Abstract
To dissect the astonishing complexity of the biomolecular machinery functioning within a cell, imaging has been an integral tool in biology, allowing researches to “view” the detailed molecular biology responsible for coordinating cellular life. To visualize the molecular components of cellular structures requires, in particular, imaging techniques capable of reaching nanoscale spatial resolutions. Such nanoimaging techniques are the focus of this volume. Chapters in the current volume are divided into four parts and include specialized techniques in the areas of light, electron, and scanning probe microscopy, as well as methodologies employing combinatorial and complementary imaging approaches.
1. Introduction
Despite the diverse physiological properties of different cell types, they are composed, generally, of the same biochemical and molecular components. The coordinated assembly of these components provides the functional characteristics of the cell and, ultimately, the viability of the organism. A common goal in biology is, therefore, to dissect the astonishing complexity of the cellular molecular machinery, seeking to understand how individual components of this machinery assemble to form higher-order structures, navigate the cell with synchronized dynamics, and interact to produce a given biological outcome. To realize this common goal, imaging has been an integral tool in biology, allowing researches to “view” the detailed molecular biology responsible for coordinating cellular life.
To directly observe the molecular components of a cell, either in isolated form or within a cellular context, biologists have at their disposal an array of established techniques in the major areas of light, electron, and scanning probe microscopy. While pivotal changes in biology have repeatedly occurred following key developments in microscopy, the quest to explore the inner workings of cells at ever-increasing levels of detail has been, at the same time, a tremendous stimulus to the development of novel and ingenious approaches to imaging. Thus, to some extent, biology has led to a continuous reinvention of the light, electron, and scanning probe microscopes, in addition to nurturing the development of completely new tools in microscopy. Today, advanced imaging modalities enable the visualization of biological specimens at a range of length scales and resolutions, in multiple dimensions (both time and space), with chemical or molecular specificity, and from the surface or bulk of the specimen, revealing the secrets of the molecular bionetwork within the cell.
Starting with the optical microscope, one of the most important historical developments in this field can be assigned to Ernst Abbe, whose rigorous description of wave-based optical theory supporting image formation in the microscope provided the rationale for the limitation of resolution and, thereby, the expected barrier to observing fine details of the molecular machinery within a specimen. The resolution limit as imposed by diffraction still holds true, but advances in instrumentation and a greater understanding of the physical photochemistry of labeling fluorophores have allowed microscopists to achieve resolutions beyond Abbe’s diffraction limit. This includes the recent development of numerous ingenious super-resolution optical imaging methods that reveal specimen details on the nanometer scale.
The legendary work by Ernst Ruska and colleagues used Abbe’s wave-based optical theory to great advantage in developing the electron microscope. The dramatically shorter wavelength of electrons employed in the electron microscope allowed biologists for the first time to examine the internal architecture of cells with nanoscale spatial resolution. The conventional view of electron microscopy, however, is that thin and exhaustively processed specimens can be observed only with rather nonspecific contrasting agents, thereby limiting the specificity of the molecular information obtained. Modern electron microscopes are based on Ruska’s prototype but have developed specialized imaging arrangements that provide greater contextual information about the sample while overcoming some of the traditional limitations of specimen preparation. The diverse modes of operation of the modern electron microscope are able to provide images of native cellular structures, in three dimensions, from large sample volumes, and containing relevant quantitative information.
Contrary to light and electron microscopy in which a magnified image of a specimen is generated by a lens-based optical system, techniques in scanning probe microscopy rely on the general principle of raster-scanning a sharp probe across a surface to produce a magnified view of the specimen topography. The hallmark scanning probe instrument is the atomic force microscope (AFM) that differs significantly from conventional lens-based microscopes in the underlying principle of image formation, and not surprisingly offers its own set of attributes and opportunities, such as the possibility to probe specific chemical and biophysical interactions between a functionalized scanning tip and the underlying sample surface. Near-field scanning optical microscopy (NSOM) is another type of scanning probe technique of current use in biology. It involves scanning with a small probe positioned less than a wavelength of light from the sample whereby light is transmitted through a small aperture in the probe tip, thus providing a means to circumvent the diffraction-limited far-field resolution of optical microscopes.
Several other specialized nanoscale imaging techniques exist that do not fall within the more general categories of light, electron, and scanning probe microscopy as highlighted above. One such technique, soft X-ray microscopy, has a history that dates back several decades, although only more recently has it become more adept at imaging biological specimens. Today, soft X-ray tomography is emerging as a powerful method to visualize entire eukaryotic cells in three dimensions with a spatial resolution that bridges that of the light and electron microscope. Another specialized technology, imaging mass spectrometry, combines attributes of both secondary ion mass spectrometry and imaging. This relatively new technique offers the exciting possibility of obtaining very specific compositional information about a sample’s surface with a lateral resolution in the tens of nanometers.
A feature common to all imaging techniques is the need to produce contrast, and in fact the success of some of the most modern techniques relies on ingenious ways in which structures can be induced to reveal themselves. Of course, an important characteristic of any imaging modality is the achievable spatial resolution, and here a diverse range of methodologies exist that cover resolutions from the nano to the macro length scales. To visualize cellular molecular components and substructures requires in particular imaging techniques capable of reaching nanoscale spatial resolutions. Such nanoimaging techniques are therefore the focus of this volume.
Chapters in the current volume are divided into four parts and include specialized techniques in the areas of light, electron, and scanning probe microscopy (Parts 1, 2, and 3, respectively), as well as methodologies employing combinatorial and complementary imaging approaches (Part 4). Nanoimaging of biological samples involves precise logistical considerations that are more acute than for conventional imaging, such as illumination timing and intensity, signal information sampling, specimen preparation and labeling, to list a few. Given the added complexity of imaging biological specimens on the nanometer scale, we hope that the chapters comprising this volume provide valuable information from successful application of a broad range of important imaging methods for the nanoscale visualization of biological structures.
2. Part 1: Light Microscopy
Light microscopy is invaluable to biologists, but its application to resolving molecular details is limited by the physical properties of light, principally the diffraction of light upon passing through a fixed aperture. Diffraction of light results in the blurring of a point object in the image and is dependent on the wavelength of light and the numerical aperture of the collecting lens. The expanded observed intensity profile of the point is known as the point spread function, with the actual object located at the center of the intensity profile. The dimensions of the point spread function are larger than the physical size of the individual molecular components within the specimen. To truly image specimen molecular details, the diffraction barrier needs to be surpassed. A collection of super-resolution optical imaging methods have been able to attain resolutions beyond the diffraction limit by employing sophisticated, and elegant, illumination parameters, optical configurations, or fluorophores with unique biophysical properties. A number of methods exploit nonlinearity in the reflected light from the specimen and often employ a timing mechanism to achieve the improved resolution.
Part 1 of this volume contains several chapters describing optical imaging methods that achieve spatial resolution beyond the practical resolution limit of conventional light microscopy. The volume begins with a description of total internal reflection fluorescence (TIRF) microscopy, which uniquely provides improved axial spatial resolution by illuminating the sample using an evanescent wave to excite fluorophores within a limited depth. Establishing the evanescent wave illumination relies on adjusting the angle of the excitation light and refractive index differences between the support substrate, usually glass coverslip, and the growth medium of the sample. The details of imaging biological samples with a TIRF microscope are highlighted in Chapter 2. Another impressive configuration that improves the axial resolution of the light microscope by aligning two opposing objective lenses in the optical path and, thereby, effectively doubling the numerical aperture is known as 4Pi microscopy. The specialized optical configuration of 4Pi microscopy enables a substantial reduction of the dimensions of the point spread function in the optical axis, and Chapter 3 describes the use of this unique optical arrangement to image subcellular architecture.
A number of relatively new developments in light microscopy are presented in Chapters 4 through 8, all of which achieve resultant spatial resolution beyond the diffraction limit as defined by the Abbe criterion. The successful implementation of the illumination-based super-resolution imaging methods of structured illumination microscopy (SIM) and stimulated emission depletion (STED) microscopy are described in Chapters 4 and 5, respectively. Among sub-diffraction limited techniques, structured illumination holds the distinction of being one of the only methods that can work with simple reflectance without the need for special dyes or fluorescence. Multiple spatially modulated illumination patterns are used to double the effective numerical aperture and, thereby, improve the lateral resolution. STED imaging uses nonlinear multistep optical illumination strategy to reduce the size of the point spread function and achieve super-resolution in the nanometer scale. The following three chapters discuss probe-based super-resolution imaging techniques based on the principle of single-molecule localization: photoactivatable localization microscopy (PALM) (Chapter 6), light microscopy with points accumulation for imaging in nanoscale topography (uPAINT) (Chapter 7), and blink microscopy (Chapter 8). Collectively, the probe-based techniques rely on the mathematically known location of the actual object within the observed intensity profile of the point spread function. Using time to acquire individual PSFs from multiple areas within the sample, the location of the objects within each PSF can be localized with high precision, and with multiple PSFs collected over time, an image map can be constructed with the location of each object determined with high spatial resolution in the final image. Several other exciting variations of the single-molecule localization method exist, but we regret that not all could possibly be included in this volume.
The successful implementation of most of the existing super-resolution imaging methods depends in great part on the biophysical properties of the fluorophores used to highlight specific molecular complexes in the cell. Therefore, the selection of appropriate fluorophores suitable, both for the sample to be imaged and the method used for imaging, is an important topic concerning super-resolution light microscopy and is presented in Chapter 9.
Being able to accurately pinpoint the location of discrete fluorescent objects is not only used to construct super-resolution images of static specimens, but can also be employed to track the trajectory of particular molecular complexes in living cells. By tracking single particles with high precision, the location of the particle at each time point can be used to build a map of the molecule’s history, and provide insight into its function. Chapter 10 details how this principle can be applied to track the dynamics of ribonucleoprotein complexes in live cells.
Collectively, the optical imaging methods, and the successful application of the methods to image biologically relevant samples, described in Part 1 of the volume highlight the many exciting possibilities available to obtain nanometer scale information from molecular assemblies within the cell.
3. Part 2: Electron Microscopy
The electron microscope has been indisputably a primary nanoscale imaging tool in biology. Traditionally, however, electron microscopy (EM) has been somewhat restricted to specialized laboratories, due to factors such as cost of instrumentation and challenges with sample preparation. Nevertheless, today some of the most advanced aspects of the technique have become readily accessible to the biological electron microscopist, thanks to technical developments in instrumentation, sample preparation and data processing. Part 2 of this volume presents some of the most important methodologies in the field of electron microscopy, with an emphasis on techniques that go beyond just basic implementations of EM.
Part 2 begins with a discussion on single-particle cryo-electron microscopy (cryo-EM), an advanced technique for generating high-resolution three-dimensional images of macromolecular assemblies. Single-particle cryo-EM does not require molecular crystals, being instead applicable to ensembles of isolated molecular complexes. This unique feature of the technique allows the three-dimensional imaging of molecular machines assuming specific functional states or having particular interactions with other molecules. Processing single-particle cryo EM data to generate a three-dimensional volume of the specimen requires careful consideration of many parameters. Chapter 11 therefore lays down a protocol to analyze single-particle cryo-EM data, guiding the reader towards each step of the data processing and analysis workflow.
Next in Part 2, Chapter 12 underlines the distinctive capability of the electron microscope in making accurate measurements of molecular mass from isolated macromolecular assemblies. Compared to other techniques such as mass spectrometry, the method of molecular mass measurement in the electron microscope is unique in that it enables determination of the mass per length of filamentous specimens or the mass per area of planar structures. Chapter 12 highlights in particular how quantitative mass per length measurements can be employed to gain important structural information on Alzheimer’s amyloid fibrils.
Chapter 13 provides another example of the capability of the electron microscope in generating quantitative information from biological specimens. Specifically, in the techniques of energy-filtering transmission electron microscopy (EFTEM) and electron energy-loss spectroscopy (EELS), electrons that have traversed a thin sample can be collected and their energies analyzed by an electron spectrometer to yield quantitative information on the nanoscale distribution of specific elements in the specimen. Chapter 13 explains some of the experimental and computational steps in EFTEM and EELS leading to the formation of quantitative elemental maps. Because phosphorus is present in abundance in DNA, these approaches enable imaging the intrinsic distribution of nucleic acids without the use of nonspecific heavy-metal stains.
Significantly, the last several years have witnessed a remarkable explosion in the application of electron tomography, a technique that allows the generation of three-dimensional images of cellular ultrastructure at nanoscale spatial resolution. While recording conventional electron tomograms from heavy-metal stained specimens is somewhat straight-forward using modern electron microscopes, imaging samples in their native state (i.e., frozen-hydrated) can be considerably more challenging. Cryo-electron tomography of native, frozen-hydrated eukaryotic cells is therefore the focus of Chapter 14.
While electron tomography can produce unique nanoscale three-dimensional views of subcellular architecture, the technique has a fundamental limitation in that samples must be electron transparent (i.e., less than about 400-nm thick). Thus, only a very small fraction of an entire eukaryotic cell or piece of tissue is amenable to analysis by electron tomography. Recently, however, there has been growing interest in developing approaches for the nanoscale reconstruction and analysis of large volumes of cellular and tissue ultrastructure. As an application for this methodology, three-dimensional EM data obtained from across several microns of brain tissue can be used to explore how patterns of circuit connectivity relate to neuronal function. Chapter 15 describes in detail how serial-section electron microscopy can be optimally implemented to generate large-volume three-dimensional reconstructions of brain tissue.
Another approach to visualizing large volumes of biological specimens in three dimensions is based on the hybrid technique of focused ion beam/scanning electron microscopy (FIB/SEM). The FIB/SEM method is the topic of Chapter 16, which illustrates primarily how a FIB can be used as a nano-scalpel to reveal subsurface microstructures that can then be examined by SEM imaging.
Despite the innumerous advantages as a nanoimaging tool, EM has an important limitation in that it does not produce images with intrinsic molecular specificity. Chapter 17 therefore underlines the use of ultrasmall gold nanoparticle conjugates as labels to localize specific intracellular proteins within sectioned cells or identify specific subunits within multi-subunit molecular assemblies. In particular, Chapter 17 explains how to synthesize a type of ultrasmall and uniform gold nanoparticle, and, in addition, how to attach antibodies to the nanoparticle allowing it to be used as a label for EM imaging of biological specimens.
4. Part 3: Scanning Probe Microscopy
Scanning probe microscopy offers unparalleled possibilities for imaging and probing model and native membranes as well as macromolecular complexes. Part 3 of this volume contains four chapters covering two distinct types of scanning probe instruments, namely the atomic force microscope and the near-field scanning optical microscope.
Part 3 starts with Chapter 18, which highlights the AFM as a tool for high resolution imaging of macromolecular complexes. Examples of imaging single DNA molecules and DNA-protein interactions are presented, together with an ample discussion of the important specimen variables and operational parameters that affect image quality and lead to reproducible imaging conditions.
The high lateral and vertical spatial resolution afforded by the AFM combined with its ability to operate in liquids has turned this instrument into a distinctive tool for the high-resolution imaging of two-dimensional crystals of membrane proteins. However, membrane proteins that are difficult to crystallize and/or express and purify are not easily amenable to high resolution AFM imaging. Chapter 19 therefore presents an attractive approach for preparing specimens of membrane protein for high resolution AFM imaging, whereby transmembrane proteins are incorporated within supported lipid bilayers at only picomolar concentrations.
Chapter 20 describes novel developments in atomic force microscopy for the high spatial resolution mapping of specific receptors on native membranes. The basic principle of this approach involves raster-scanning a functionalized tip onto the specimen and recording force spectroscopy measurements at every pixel of the topographic image. Chapter 20 explains the implementation of this exciting approach, termed simultaneous topography and recognition imaging technique (TREC), showing as an example how it can be used to map the nanoscale distribution of Fcγ receptors on macrophages.
Part 3 ends with a chapter on near-field scanning optical microscopy (NSOM). In this technique, an optical probe of subwavelength diameter is raster-scanned across a sample keeping very close proximity to its surface. Lateral resolution becomes independent of far-field diffraction, being instead a function of the diameter of the probe aperture and its distance to the sample surface. Chapter 21 explains how scanning probe microscopes can be modified in-house to implement NSOM, and provides examples of NSOM fluorescence imaging of model and natural biological membranes.
5. Part 4: Complementary Techniques
The imaging techniques discussed so far possess distinct advantages in obtaining specific information from biological samples. Combining multiple imaging methods provides a means to acquire specific information within a contextual structure, thus enhancing the collective information from the sample. For example, the specificity of the labeling used in fluorescence microscopy can be used to great advantage to complement the architectural information available from electron microscopy. Correlative fluorescence and electron microscopy methods not only provide specificity along with ultrastructural detail but also help reduce the burden of screening the sample for imaging, thereby increasing the probability of obtaining information from samples in the desired growth state, or biochemical process.
A multitude of correlative microscopy combinations have been developed with a few interesting examples described in Part 4 of the volume. A description of two correlative fluorescence and electron microscopy techniques are presented in the first two chapters of Part 4, with the combination of fluorescence microscopy and energy-filtering transmission electron microscopy used to reveal the ultrastructure and biochemical composition of interesting subnuclear structures in Chapter 22, and the three-dimensional high data output correlative light and electron microscopy imaging of cryosections in Chapter 23.
Uniquely, the combination of fluorescence microscopy with atomic force microscopy provides the specificity of fluorophore labeling with the fine topographical details available using AFM. The description of an integrated AFM and fluorescence instrument is presented in Chapter 24 that has been successfully employed in imaging specific interactions occurring at membrane interfaces. Interestingly, the multimodal instrument is amenable to combining super-resolution optical methods with scanning probe microscopy, with great promise for data-rich nanoimaging in biology.
An exciting method for imaging small prokaryotes or eukaryotic cells in three dimensions has emerged by applying the principle of soft X-ray microscopy in a tomographic imaging configuration. Soft X-ray microscopy uses illuminating photons in the water window of the electromagnetic spectrum, meaning that these photons are much more efficiently absorbed by organic mass than water. The technique enables imaging intact frozen-hydrated cells with a spatial resolution in the tens of nanometers, bridging an important gap between light and electron microscopy. Chapter 25 describes experimental and computational procedures in soft X-ray tomography leading to the nanoscale three-dimensional reconstruction of frozen-hydrated cells.
The ability to obtain chemically specific information at nanoscale spatial resolution within cellular or model membranes is an exceptional feature of secondary ion mass spectrometry imaging. In this technique, a focused ion beam raster scans across a specimen producing secondary ions that are then analyzed by a mass spectrometer. A map of the nanoscale distribution of specific membrane lipids can be generated by detecting the secondary ion signal from those lipid species that have been isotopically labeled. In Chapter 26, basic protocols and methodologies are described for the secondary ion mass spectrometry imaging of sphingolipids in the plasma membrane.
6. Conclusion
Collectively, the nanoimaging methods presented in this volume of Methods in Molecular Biology we expect will provide valuable information on how to apply and adapt the methods for imaging other samples of interest, and guide the use of nanoimaging in the exploration of the molecular machinery responsible for regulating cellular life.