Intravital microscopy: Imaging host–parasite interactions in lymphoid organs

Intravital microscopy allows imaging of biological phenomena within living animals, including host–parasite interactions. This has advanced our understanding of both, the function of lymphoid organs during parasitic infections, and the effect of parasites on such organs to allow their survival. In parasitic research, recent developments in this technique have been crucial for the direct study of host–parasite interactions within organs at depths, speeds and resolution previously difficult to achieve. Lymphoid organs have gained more attention as we start to understand their function during parasitic infections and the effect of parasites on them. In this review, we summarise technical and biological findings achieved by intravital microscopy with respect to the interaction of various parasites with host lymphoid organs, namely the bone marrow, thymus, lymph nodes, spleen and the mucosa‐associated lymphoid tissue, and present a view into possible future applications.     

Photonic detection of bacterial pathogens in living hosts

The study of pathogenic is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurium that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bio-luminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that the real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo .     

Quantitative Analysis of Monocyte Subpopulations in Murine Atherosclerotic Plaques by Multiphoton Microscopy

The progressive accumulation of monocyte-derived cells in the atherosclerotic plaque is a hallmark of atherosclerosis. However, it is now appreciated that monocytes represent a heterogeneous circulating population of cells that differ in functionality. New approaches are needed to investigate the role of monocyte subpopulations in atherosclerosis since a detailed understanding of their differential mobilization, recruitment, survival and emigration during atherogenesis is of particular importance for development of successful therapeutic strategies. We present a novel methodology for the in vivo examination of monocyte subpopulations in mouse models of atherosclerosis. This approach combines cellular labeling by fluorescent beads with multiphoton microscopy to visualize and monitor monocyte subpopulations in living animals. First, we show that multiphoton microscopy is an accurate and timesaving technique to analyze monocyte subpopulation trafficking and localization in plaques in excised tissues. Next, we demonstrate that multiphoton microscopy can be used to monitor monocyte subpopulation trafficking in atherosclerotic plaques in living animals. This novel methodology should have broad applications and facilitate new insights into the pathogenesis of atherosclerosis and other inflammatory diseases.     

Nonlinear vibrational microscopy applied to lipid biology

Optical microscopy is an indispensable tool that is driving progress in cell biology. It still is the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Most prominently, fluorescence microscopy based on dye-labeling or protein fusions with fluorescent tags is a highly sensitive and specific method of visualizing biomolecules within sub-cellular structures. It is however severely limited by labeling artifacts, photo-bleaching and cytotoxicity of the labels. Coherent Raman Scattering (CRS) has emerged in the last decade as a new multiphoton microscopy technique suited for imaging unlabeled living cells in real time with high three-dimensional spatial resolution and chemical specificity. This technique has proven to be particularly successful in imaging unstained lipids from artificial membrane model systems, to living cells and tissues to whole organisms. In this article, we will review the experimental implementations of CRS microscopy and their application to imaging lipids. We will cover the theoretical background of linear and non-linear vibrational micro-spectroscopy necessary for the understanding of CRS microscopy. The different experimental implementations of CRS will be compared in terms of sensitivity limits and excitation and detection methods. Finally, we will provide an overview of the applications of CRS microscopy to lipid biology.     

Two-photon microscopy: shedding light on the chemistry of vision

Two-photon microscopy (TPM) has come to occupy a prominent place in modern biological research with its ability to resolve the three-dimensional distribution of molecules deep inside living tissue. TPM can employ two different types of signals, fluorescence and second harmonic generation, to image biological structures with subcellular resolution. Two-photon excited fluorescence imaging is a powerful technique with which to monitor the dynamic behavior of the chemical components of tissues, whereas second harmonic imaging provides novel ways to study their spatial organization. Using TPM, great strides have been made toward understanding the metabolism, structure, signal transduction, and signal transmission in the eye. These include the characterization of the spatial distribution, transport, and metabolism of the endogenous retinoids, molecules essential for the detection of light, as well as the elucidation of the architecture of the living cornea. In this review, we present and discuss the current applications of TPM for the chemical and structural imaging of the eye. In addition, we address what we see as the future potential of TPM for eye research. This relatively new method of microscopy has been the subject of numerous technical improvements in terms of the optics and indicators used, improvements that should lead to more detailed biochemical characterizations of the eyes of live animals and even to imaging of the human eye in vivo.     

Deep tissue two-photon microscopy

With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional-including confocal-fluorescence microscopy. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.     

Are pathogenic bacteria just looking for food? Metabolism and microbial pathogenesis

It is interesting to speculate that the evolutionary drive for microbes to develop pathogenic characteristics was to access the nutrient resources that animals provided. Animal environments that pathogens colonize have likely driven the evolution of new bacterial characteristics to maximize these new nutritional opportunities. This review focuses on genomic and functional aspects of pathogen metabolism that allow efficient utilization of nutrient resources provided by animals. Similar to genes encoding specific virulence traits, genes encoding metabolic functions have been horizontally acquired by pathogens to provide a selective advantage in host tissues. Selective advantage in host tissues can also be gained by loss of function mutations that alter metabolic capabilities. Greater understanding of bacterial metabolism within host tissues should be important for increased understanding of host-pathogen interactions and the development of future therapeutic strategies.     

Simultaneous imaging of GFP,CFP and collagen in tumors <Emphasis Type="Italic">in vivo</Emphasis>using multiphoton microscopy

Background  

The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy.     

The immunology of myiasis: parasite survival and host defense strategies

Infestations by dipterous larvae that feed on dead or living vertebrate tissues for a variable period are known as myiases; these infestations reduce host physiological functions, destroy host tissues and cause significant economic losses to livestock worldwide. Recent advances in understanding the specific and nonspecific immune responses of hosts to infestation by myiasis-causing larvae and the immunological strategies evolved by larvae against the host are reviewed here. The practical implications of immunological knowledge for diagnostic and vaccination strategies are also discussed, with a view to developing environmentally sustainable control methods to be used as an alternative to chemical treatments.     

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.     

Progression of bacterial infections studied in real time--novel perspectives provided by multiphoton microscopy

The holy grail of infection biology is to study a pathogen within its natural infectious environment, the living host. Advances in in vivo imaging techniques have begun to introduce the possibility to visualize, in real time, infection progression within a living model. In this review we detail the current advancements and knowledge in multiphoton microscopy and how it can be related to the field of microbial infections. This technology is a new and very valuable tool for in vivo imaging, and using this technique it is possible to begin to study various microbes within their natural infectious environment - the living host.     

Dynamics of bacterial growth and distribution within the liver during Salmonella infection

Salmonella enterica causes severe systemic diseases in humans and animals and grows intracellularly within discrete tissue foci that become pathological lesions. Because of its lifestyle Salmonella is a superb model for studying the in vivo dynamics of bacterial distribution. Using multicolour fluorescence microscopy in the mouse typhoid model we have studied the interaction between different bacterial populations in the same host as well as the dynamic evolution of foci of infection in relation to bacterial growth and localization. We showed that the growth of Salmonella in the liver results in the spread of the microorganisms to new foci of infection rather than simply in the expansion of the initial ones. These foci were associated with independently segregating bacterial populations and with low numbers of bacteria in each infected phagocyte. Using fast-growing and slow-growing bacteria we also showed that the increase in the number of infected phagocytes parallels the net rate of bacterial growth of the microorganisms in the tissues. These findings suggest a novel mechanism underlying growth of salmonellae in vivo with important consequences for understanding mechanisms of resistance and immunity.     

Preservation of immunoreactivity and fine structure of adult C. elegans tissues using high-pressure freezing.

The location of a protein labeled by immunogold techniques can be resolved under an electron beam to within nanometers of its epitope, a resolution that makes immunoelectron microscopy a valuable tool for studies of cell biology. However, tissues in the nematode Caenorhabditis elegans are difficult to preserve for immunoelectron microscopic studies. The animal's cuticle slows the diffusion of solutions into the animal and thus makes it difficult to preserve both immunoreactivity and cell morphology. Here we describe a protocol that circumvents these problems. Specifically, we instantly immobilized tissue in vitreous ice by freezing living adult animals under high pressure. Frozen specimens were then chemically fixed, dehydrated, and embedded at low temperatures. As a result, chemical diffusion across the cuticle could occur over an extended period without morphological deterioration. We show that this method is capable of preserving both cell morphology, including fine structures, and immunoreactivity. Therefore, it provides a means to characterize the localization of endogenous proteins and exogenous proteins, such as the green fluorescent protein (GFP), with respect to subcellular compartments in C. elegans tissues by using postembedding immunogold labeling.     

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.     

Epoxide hydrolases: their roles and interactions with lipid metabolism

The epoxide hydrolases (EHs) are enzymes present in all living organisms, which transform epoxide containing lipids by the addition of water. In plants and animals, many of these lipid substrates have potent biologically activities, such as host defenses, control of development, regulation of inflammation and blood pressure. Thus the EHs have important and diverse biological roles with profound effects on the physiological state of the host organisms. Currently, seven distinct epoxide hydrolase sub-types are recognized in higher organisms. These include the plant soluble EHs, the mammalian soluble epoxide hydrolase, the hepoxilin hydrolase, leukotriene A4 hydrolase, the microsomal epoxide hydrolase, and the insect juvenile hormone epoxide hydrolase. While our understanding of these enzymes has progressed at different rates, here we discuss the current state of knowledge for each of these enzymes, along with a distillation of our current understanding of their endogenous roles. By reviewing the entire enzyme class together, both commonalities and discrepancies in our understanding are highlighted and important directions for future research pertaining to these enzymes are indicated.     

Tyramide Signal Amplification Method in Multiple-Label Immunofluorescence Confocal Microscopy

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.     

Characterization of schistosome tegumental alkaline phosphatase (SmAP)

Schistosomes are parasitic platyhelminths that currently infect over 200 million people globally. The parasites can live for years in a putatively hostile environment - the blood of vertebrates. We have hypothesized that the unusual schistosome tegument (outer-covering) plays a role in protecting parasites in the blood; by impeding host immunological signaling pathways we suggest that tegumental molecules help create an immunologically privileged environment for schistosomes. In this work, we clone and characterize a schistosome alkaline phosphatase (SmAP), a predicted ～60 kDa glycoprotein that has high sequence conservation with members of the alkaline phosphatase protein family. The SmAP gene is most highly expressed in intravascular parasite life stages. Using immunofluorescence and immuno-electron microscopy, we confirm that SmAP is expressed at the host/parasite interface and in internal tissues. The ability of living parasites to cleave exogenous adenosine monophosphate (AMP) and generate adenosine is very largely abolished when SmAP gene expression is suppressed following RNAi treatment targeting the gene. These results lend support to the hypothesis that schistosome surface enzymes such as SmAP could dampen host immune responses against the parasites by generating immunosuppressants such as adenosine to promote their survival. This notion does not rule out other potential functions for the adenosine generated e.g. in parasite nutrition.     

Single-embryo metabolomics and systematic prediction of developmental stage in zebrafish

Metabolites, the end products of gene expression in living organisms, are tightly correlated with an organism's development and growth. Thus, metabolic profiling is a potentially important tool for understanding the events that have occurred in cells, tissues, and individual organisms. Here, we present a method for predicting the developmental stage of zebrafish embryos using novel metabolomic non-target fingerprints of "single-embryos". With this method, we observed the rate of development at different temperatures. Our results suggest that this method allows us to analyse the condition, or distinguish the genotype, of single-embryos before expression of their ultimate phenotype.     

What Ticks Do Under Your Skin: Two-Photon Intravital Imaging of Ixodes Scapularis Feeding in the Presence of the Lyme Disease Spirochete

Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding.