Intravital microscopy: a novel tool to study cell biology in living animals

Abstract

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criteria. Indeed, first we will focus on those studies in which organs where imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.

Introduction

In the last two decades the field of mammalian cell biology has developed extraordinarily, and this is in part due to the advancements in the ability to image almost any cellular process in cultured cells. Indeed, the level of resolution currently achieved allows from imaging the movements of a single cell to tracking subcellular organelles or even single molecules. In addition, cell cultures are extremely flexible systems that offer the advantage of being easily manipulated both pharmacologically and genetically, thus enabling the acquisition of detailed molecular information about the machinery regulating the process of interest. However, a major limitation of in vitro models is that they do not always reconstitute the tissue environment as found in animal tissues under physiological conditions. For example, cell cultured on plastic or glass surfaces, which are the most utilized experimental models in cell biology, lack the three-dimensional organization, which is crucial for many cellular processes (Cukierman et al., 2001). On the other hand, three-dimensional cell culture models, which are now widely adopted and utilize various purified components of the extracellular matrix, lack most of the key components that play an active role in the environment of any native tissue, such as other cell types and signaling molecules (Cukierman et al., 2001; Ghajar and Bissell, 2008; Xu et al., 2009). To overcome these limitations, a large effort has been directed towards developing techniques and tools to image and study cellular events in living animals, with the goal of achieving the same depth of analysis that is currently available for in vitro models. Although in live animals organs have been imaged since the early 1930’s (Beck and Berg, 1931), the major breakthroughs in this field occurred in the last decade with the improvement of conventional microscopes and more importantly with the development of microscopes based on the “nonlinear” excitation of the specimen, which has opened the door to deep tissue imaging (Helmchen and Denk, 2005; Mertz, 2004; Zipfel et al., 2003b).

The focus of this review is primarily on intravital microscopy (IVM), which includes all those optical microscopy techniques that are utilized to perform kinetic and functional studies in living animals. Although IVM has been utilized also in smaller organisms such as drosophila, zebra fish, and C. elegans (Gualda et al., 2008; O’Brien et al., 2009; Vinegoni et al., 2009; Yaniv et al., 2006), we will discuss primarily those studies performed in small rodents. First, we will present a brief overview of some of the techniques based on non-linear optical microscopy, suggesting more specialized articles to the reader for the technical details. Next, we will review how IVM has been applied to the imaging of biological processes at the level of either tissues or single cells, leading to tremendous advancements in fields such as neurobiology, cancer biology and immunology. Finally, we will review recent studies in which IVM has been utilized to image processes at a subcellular level, discussing how this area has opened a new era of investigations in cell biology.

Non-linear Optical Microscopy

Non-linear optical microscopy techniques generate contrast by using higher-order interactions between light and biological matter. Processes whose dependence from the incident light is nonlinear typically involve the absorption or the scattering and recombination of two or more photons by the specimen (Campagnola and Loew, 2003; Helmchen and Denk, 2005; Mertz, 2004; Oheim et al., 2006; Rubart, 2004; So et al., 2000; Stutzmann and Parker, 2005; Svoboda and Yasuda, 2006; Zipfel et al., 2003b).

Two- and Three-Photon Microscopy

The theoretical formulation of multi-photon excitation was proposed for the first time by Maria Goppert-Mayer in 1931 (Göppert-Mayer, 1931). However, it took 30 years to be experimentally proven with the invention of the first laser, and almost sixty years for the first multi-photon microscope to be built (Denk et al., 1990). Two- (three-) photon excitation is based on the fact that a molecule of a fluorophore can be excited by the almost simultaneous absorption (between atto- and femto-seconds, 10⁻¹⁸-10⁻¹⁵ sec) of two (three) photons that have half (a third) of the energy that would be required to fill the gap between two of its energetic levels (Fig. 1a and 1b) (Helmchen and Denk, 2005; Zipfel et al., 2003b). This implies that multi-photon excitation requires near infrared light (NIR) or infrared light (IR) that have the ability to penetrate biological tissues deeper than UV or visible light, making it the ideal choice for deep tissue imaging (Oheim et al., 2001). Indeed, whereas in single confocal microscopy biological specimen can be imaged up to a depth of 50–60 μm, in multi-photon microscopy (MPM) this range can be extended up to 1 mm either using tissues that exhibit a lower light scattering such as the brain (Theer et al., 2003) or by utilizing longer excitation wavelengths (Andresen et al., 2009; Kobat et al., 2009). The probability of multi-photon transitions to occur is extremely low and requires very high light intensities concentrated in space and in time. This is achieved by using lasers that emit NIR/IR light in very short pulses (typically in the order of 100 fs) at high repetition rates (80–100 MHz), and by using high numerical aperture lenses that focus the light to the excitation spot. In ideal conditions the absorption and the emission occur in a very small volume (1 fl) (Helmchen and Denk, 2005; Zipfel et al., 2003b) reducing significantly both photo-toxicity and photo-bleaching. Furthermore, this avoids the issue of off-focus emission, which in confocal microscopy is eliminated through the use of a pinhole (Fig. 1d). Finally, another important feature of multi-photon excitation is that for all the fluorophores characterized so far, the absorption spectra are much broader than in single photon excitation. This enables the imaging of multiple fluorophores using a single excitation wavelength (Fig. 1f) (Bestvater et al., 2002; Spiess et al., 2005).

Figure 1. Non linear optical microscopy.

a–d Jablonski diagram illustrating (a) single photon (1P), (b) two-photon (2P) and three-photon (3P) excitation, (c) second (SHG) and third (THG) harmonic generation, and, (d) Coherent Anti-Stokes Raman Spectroscopy (CARS). a,b - In both single and MP microscopy, the emitted photons have a lower energy than the sum of the incident ones, due to some energy loss (yellow arrow). c – In SHG and THG the incident photons are scattered and recombine in a single one, without energy loss. d- In CARS microscopy two beams are used: the pump (ω_p) and the stokes (ω_s). When they are tuned to match a vibrational energy gap (ω_vib), a strong anti-stokes signal is generated (ω_CARS). Note that both in the harmonic emission and in CARS no electronic transitions occur. e) Non-linear emission occurs at the focal spot. f) Multiple fluorophores can be imaged using a single excitation wavelength. Alexa 488-dextran transferrin (green) and Texas Red–dextran (red) were injected in the submandibular glands of male rats and internalized into endosomal vesicles by fibroblast located in the stroma. After 1 hour, the glands were imaged by MPM using 750 nm as excitation wavelength. The endogenous fluorescence highlights the acinar structures (cyan). Note that both transferrin and dextran bind to the extracellular matrix surrounding the acini. Scale bar - 20 μm

Second and Third Harmonic Generation Microscopy

Second harmonic generation (SHG) and third harmonic generation (THG) are processes that do not involve any energy absorption since the incident photons are scattered, recombining into a single photon in a process without energy loss (Fig. 1c). For this reason they are suitable for imaging biological specimen with even lower photo-toxicity than MPM (Campagnola and Loew, 2003). Although the major harmonic signals are produced in the forward direction and thus more suited for imaging slices, the backward scattering signal is still sufficient for imaging thick tissue in live organisms. Several molecules are able to generate harmonic signals, especially when assembled in highly ordered structures. Among them are collagen, microtubules, and muscle myosin (Campagnola and Loew, 2003; Zipfel et al., 2003a; Zoumi et al., 2002). Recently, lipids forming lipid bodies have also been successfully imaged in various living organisms by using THG (Debarre et al., 2006). Due to the different nature of the harmonic emission, SHG and THG are often combined with MPM, expanding the repertoire of information that can be acquired. Recently, using spectral un-mixing techniques, up to six intrinsic signals coming from both multi-photon and harmonic emissions were resolved, providing very detailed information about the architecture of the skin in live nude mice (Radosevich et al., 2008).

Coherent Anti-Stokes Raman Scattering (CARS)

CARS microscopy is based on the use of two laser pulses (the pump and the Stokes) that are tuned to match the energetic gap between two vibrational levels in the molecule of interest (Muller and Zumbusch, 2007) (Fig. 1d). Under these conditions a strong anti-Stokes signal is emitted, which generates the contrast to image the specimen. One of the major applications of CARS microscopy comes from tuning the wavelengths to the CH₂ vibrational bands that enables imaging various molecules such as myelin fibers, and lipids, either within the membrane bilayer or in intracellular storage compartments. Furthermore, CARS has been utilized to image arterial walls and atherosclerotic lesions (Evans et al., 2005; Fu et al., 2008; Wang et al., 2009). Although the use of CARS in live animals has been very limited thus far, this is a very promising technique that has a strong potential for live animal studies especially when complemented with MPM and SHG.

Fluorescence Lifetime Imaging (FLIM)

FLIM is based on the measurement of the lifetime that a given molecule spends in an excited state. This parameter is a characteristic of each molecule, and does not depend on its concentration. Since the lifetime is sensitive to modifications of the environment, FLIM has become a powerful tool in the quantitative analysis of cellular parameters such as pH, oxygen levels, ions concentration, and the metabolic state of various biomolecules. When FLIM is combined with multi-photon excitation and harmonic generation, it becomes suitable for deep tissue imaging, providing valuable information on the tissue microenvironment (Levitt et al., 2009; Niesner et al., 2008; Provenzano et al., 2009).

Imaging tissue architecture and function in vivo

Intrinsic or endogenous fluorescence

Several endogenous molecules are excited using either non-linear optical techniques or single photon microscopy, providing valuable information on the tissue architecture without the need for exogenous labeling (Zipfel et al., 2003a). Although several studies have been performed utilizing endogenous emissions in explanted tissues, only few have been carried out in live animals. One of the molecules that has been exploited for this purpose is NAD(P)H that emits in the visible range upon either single photon (360 nm) or two-photon excitation (710–760 nm). Although its two-photon cross section is very low, its abundance within the cell makes it a suitable endogenous label for both metabolic and structural studies (Fig. 2a–i). Changes in the levels of NAD(P)H were measured in live mice during ischemia and reperfusion in the jejunum (Guan et al., 2009), microcirculatory failure in the liver (Paxian et al., 2004) or in the kidney after LPS-induced sepsis (Wu et al., 2007), providing novel data on the metabolic state of the tissue under pathological conditions. Recently, levels of NAD(P)H were measured in the skin and in the liver using FLIM (Roberts et al., 2008). NAD(P)H is distributed both in the cytoplasm and in the mitochondria, and at a relatively low magnification, its signal highlights the details of the architecture of various tissues. For example, in the brain, the astrocytes show a characteristic NAD(P)H pattern that distinguishes them from other cell populations (Kasischke et al., 2004); in skeletal muscles of both live animals and humans the architecture and the contractions of the sarcomere have been imaged taking advantage of the high concentration of mitochondria along the sarcomere Z-discs (Llewellyn et al., 2008; Rothstein et al., 2005); in the salivary glands of both live rats and mice, the architecture of the acini have been resolved by exciting the intrinsic emission of NAD(P)H (Fig. 2i and 2i′); and the fine details of the structure of the large ducts, which are highly enriched in mitochondria on the basolateral pole of the epithelium, have also been imaged at a level of resolution almost comparable to that obtained by classical immunohistochemistry (Masedunskas and Weigert, 2008; Sramkova et al., 2009) (Fig. 2i′, 2i″ and 2j). Another molecule whose intrinsic fluorescence has been exploited for in vivo imaging is collagen, which when arranged in fibers generates a strong SHG signal (Campagnola and Loew, 2003; Zoumi et al., 2002). Due to its very low turnover and stability, several studies have been focusing on analyzing the architecture of collagen fibers in various explanted organs under both physiological and pathological conditions (Cox et al., 2003; Megens et al., 2007; Morishige et al., 2006; Pena et al., 2007; Schenke-Layland et al., 2008). Moreover, imaging collagen fibers in live animals has proven to be a valuable reference point within the tissue, particularly in the context of tumor migration and invasion where an important issue is to correctly locate and orient tumors that are repeatedly imaged over long period of times (Brown et al., 2003; Perentes et al., 2009; Wyckoff et al., 2007). Furthermore, imaging the organization of collagen fibers has been extremely valuable in studies related to skin diseases both in live rodents and in patients (Konig et al., 2007). Finally, in order to highlight various structural features in live animals, other molecules have been imaged by using different modalities, such as elastin in the skin (TPM), myosin fibers in the skeletal muscle (SHG), myelin fibers in the corpus callosum (CARS) or lipid-enriched structures (CARS) (Evans et al., 2005; Fu et al., 2008; Konig et al., 2007; Llewellyn et al., 2008).

Figure 2. Imaging the architecture of the tissues in live animals.

a–i Excitation of intrinsic fluorescence to image tissue architecture. Rats were anesthetized and various organs such as liver (a), kidney (b), brain cortex (c), skeletal muscle (d), epididymis (f), bladder (g), prostate (h) and lacrimal glands (i) were imaged at a low magnification by using 740 nm as excitation wavelength. Scale bar - 100 μm. (i) The submandibular glands were imaged at a higher magnification (i) and details of the structure the acini (i′) and the large striated ducts (i″) are compared with the classical H&E staining (j). Scale bars - 10 μm. k–m Imaging the vasculature in live animal. Texas-Red dextran was systemically injected in anesthetized rats and the liver (k), the kidney (l) and the brain cortex (m) were imaged using 740 nm (k,l) or 920 nm (m) as excitation wavelength. n- Vasculature and salivary ducts in live animals. FITC dextran was injected systemically in anesthetized rats, whereas Texas-Red dextran was injected into the Wharton’s duct as described in Sramkova et al. 2009. The salivary glands were imaged by MPM using 920 nm as excitation wavelength. Scale bars 20 μm.

These examples show that the use of intrinsic fluorescence in a given tissue can be a powerful tool not only for basic research but also for diagnostic purposes, since it does not require the use of exogenous labeling (Konig et al., 2007). Hence, characterizing the patterns of intrinsic fluorescence generated in different tissues is a crucial direction to explore and develop further.

Exogenous labeling of the tissues

Another approach to image tissue architecture and function is to either administer exogenous dyes or to genetically introduce fluorescent proteins selectively targeted to the tissue of interest (Fig. 2k–n). For example, systemic injections of fluorescently labeled bovine serum albumin (BSA) or dextrans of different sizes have enabled studying and measuring both glomerular filtration and tubular reabsorption in the kidney (Kang et al., 2006; Yu et al., 2005, 2007) (Fig. 2l and supplementary movie 2). In the pancreas of live mice, by imaging with a millisecond temporal resolution, blood flow patterns were determined in the islet vasculature bed (Nyman et al., 2008). This approach has been extensively used in neuroscience where vasculature flow has been imaged and measured either in normal conditions or under acute ischemic damage in the brain cortex (Levene et al., 2004; Theer et al., 2003; Zhang and Murphy, 2007) (Fig. 2m) or in the olfactory bulb (Chaigneau et al., 2003; Chaigneau et al., 2007; Stefanovic et al., 2008). Imaging the vasculature both acutely and chronically has been an extremely important tool in the context of cancer biology to address key questions such as the contribution of the local microenvironment to tumor-induced angiogenesis (Fukumura and Jain, 2008; Koehl et al., 2009), tumor-induced vascular permeability (Gavard et al., 2009), and to follow the bio-distribution of drugs or other molecules in the tumoral mass (Bhirde et al., 2009; Smith et al., 2008). Moreover, exogenous dyes can also be locally administered in different tissues to highlight various structural features. For example, sulforhodamine B has been injected into the muscles of mice to image elastin fibers, or to selectively stain the astrocytes in the brain (Ricard et al., 2007; Verant et al., 2008); dextran has been injected into the salivary ducts of rats to image dynamically the ductal system (Masedunskas and Weigert, 2008; Sramkova et al., 2009) (Fig. 2n and supplementary movie 3); curcumin and metoxy-04 has been injected to label amyloid plaques in tg2576 mice, a model for Alzheimer’s disease (Garcia-Alloza et al., 2007; Spires et al., 2005); and Ru(phen3)2+ has been used to image the level of oxygen in the liver (Paxian et al., 2004). Finally, significant information about the architecture of tissues in vivo has been generated through the use of transgenic models expressing fluorescent reporters under the control of specific tissue promoters. The field of neuroscience has pioneered this approach with the generation of mice with specific neuronal populations expressing GFP or YFP, and recently, using a combinatorial strategy, the so-called “brainbow” mice were generated in which neurons are labeled with different colors, providing an experimental tool to analyze the neuronal circuitry (Livet et al., 2007; Svoboda and Yasuda, 2006). The use of fluorescent transgenic models is now rapidly expanding to address biological questions in other fields. For example, mice have been generated to image the pancreatic beta cells (Nyman et al., 2008), the endothelium in various organs such as the kidney and the spleen, or in the presence of implanted tumors (Grayson et al., 2003; Hillen et al., 2008; Sutton et al., 2003), and many more are becoming available.

Imaging single cells in vivo

The ability to follow over time the fate of single cells within a given organ in live animals has contributed to major breakthroughs in fields such as cancer biology, immunology, microbiology, and recently in stem cell research. In cancer biology several experimental systems have been developed to track the motility of cancer cells within a tumor in vivo. For example, mammary tumors have been imaged in situ in mice models highlighting the role of the macrophages during the intravasation process (Wang et al., 2007; Wyckoff et al., 2007), and the migration of highly invasive melanomas have been tracked dynamically, leading to the determination of interesting correlations between the differentiation state of the cells and their migration ability (Pinner et al., 2009; Pinner and Sahai, 2008). Notably, a lot of effort has been also placed to perform long term imaging of tumors in the same animal in order to provide valuable information on the invasive process of slowly migrating tumors. This has become possible by the development of procedures to install optical windows in various areas of the body. Two successful examples are the dorsal skin chamber, installed in the back of immunocompromised mice, and the optical window installed in the mammary fat pad (Alexander et al., 2008; Gligorijevic et al., 2009; Kedrin et al., 2008). These techniques have been combined with the use of novel fluorescent proteins that can be either photo-activated or photo-switched (Gligorijevic et al., 2009; Lippincott-Schwartz and Patterson, 2008; Patterson and Lippincott-Schwartz, 2002), and have provided a plethora of novel information about the modality of migration of invasive cells in vivo and their relationship with the local microenvironment, particularly the vasculature and the lymphatic system.

Imaging the cells of the immune system in a live animal has revealed novel aspects of the dynamics of cellular immunity. Most of the experimental systems are based on transferring of fluorescently labeled isolated cells into recipient animals. From the first pioneering studies looking at the movements of B lymphocytes and T cells in the intact lymph-nodes (Bousso and Robey, 2003; Mempel et al., 2004; Miller et al., 2002; Stoll et al., 2002), a number of immunological questions have been addressed, spanning from T Cell activation (Hickman et al., 2008; Miller et al., 2004), the formation of mycobacterium-induced granulomas in the liver (Egen et al., 2008), T cell infiltration and elimination of solid tumors (Boissonnas et al., 2007; Breart et al., 2008), migration of dendritic cells (Roediger et al., 2008), and the extrafollicular activation of B cells (Qi et al., 2006). For a more complete overview of these processes we suggest more focused reviews (Cahalan and Parker, 2008; Germain et al., 2005; Hickman et al., 2009; Nitschke et al., 2008). Another field that has benefited from the development of IVM microscopy is the biology of pathogen infection. One of the first studies conducted to image the progression of bacterial infections in live tissue was performed in the kidney, where the proliferation of a GFP-expressing uro-pathogenic Escherichia Coli was studied (Mansson et al., 2007). Several other studies were performed using different approaches and strategies to image either later stages of the infectious process as in the case of the Staphylococcus aureus and the Borrelia burgdorferi (Laschke et al., 2005; Norman et al., 2008) or focusing on the site of the infection as in the case of the Leishmania major, where the interaction with CD4⁺ T cells was analyzed (Filipe-Santos et al., 2009).

Finally, IVM has been recently utilized in stem cell research to track individual hematopoietic stem cells over time in the calvarium bone marrow of living mice, thus opening the field to new and exciting discoveries (Lo Celso et al., 2009).

Imaging in vivo at the subcellular level: a new approach to cell biology

In a live animal, the major challenge in performing imaging at a subcellular level is represented by the motion artifacts due to the respiration and the heartbeat. The use of stereotactic devices that completely immobilize the head of the animal has been instrumental in achieving this high level of resolution in the brain. Indeed, the first structures that were resolved in vivo at a submicron resolution were the dendritic spines that can be imaged for over a month in transgenic mice expressing YFP or GFP in a subset of layer V pyramidal neurons (Mizrahi et al., 2004; Pan and Gan, 2008; Svoboda and Yasuda, 2006). In the last few years, both surgical procedures and novel devices ensuring the stabilization of the organ of interest have been developed. The first application of IVM for the imaging of fast moving intra-cellular organelles in organs other than the brain was pioneered by the Molitoris group who has studied the internalization of fluorescently labeled dextrans and folic acid in the externalized kidney, establishing also a methodology to extract quantitative information (Dunn et al., 2002; Sandoval et al., 2004; Sandoval and Molitoris, 2008). More recently, the endocytosis of different systemically injected fluorescent molecules and their trafficking through the endosomal and the lysosomal system has been imaged in the submandibular glands of live rats at a much higher resolution than previously reported (Masedunskas and Weigert, 2008). In this study, the use of a custom-made holder designed to stabilize the externalized glands has enabled to continuously follow the fate of the injected molecules from the internalization at the plasma membrane to and throughout the endosomal system for a long period of time (Fig. 3a and supplementary movie 4). Remarkably, intracellular events such as endosomal or lysosomal fusion were imaged at a resolution comparable to that achieved in cell cultures (Fig 3b. and supplementary movie 5). Using a similar approach, compensatory endocytosis, a process of membrane retrieval that is associated with agonist-induced secretory granule exocytosis was also imaged in the acini of the salivary glands of live rats (Sramkova et al., 2009). Another tool that has increased the ability to detect small organelles in living animals are the quantum-dots (Qdots), which are semiconductor nanocrystals encapsulated with biopolymers that exhibit a very bright and stable fluorescence, and can be coupled to any biological molecule (Li et al., 2007; Lidke et al., 2004; Michalet et al., 2005). Qdots internalization has been shown in dendritic cells in the lymph-nodes of live mice (Sen et al., 2008), whereas Qdots conjugated to nanotubes linked to EGF have been imaged during their internalization in head and neck tumor cells transplanted in the back of immunocompromised mice (Bhirde et al., 2009). Exocytosis is another subcellular process that has been imaged in the kidney, where the release of renin from the granular cells of the glomeruli was studied by using quinacrine to label a population of secretory granules (Toma et al., 2006). Other subcellular compartments, such as mitochondria, were imaged dynamically in live animals either in the liver during ischemia-reperfusion using Rhodamine123 (Zhong et al., 2008), or in the kidney using tetra-methyl rhodamine methyl ester (TMRM) (Hall et al., 2009) (Fig. 3b and supplementary movie 5).

Figure 3. Imaging subcellular structures in live animals.

a) Endocytosis of fluorescently labeled dextrans in the salivary glands of live rats. Anesthetized rats were injected with Hoechst to label the nuclei (blue), and imaged in time-lapse by using two-photon microscopy. After 2:30 minutes, a 500 kDa FITC-dextran was injected to label the vasculature (green) and after 6:00 min a 70 kDa Texas-red dextran was injected to image the endocytic process. Endocytic structures appeared right after the injection and they increased in number and in size over time (see supplementary movie 4). Excitation wavelength 820 nm. Scale bar - 20 μm. b) Imaging lysosomal fusion in a live animal. Rats were injected with Alexa 488 dextran (green) and Mitotracker (red) and after 4 hours the submandibular glands were imaged in time-lapse by using single photon confocal microscopy. Two lysosomal structures were caught during a fusion event (inset). Note the dynamics of both the lysosomes and the mitochondria in supplementary movie 5. Scale bar- 5 μm. c-e Gene transduction in live animal. The acinar cells of the salivary glands of live rats were transduced by using plasmid DNA encoding for different genes as described in Sramkova et al., 2009. c) Cell expressing TGN38-mCherry, which show the typical TGN ribbon-like structure (red, arrows), and the water channel Aquaporin5-YFP (arrowheads), localized both at the apical plasma membrane and in vesicular structures (arrowheads). d) Cell expressing Life Act-GFP to label F-actin (Riedl et al., 2008). Note the enrichment of F-actin at the apical pole of the plasma membrane. e) Cell expressing LifeAct-GFP (green) and TGN-mCherry (red, arrow). Texas red dextran was also injected systemically in the rat and appeared in a blood vessel (arrowheads, supplementary movie 6). Scale bars - 5 μm.

A major breakthrough that has allowed extending IVM to many other areas of cell biology is the ability to rapidly transduce fluorescently tagged genes in specific cell populations of the organ of interest. One of the first studies in this direction was aimed at studying the actin cytoskeleton and was performed in the endothelial cells of the kidney that were transduced with either GFP-actin or GFP-cofilin by using micro-puncture techniques and adenoviral vectors (Ashworth and Tanner, 2006; Tanner et al., 2005). The skeletal muscle in live mice is also an organ suitable to transduce genes and has been exploited to study various cellular functions. For example, the activity of the protease calpain was measured by using the fluorescence resonance energy transfer (FRET) signal generated by a calpain sensor (Stockholm et al., 2005); the calcium sensor Cameleon was transduced to image and measure the change in calcium levels in the sacroplasmic reticulum; whereas the cAMP sensor Epac was utilized to look at changes in cAMP levels during beta-adrenergic stimulation (Rudolf et al., 2006). Finally, the dynamics of the translocation of the glucose transporter GLUT4 to the sarcolemma in response to insulin was analyzed with respect to the metabolism of phosphoinositides (Lauritzen et al., 2006). Recently, using the salivary glands as a model organ and plasmid DNA, it was shown that genes can be selectively targeted and robustly expressed in the different subpopulations of cells forming the parenchyma of the salivary glands (Sramkova et al., 2009) (Fig. 3c–e and supplementary movie 6). By using high resolution TPM, different subcellular organelles were imaged dynamically such as clathrin-coated vesicles, the trans-Golgi network (Fig. 3c and 3e), and early endosomal compartments, with a resolution comparable to that achieved in cell culture by single photon confocal microscopy. Furthermore, the dynamics of the actin cytoskeleton (Fig. 3d, 3e and supplementary movie 6) and the distribution of the water channel Aquaporin 5 (Fig. 3c) at the plasma membrane was also analyzed, showing that this technology can be applied to address and study different cellular processes.

Finally, we would like to highlight the fact that when the cells of interest are located in the first 30–50 μm from the surface of the organ and the labeled fluorophores that are utilized are particularly bright, single photon confocal microscopy can also be utilized, enabling to perform imaging at a much higher resolution (compare fig 3a and 3b, and supplementary movies 4 and 5).

Outlook

In conclusion, IVM is a powerful approach that is now mature to be fully exploited in many different areas of cell biology to answer very specific questions in the proper physiological context. In terms of improving the imaging of subcellular structures, we envision that the future directions should focus on refining surgical procedures, developing tools to minimize the motion of the organs without compromising their physiology, routinely introducing miniaturized lenses and micro-endoscopes that can be properly oriented in the tissue, and developing novel fluorophores specifically designed for deep tissue imaging. Furthermore, the generation of more sophisticated molecular and genetic tools will increase the repertoire of intracellular processes that can be imaged and provide effective tools to dissect the molecular machinery regulating the process of interest. This is clearly the beginning of a new era of novel and exciting discoveries in the biomedical field.