluorescent proteins, single standard organic fluorophores, or conventional DNA-binding dye molecules.

  • Imaging and quantitative analyses of nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution.

  • “Molecular optics” approaches to study the nuclear landscape and the functional genome architecture directly in individual cells down to the single molecule level.

Abstract

The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca. 200 nm). After a brief review of available super-resolution microscopy (SRM) methods, we focus on a specific SRM approach to study nuclear genome structure at the single cell/single molecule level, Spectral Precision Distance/Position Determination Microscopy (SPDM). SPDM, a variant of localization microscopy, makes use of conventional fluorescent proteins or single standard organic fluorophores in combination with standard (or only slightly modified) specimen preparation conditions; in its actual realization mode, the same laser frequency can be used for both photoswitching and fluorescence read out. Presently, the SPDM method allows us to image nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution, and to perform quantitative analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. As a biomedical research application, using dual-color SPDM, it became possible to monitor in mouse cardiomyocyte cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA). These novel “molecular optics” approaches open an avenue to study the nuclear landscape directly in individual cells down to the single molecule level and thus to test models of functional genome architecture at unprecedented resolution.

Introduction

For more than a century it was a generally accepted truth that structural details inside cells could only be detected if they were larger than about 0.2 µm (200 nm). Below this optical resolution limit of 200 nm (object plane) and about 600 nm along the optical axis obtained at the maximum numerical aperture (NA) available, the microscopic images were blurred to the observer like the letters of a newspaper seen from a large distance: Only the principal headlines could be read but not the decisive details. The example in Fig. 1 shows a conventional wide field image of a human cell nucleus where two types of nuclear proteins had been labeled with two different colors. The image suggests that the nuclear distribution of these two proteins might be somewhat different but a quantitative detailed analysis is extremely difficult. All attempts to substantially extend these resolution limits of “conventional” light microscopy failed. However, fast growing evidence indicates that the genome in mammalian cell nuclei has a highly complex spatial organization; for reviews see [38], [39], [48], [123], [27]. Hence, it will be highly desirable to obtain more information about its nanostructure.

About 140 years ago, Ernst Abbe, the pioneer of modern high resolution microscopy, has dealt extensively with this amazing and disturbing phenomenon and found the solution: It was no longer a technical issue of making better lenses, but a fundamental limit of knowledge using imaging; the limitation was a direct consequence of the nature of light waves; thus it was intimately connected with the fundamental laws of Physics. In 1873 Abbe summarized his findings in one of the most famous texts of optical Physics. Surprisingly, it did not contain one single mathematical formula. Nonetheless, the physical content was so fundamental that since then its main results have been presented in every basic Physics and Science course all over the world. Abbe postulated [1]

that the limit of discrimination will never pass significantly beyond half the wavelength of blue light”1

“Half the wavelength of blue light“ is about 0.2 µm or 200 nm (200·10−9 m). In general terms, Abbe stated a relationship for the smallest distance dmin that two point-like object details (or more correctly thin lines) can have so that they could still be discriminated from each other (“resolved”) by microscopy. ‘Point-like’ means that the object dimensions (or the thickness of the lines) are much smaller than the wavelength used for imaging. According to his relationship expressed in formula (1), the smallest distance dmin is essentially determined by the (vacuum) wavelength λ of the light used for imaging and the factor , the numerical nsinα aperture NA (n is the refractive index of the sample, and α is the half- aperture angle of the objective lens).dmin=λ2nsinα

This means that the optical resolution dmin gets better if a shorter wavelength is used for imaging, and if the microscope objective lens has a larger numerical aperture. While the shortest wavelength in visible light microscopy is about 400 nm, the numerical aperture nsinα cannot be larger than n (the largest half aperture angle possible for any single objective lens is 90°, and sin90° = 1). The refractive index n is an optical material “constant”; in cell biology, the typical maximum is around 1.5; useful values for the numerical aperture in advanced light microscopes are around 1.4 (in some cases up to 1.6). Altogether, even assuming the best optical conditions, this limited the best optical resolution of any microscope to a value of about 1/3 to 1/2 of the (vacuum) wavelength of light used, or dmin  150 to 200 nm in the object plane.

Abbe was concerned with the limits of resolution imposed for transmitted light; this means the object contrast was given by local differences in absorption; for example, if a cell is stained with a DNA binding dye, the nucleus absorbs UV-light strongly, while the cytoplasm does not; this method is still widely used in cellular pathology for chromatin texture analyses. However, in many fields of modern biology and medicine, fluorescence microscopy has become more and more important. In fluorescence microscopy, fluorescent molecules can be specifically attached to the cellular structures to be imaged (or molecules can emit fluorescent light by themselves, such as various types of Fluorescent Proteins); ‘labelling’ presently allows to visualize in a specific way almost any type of macromolecules in the cell; thus fluorescence microscopy has emerged as one of the most important tools of modern biology. The labelling required can be done by a large variety of methods. If the labeled cells (or other biostructures, such as viruses or bacteria) are illuminated with light of an appropriate wavelength, fluorescence is excited at given wavelengths different from the excitation light; the fluorescence emission is separated from the excitation, and a fluorescence image is obtained. Due to the underlying molecular Physics, the light emitted by these ‘fluorochromes’ is ‘incoherent’, i.e. from the optical point of view the individual fluorescent molecules emit light independently from each other, like the stars in the sky. Consequently, the same limits of resolution are valid for all these ‘self-luminous’ sources as for the telescopes used in Astronomy.

These limits were stated by a famous contemporary of Ernst Abbe in England, Lord Rayleigh. In 1896 [100] he came to the conclusion that the smallest distance dmin between two self-luminous ‘point sources’ is proportional to the image producing wavelength, and inversely proportional to the numerical aperture NA =nsinα, with a factor 0.61:dmin=0.61λ/NA

In fluorescence microscopy, for a numerical aperture NA = 1.4, this amounts to 43% of the imaging wavelength, i.e. again roughly to about half the vacuum wavelength, as stated by Ernst Abbe in 1873.

As the only possibility to further enhance the resolution, Rayleigh suggested the use of shorter wavelengths, such as ultraviolet light. This has also been implemented; however, glass becomes quickly opaque in the ultraviolet region, i.e. with shorter wavelengths, so that the resolution cannot easily be enhanced in this way by a large amount.

For many decades to come, the resolution enhancement needed in biology to image the cellular nanostructures underlying the extremely complex spatial organization of life was achieved by shortening the wavelength not using light but using electrons. Electron microscopy played a major role in the discovery of essential elements of chromatin nanostructure, such as the nucleosomes [92], [93] and other important features (for review see [104]).

Nevertheless, it remained of the greatest importance to find ways to enhance the optical resolution also in light microscopy. Notwithstanding the immense contributions of electron microscopy, there are a large number of practical reasons for the use of visible light microscopy at enhanced resolution (if possible). For example, observations of nuclear nanostructure using electron microscopes are very complex and time consuming; typically, they require long and sophisticated fixation protocols to avoid artefacts; they are limited to the imaging of surfaces, i.e. to look into the interior of a nucleus, the cell has to be cut into very thin slices, each with a thickness of few tens of nm; or one thin layer after the other has to be imaged and then peeled off; simultaneous labelling of more than two molecular targets is extremely difficult; live cell observations are impossible. In nuclear Biology, it should be extremely advantageous to analyze the nanoscale distribution of transcription and splicing factors, polymerases, repair proteins etc. with respect to chromatin; the spatial arrangement of histone markers with respect to specific DNA sequences; or the condensation/decondensation of silenced/transcribed genes in the nucleus as a function of epigenetic modifications [38].

Already Abbe thought it possible that one day, new methods might be discovered that could make possible optical imaging far beyond the resolution limit stated by him. In his 1873 publication he stated that the limit of about one-half wavelength is valid only

as long as no moments are considered that lie entirely outside the scope of established theory2

Today this prophecy is realized, and its revolutionary importance for Biology and Medicine has recently been honored by the Nobel Prize in Chemistry 2014 to pioneers in the fields of focused nanoscopy, localization microscopy based on photoswitchable molecules, and single molecule Physics [42]. This bypass of the Abbe-Limit became possible by new discoveries in physics, photochemistry, optoelectronics, and information technology which have made possible to extend very substantially our knowledge of the ‘nanoworld’: Contributions from the field of Physics are in particular the insight that fluorescence based imaging provides possibilities for super-resolution not given in transmitted light; the development of lasers in the visible spectral range; the realization of highly sensitive light detectors/sensor arrays; the insight gained from the wave theory of light that an appropriate combination of images taken under different conditions allows to stretch the resolution limit even if the individual image is subject to the conventional resolution limit; in the field of Computer Sciences the development of fast digital image processing; from Physical Chemistry the exploration of organic molecules and the possibility to switch their optical characteristics; the rapid evolution in the field of Molecular Cell Biology with its many approaches to specific fluorescence labelling of (in principle) any kind of protein or nucleic acid. On the basis of these discoveries and multi-disciplinary developments, in the last two decades a variety of super-resolution microscopy methods have been devised. Here, the term ‘super-resolution’ is understood in its original meaning [121] to denote light-optical techniques with an enhanced spatial resolution beyond the limits achieved in conventional microscopy (as given by the Abbe/Rayleigh formulas cited above) prior to the introduction of the ‘nanoscopy’ approaches described below. In the following, after a short overview on these new developments, we focus on the principles of localization microscopy based elucidations of nuclear nanostructures. A detailed historical account of these developments is given in [37].

Section snippets

4Pi-Microscopy

Confocal microscopy presented the first idea to effectively overcome the resolution limit at least a little bit, and paved the way for further advances. The principal idea has been to scan the object point by point with a focused light beam [88], [95], [110], and to construct from the point-by-point response an image. In its present application to cell biology, it is based on excitation of fluorescence using laser sources [24], [29]; for review see [85]. Theoretically, the gain in resolution

Super-Resolution based on Structured Illumination Excitation (SIE) Microscopy

Scanning of the object with a strongly focused laser beam (with or without switching ‘dark’ the molecules in the vicinity of the focal center of the scanning beam) is not the only possibility to achieve super-resolution in fluorescence microscopy. Instead, the object may be scanned also with an extended pattern of excitation light. In this way, enhanced resolution has become possible even with photostable dyes, i.e. fluorochromes which under appropriate conditions emit light at constant

Localization microscopy

For many decades it has been regarded to be next to impossible to enhance the resolution of light microscopy in such a way that individual molecules with mutual distances far below the Abbe/Rayleigh limit obtained with high NA lenses can be resolved. This has now been achieved in surprisingly simple ways by various methods of localization microscopy. For the development of one of these methods, Photo-Activated Localization Microscopy (PALM) using specific Green Fluorescent Proteins with

Perspectives for Super-Resolution Microscopy of nuclear Nanostructures

Super-resolution microscopy (SRM) methods are rapidly extending their application potential [30], [33] to various nanostructures in the cell nucleus, including the complexes for DNA transcription, repair, and gene regulation. This allows to test quantitatively models of functional spatial nuclear architecture as well as allow manifold applications in cellular pathology. In this report, we briefly reviewed some of the presently available SRM approaches (focused nanoscopy; structured

Concluding remarks

Super-resolution light microscopy/nanoscopy (SRM) methods already now have contributed a wealth of new insights in the architecture of the cell nucleus on the nanoscale, from the nuclear boundary to the molecular machines inside the nucleus and to nuclear genome structure. It is anticipated that SRM approaches in combination with molecular biology, biochemistry methods and theoretical biocomputing efforts will make it possible to gain a full quantitative insight into the mechanisms of nuclear

Acknowledgement

We gratefully acknowledge the input of many members of our research group (for further information see www.optics.imb-mainz.de), especially Dr. David Baddeley (Yale University), Dr. Antonio Virgilio Failla (University Medical Center Hamburg), as well as Drs. Marion & Thomas Cremer (LMU München) for stimulating discussions. We thank for the generous support by various funding institutions, in particular University Heidelberg, the Deutsche Forschungsgemeinschaft; the German Bundesministerium für

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