Phase-plate electron microscopy: a novel imaging tool to reveal close-to-life nano-structures

After slow progress in the efforts to develop phase plates for electron microscopes, functional phase plate using thin carbon film has been reported recently. It permits collecting high-contrast images of close-to-life biological structures with cryo-fixation and without staining. This report reviews the state of the art for phase plates and what is innovated with them in biological electron microscopy. The extension of thin-film phase plates to the material-less type using electrostatic field or magnetic field is also addressed.     

Functional studies in living animals using multiphoton microscopy

In vivo microscopy is a powerful method for studying fundamental issues of physiology and pathophysiology. The recent development of multiphoton fluorescence microscopy has extended the reach of in vivo microscopy, supporting high-resolution imaging deep into the tissues and organs of living animals. As compared with other in vivo imaging techniques, multiphoton microscopy is uniquely capable of providing a window into cellular and subcellular processes in the context of the intact, functioning animal. In addition, the ability to collect multiple colors of fluorescence from the same sample makes in vivo microscopy uniquely capable of characterizing up to three parameters from the same volume, supporting powerful correlative analyses. Since its invention in 1990, multiphoton microscopy has been increasingly applied to numerous areas of medical investigation, providing invaluable insights into cell physiology and pathology. However, researchers have only begun to realize the true potential of this powerful technology as it has proliferated beyond the laboratories of a relatively few pioneers. In this article we present an overview of the advantages and limitations of multiphoton microscopy as applied to in vivo imaging. We also review specific examples of the application of in vivo multiphoton microscopy to studies of physiology and pathology in a variety of organs including the brain, skin, skeletal muscle, tumors, immune cells, and visceral organs.     

Structured illumination microscopy of a living cell

Due to diffraction, the resolution of imaging emitted light in a fluorescence microscope is limited to about 200 nm in the lateral direction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Here we demonstrate that with the help of a spatial light modulator, this technique can be used for imaging slowly moving structures in living cells. This article has been submitted as a contribution to the Festschrift entitled “Uncovering cellular sub-structures by light microscopy” in honour of Professor Cremer’s 65th birthday.     

A quick guide to light microscopy in cell biology

Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: the resolution is well-matched to the sizes of subcellular structures, a diverse range of available fluorescent probes makes it possible to mark proteins, organelles, and other structures for imaging, and the relatively nonperturbing nature of light means that living cells can be imaged for long periods of time to follow their dynamics. Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments.     

Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels

Background

The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years.

Methodology/Principal Findings

We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization.Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology.Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces.

Conclusions

To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.     

Firefly luciferase as a tool in molecular and cell biology

The unique properties of firefly luciferase and the cloning of the gene for this enzyme have spawned a number of novel applications of this protein. We summarize a few of these applications including its use as a reporter gene, as a model for the study of protein import into peroxisomes, and as a component of a heterologous gene expression system.     

Confocal fluorescence microscopy in modern cell biology

Confocal fluorescence microscopy has become a major tool in modern cell biology. The paper explains the basic principles and especially the depth discrimination properties of confocal microscopy. An important application is described briefly and outlined with some figures. The paper concludes with remarks on features to be expected in the near future.     

Feeling the forces: atomic force microscopy in cell biology

Atomic force microscopy allows three-dimensional imaging and measurements of unstained and uncoated biological samples in air or fluid. Using this technology it offers resolution on the nanometer scale and detection of temporal changes in the mechanical properties, i.e. surface stiffness or elasticity in live cells and membranes. Various biological processes including ligand-receptor interactions, reorganization, and restructuring of the cytoskeleton associated with cell motility that are governed by intermolecular forces and their mode of detection will be discussed.     

Electron microscopy in cell biology: integrating structure and function

Electron microscopy (EM) is at the highest-resolution limit of a spectrum of complementary morphological techniques. When combined with molecular detection methods, EM is the only technique with sufficient resolution to localize proteins to small membrane subdomains in the context of the cell. Recent procedural and technical developments have increasingly improved the power of EM as a cell-biological tool.     

Imaging cell biology in live animals: Ready for prime time

Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.The discovery of GFP combined with the ability to engineer its expression in living cells has revolutionized mammalian cell biology (Chalfie et al., 1994). Since its introduction, several light microscopy–based techniques have become invaluable tools to investigate intracellular events (Lippincott-Schwartz, 2011). Among them are: time-lapse confocal microscopy, which has been instrumental in studying the dynamics of cellular and subcellular processes (Hirschberg et al., 1998; Jakobs, 2006; Cardarelli and Gratton, 2010); FRAP, which has enabled determining various biophysical properties of proteins in living cells (Berkovich et al., 2011); and fluorescence resonance energy transfer (FRET), which has been used to probe for protein–protein interactions and the local activation of specific signaling pathways (Balla, 2009). The continuous search for improvements in temporal and spatial resolution has led to the development of more sophisticated technologies, such as spinning disk microscopy, which allows the resolution of fast cellular events that occur on the order of milliseconds (Nakano, 2002); total internal reflection microscopy (TIRF), which enables imaging events in close proximity (100 nm) to the plasma membrane (Cocucci et al., 2012); and super-resolution microscopy (SIM, PALM, and STORM), which captures images with resolution higher than the diffraction limit of light (Lippincott-Schwartz, 2011).Most of these techniques have been primarily applied to in vitro model systems, such as cells grown on solid substrates or in 3D matrices, explanted embryos, and organ cultures. These systems, which are relatively easy to maintain and to manipulate either pharmacologically or genetically, have been instrumental in providing fundamental information about cellular events down to the molecular level. However, they often fail to reconstitute the complex architecture and physiology of multicellular tissues in vivo. Indeed, in a live organism, cells exhibit a 3D organization, interact with different cell types, and are constantly exposed to a multitude of signals originated from the vasculature, the central nervous system, and the extracellular environment. For this reason, scientists have been attracted by the possibility of imaging biological processes in live multicellular organisms (i.e., intravital microscopy [IVM]). The first attempt in this direction was in 1839, when Rudolph Wagner described the interaction of leukocytes with the walls of blood vessels in the webbed feet of a live frog by using bright-field transillumination (Wagner, 1839). Since then, this approach has been used for over a century to study vascular biology in thin areas of surgically exposed organs (Irwin and MacDonald, 1953; Zweifach, 1954) or by implanting optical windows in the skin or the ears (Clark and Clark, 1932). In addition, cell migration has also been investigated using transparent tissues, such as the fin of the teleost (Wood and Thorogood, 1984; Thorogood and Wood, 1987). The introduction of epifluorescence microscopy has enabled following in more detail the dynamics of individual cells in circulation (Nuttall, 1987), in tumors (MacDonald et al., 1992), or in the immune system (von Andrian, 1996), and the spatial resolution has been significantly improved by the use of confocal microscopy, which has made it possible to collect serial optical sections from a given specimen (Villringer et al., 1989; O’Rourke and Fraser, 1990; Jester et al., 1991). However, these techniques can resolve structures only within a few micrometers from the surface of optically opaque tissues (Masedunskas et al., 2012a). It was only in the early nineteen nineties, with the development of multiphoton microscopy, that deep tissue imaging has become possible (Denk et al., 1990; Zipfel et al., 2003b), significantly contributing to several fields, including neurobiology, immunology, and cancer biology (Fig. 1; Svoboda and Yasuda, 2006; Amornphimoltham et al., 2011; Beerling et al., 2011). In the last few years, the development of strategies to minimize the motion artifacts caused by the heartbeat and respiration has made it possible to successfully image subcellular structures with spatial and temporal resolutions comparable to those achieved in in vitro model systems, thus providing the opportunity to study cell biology in live mammalian tissues (Fig. 1; Weigert et al., 2010; Pittet and Weissleder, 2011).Open in a separate windowFigure 1.Spatial resolution and current applications of intravital microscopy. IVM provides the opportunity to image several biological processes in live animals at different levels of resolution. Low-magnification objectives (5–10×) enable visualizing tissues and their components under physiological conditions and measuring their response under pathological conditions. Particularly, the dynamics of the vasculature have been one of topic most extensively studied by IVM. Objectives with higher magnification (20–30×) have enabled imaging the behavior of individual cell over long periods of time. This has led to major breakthroughs in fields such as neurobiology, immunology, cancer biology, and stem cell research. Finally, the recent developments of strategies to minimize the motion artifacts caused by the heartbeat and respiration combined with high power lenses (60–100×) have opened the door to image subcellular structures and to study cell biology in live animals.The aim of this review is to highlight the power of IVM in addressing cell biological questions that cannot be otherwise answered in vitro, due to the intrinsic limitations of reductionist models, or by other more classical approaches. Furthermore, we discuss limitations and areas for improvement of this imaging technique, hoping to provide cell biologists with the basis to assess whether IVM is the appropriate choice to address their scientific questions.

Imaging techniques currently used to perform intravital microscopy

Confocal and two-photon microscopy are the most widely used techniques to perform IVM. Confocal microscopy, which is based on single photon excitation, is a well-established technique (Fig. 2 A) that has been extensively discussed elsewhere (Wilson, 2002); hence we will only briefly describe some of the main features of two-photon microscopy and other nonlinear optical techniques.Open in a separate windowFigure 2.Fluorescent light microscopy imaging techniques used for intravital microscopy. (A) Confocal microscopy. (top) In confocal microscopy, a fluorophore absorbs a single photon with a wavelength in the UV-visible range of the spectrum (blue arrow). After a vibrational relaxation (orange curved arrow), a photon with a wavelength shifted toward the red is emitted (green arrow). (center) In thick tissue, excitation and emission occur in a relative large volume around the focal plane (F.P.). The off-focus emissions are eliminated through a pinhole, and the signal from the focal plane is detected via a photomultiplier (PMT). Confocal microscopy enables imaging at a maximal depth to 80–100 µm. (bottom) Confocal z stack of the tongue of a mouse expressing the membrane marker m-GFP (green) in the K14-positive basal epithelial layer, and the membrane marker mTomato in the endothelium (red). The xy view shows a maximal projection of 40 z slices acquired every 2.5 µm, whereas the xz view shows a lateral view of the stack. In blue are the nuclei labeled by a systemic injection of Hoechst. Excitation wavelengths: 450 nm, 488 nm, and 562 nm. (B) Two- and three-photon microscopy. (top) In this process a fluorophore absorbs almost simultaneously two or three photons that have half (red arrow) or a third (dark red arrow) of the energy required for its excitation with a single photon. Two- or three-photon excitations typically require near-IR or IR light (from 690 to 1,600 nm). (center) Emission and excitation occur only at the focal plane in a restricted volume (1.5 fl), and for this reason a pinhole is not required. Two- and three-photon microscopy enable imaging routinely at a maximal depth of 300–500 µm. (bottom) Two-photon z stack of an area adjacent to that imaged in A. xy view shows a maximal projection of 70 slices acquired every 5 µm. xz view shows a lateral view of the stack. Excitation wavelength: 840 nm. (C) SHG and THG. (top) In SHG and THG, photons interact with the specimen and combine to form new photons that are emitted with twice or three times their initial energy without any energy loss. (center) These processes have similar features to those described for two- and three-photon microscopy and enable imaging at a maximal depth of 200–400 µm. (bottom) z stack of a rat heart excited by two-photon microscopy (740 nm) to reveal the parenchyma (green), and SHG (930 nm) to reveal collagen fibers (red). xy shows a maximal projection of 20 slices acquired every 5 µm. xz view shows a lateral view of the stack. Bars: (xy views) 40 µm; (xz views) 50 µm.The first two-photon microscope (Denk et al., 1990) was based on the principle of two-photon excitation postulated by Maria Göppert-Mayer in her PhD thesis (Göppert-Mayer, 1931). In this process a fluorophore is excited by the simultaneous absorption of two photons with wavelengths in the near-infrared (IR) or IR spectrum (from 690 to 1,600 nm; Fig. 2 B). Two-photon excitation requires high-intensity light that is provided by lasers generating very short pulses (in the femtosecond range) and is focused on the excitation spot by high numerical aperture lenses (Zipfel et al., 2003b). There are three main advantages in using two-photon excitation for IVM. First, IR light has a deeper tissue penetration than UV or visible light (Theer and Denk, 2006). Indeed, two-photon microscopy can resolve structures up to a depth of 300–500 µm in most of the tissues (Fig. 2 B), and up to 1.5 mm in the brain (Theer et al., 2003; Masedunskas et al., 2012a), whereas confocal microscopy is limited to 80–100 µm (Fig. 2 A). Second, the excitation is restricted to a very small volume (1.5 fl; Fig. 2 B). This implies that in two-photon microscopy there is no need to eliminate off-focus signals, and that under the appropriate conditions photobleaching and phototoxicity are negligible (Zipfel et al., 2003b). However, confocal microscopy induces out-of-focus photodamage, and thus is less suited for long-term imaging. Third, selected endogenous molecules can be excited, thus providing the contrast to visualize specific biological structures without the need for exogenous labeling (Zipfel et al., 2003a). Some of these molecules can also be excited by confocal microscopy using UV light, although with the risk of inducing photodamage.More recently, other nonlinear optical techniques have been used for IVM, and among them are three-photon excitation, and second and third harmonic generation (SHG and THG; Campagnola and Loew, 2003; Zipfel et al., 2003b; Oheim et al., 2006). Three-photon excitation follows the same principle as two-photon (Fig. 2 B), and can reveal endogenous molecules such as serotonin and melatonin (Zipfel et al., 2003a; Ritsma et al., 2013). In SHG and THG, photons interact with the specimen and combine to form new photons that are emitted with two or three times their initial energy (Fig. 2 C). SHG reveals collagen (Fig. 2 C) and myosin fibers (Campagnola and Loew, 2003), whereas THG reveals lipid droplets and myelin fibers (Débarre et al., 2006; Weigelin et al., 2012). Recently, two other techniques have been used for IVM: coherent anti-Stokes Raman scattering (CARS) and fluorescence lifetime imaging (FLIM). CARS that is based on two laser beams combined to match the energy gap between two vibrational levels of the molecule of interest, has been used to image lipids and myelin fibers (Müller and Zumbusch, 2007; Fu et al., 2008; Le et al., 2010). FLIM, which measures the lifetime that a molecule spends in the excited state, provides quantitative information on cellular parameters such as pH, oxygen levels, ion concentration, and the metabolic state of various biomolecules (Levitt et al., 2009; Provenzano et al., 2009; Bakker et al., 2012).We want to emphasize that two-photon microscopy and the other nonlinear techniques are the obligatory choice when the imaging area is located deep inside the tissue, endogenous molecules have to be imaged, or long-term imaging with frequent sampling is required. However, confocal microscopy is more suited to resolve structures in the micrometer range, because of the possibility of modulating the optical slice (Masedunskas et al., 2012a).

IVM to investigate biological processes at the tissue and the single cell level

The main strength of IVM is to provide information on the dynamics of biological processes that otherwise cannot be reconstituted in vitro or ex vivo. Indeed, IVM has been instrumental in studying several aspects of tissue physiopathology (Fig. 3, A and B). Although other approaches such as classical immunohistochemistry, electron microscopy, and indirect immunofluorescence may provide detailed structural and quantitative information on blood vessels, IVM enables measuring events such as variations of blood flow at the level of the capillaries or local changes in blood vessel permeability. These data have been instrumental in understanding the mechanisms of ischemic diseases and tumor progression, and in designing effective anticancer treatments.

Table 1.

IVM to study tissue physiopathology

Learning cell biology as a team: a project-based approach to upper-division cell biology

To help students develop successful strategies for learning how to learn and communicate complex information in cell biology, we developed a quarter-long cell biology class based on team projects. Each team researches a particular human disease and presents information about the cellular structure or process affected by the disease, the cellular and molecular biology of the disease, and recent research focused on understanding the cellular mechanisms of the disease process. To support effective teamwork and to help students develop collaboration skills useful for their future careers, we provide training in working in small groups. A final poster presentation, held in a public forum, summarizes what students have learned throughout the quarter. Although student satisfaction with the course is similar to that of standard lecture-based classes, a project-based class offers unique benefits to both the student and the instructor.     

Embryonic stem-cell culture as a tool for developmental cell biology

The cell biology of the early processes of mammalian embryogenesis, such as germ-layer formation, has been technically challenging to study owing to the size and accessibility of mammalian embryos. Embryonic stem cells, which can generate the three germ layers in vitro, are useful for studying embryogenesis at the cellular level. So, how can the study of embryonic stem cells and their differentiation provide a deeper understanding of the cell biology of early development?     

Coherent phase microscopy in cell biology: visualization of metabolic states

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image (‘phase thicknesses’ Δh) and cell energization. It was demonstrated that the phase thickness Δh decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Δh and cell diameter d demonstrated that a decrease in the phase thickness Δh could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.     

Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB–SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce “keyframe imaging” as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10⁹ in a single automated experimental run.