Quantifying and visualising the nuances of cellular dynamics in vivo using intravital imaging

Intravital imaging is a powerful technology used to quantify and track dynamic changes in live cells and tissues within an intact environment. The ability to watch cell biology in real-time ‘as it happens’ has provided novel insight into tissue homeostasis, as well as disease initiation, progression and response to treatment. In this minireview, we highlight recent advances in the field of intravital microscopy, touching upon advances in awake versus anaesthesia-based approaches, as well as the integration of biosensors into intravital imaging. We also discuss current challenges that, in our opinion, need to be overcome to further advance the field of intravital imaging at the single-cell, subcellular and molecular resolution to reveal nuances of cell behaviour that can be targeted in complex disease settings.     

Stretching the timescale of intravital imaging in tumors

Since the time it was pioneered in 1992, intravital imaging of tumors at cellular resolution has offered us the extremely important opportunity of “seeing biology”. However, until now, most studies were monitoring tumor cell behavior in the same animal over short times, requiring the combining of acquired data into a hypothesis via statistical analysis. In the last several months, different groups have independently developed techniques to extend the time scale of intravital imaging to several days. This improvement allows one to address the connection between tumor cell behavior and the microenvironment which surrounds them. We can now assess dynamics of the cell-cell interactions in tumors, analyze tumor cell fate and changes in the tumor extracellular matrix which accompany tumor progression.     

Functional studies of the kidney of living animals using multicolor two-photon microscopy

Optical microscopy, when applied to livinganimals, provides a powerful means of studying cell biology in the mostphysiologically relevant setting. The ability of two-photon microscopyto collect optical sections deep into biological tissues has opened upthe field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way inwhich two-photon microscopy can be applied to intravital studies ofkidney physiology. Because the kidney is easily externalized withoutcompromising its function, microscopy can be used to evaluate variousaspects of renal function in vivo. These include cell vitality andapoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes andsimultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of imagescontaining uncharacterized autofluorescence. The studies described heredemonstrate the way in which two-photon microscopy can provide a levelof resolution previously unattainable in intravital microscopy,enabling kinetic analyses and physiological studies of the organs ofliving animals with subcellular resolution.

     

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

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 criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.     

Multimodal optical imaging

The recent resurgence of interest in the use of intravital microscopy in lung research is a manifestation of extraordinary progress in visual imaging and optical microscopy. This review evaluates the tools and instrumentation available for a number of imaging modalities, with particular attention to recent technological advances, and addresses recent progress in use of optical imaging techniques in basic pulmonary research.1 Limitations of existing methods and anticipated future developments are also identified. Although there have also been major advances made in the use of magnetic resonance imaging, positron emission tomography, and X-ray and computed tomography to image intact lungs and while these technologies have been instrumental in advancing the diagnosis and treatment of patients, the purpose of this review is to outline developing optical methods that can be evaluated for use in basic research in pulmonary biology.     

Single Cell Analysis of Drug Distribution by Intravital Imaging

Recent advances in the field of intravital imaging have for the first time allowed us to conduct pharmacokinetic and pharmacodynamic studies at the single cell level in live animal models. Due to these advances, there is now a critical need for automated analysis of pharmacokinetic data. To address this, we began by surveying common thresholding methods to determine which would be most appropriate for identifying fluorescently labeled drugs in intravital imaging. We then developed a segmentation algorithm that allows semi-automated analysis of pharmacokinetic data at the single cell level. Ultimately, we were able to show that drug concentrations can indeed be extracted from serial intravital imaging in an automated fashion. We believe that the application of this algorithm will be of value to the analysis of intravital microscopy imaging particularly when imaging drug action at the single cell level.     

Three‐dimensional intravital imaging in bone research

Intravital imaging has emerged as a novel and efficient tool for visualization of in situ dynamics of cellular behaviors and cell‐microenvironment interactions in live animals, based on desirable microscopy techniques featuring high resolutions, deep imaging and low phototoxicity. Intravital imaging, especially based on multi‐photon microscopy, has been used in bone research for dynamics visualization of a variety of physiological and pathological events at the cellular level, such as bone remodeling, hematopoiesis, immune responses and cancer development, thus, providing guidance for elucidating novel cellular mechanisms in bone biology as well as guidance for new therapies. This review is aimed at interpreting development and advantages of intravital imaging in bone research, and related representative discoveries concerning bone matrices, vessels, and various cells types involved in bone physiologies and pathologies. Finally, current limitations, further refinement, and extended application of intravital imaging in bone research are concluded.      

A novel method to analyze leukocyte rolling behavior <Emphasis Type="Italic">in vivo</Emphasis>

Leukocyte endothelial cell interaction is a fundamentally important process in many disease states. Current methods to analyze such interactions include the parallel-plate flow chamber and intravital microscopy. Here, we present an improvement of the traditional intravital microscopy that allows leukocyte-endothelial cell interaction to be studied from the time the leukocyte makes its initial contact with the endothelium until it adheres to or detaches from the endothelium. The leukocyte is tracked throughout the venular tree with the aid of a motorized stage and the rolling and adhesive behavior is measured off-line. Because this method can involve human error, methods to automate the tracking procedure have been developed. This novel tracking method allows for a more detailed examination of leukocyte-endothelial cell interactions. Published: August 27, 2004.     

Intravital imaging of fluorescent markers and FRET probes by DNA tattooing

Background  

Advances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals. However, the generation of transgenic mice is a lengthy process and intravital imaging requires specialized knowledge and equipment. Here, we report a rapid and undemanding intravital imaging method using generally available equipment.     

Tissue distribution of chelants during life

Injection of dithizone and quinoline compounds to animals leads to the intravital zinc chelate granule formation detectable on frozen tissue sections. The intensity of intravital histochemical reaction depends on be complexing ability, the dose, the ways of the agent injection, and also on the presence of other ligands lowering the agent's coordination volume use.     

Viral nanoparticles as tools for intravital vascular imaging

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.     

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.     

The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation

Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation.     

Treadmill Exercise Induces Neutrophil Recruitment into Muscle Tissue in a Reactive Oxygen Species-Dependent Manner. An Intravital Microscopy Study

Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91^phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process.     

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.     

3D and 4D imaging of immune cells in vitro and in vivo

Analyzing the dynamics of cellular immune responses, although performed for decades in immunologic research, has seen an enormous increase in the number of studies using this approach since the development of intravital 2-photon microscopy. Meanwhile, new insights into the dynamics of cellular immunity are being published on a daily basis. This review gives a short overview of the currently most widely used techniques, both on the microscopy side as well as on the experimental part. Difficulties and promises will be discussed. Finally, a personal selection of the most interesting findings of the first 6 years of intravital 2-photon microscopy for immunological questions will be given. The overall aim is to get the reader interested into this fascinating way of investigating the immune response by means of “dynamic histology”. This already has and will continue to broaden our view on how immune cells work in real life.     

A novel model for the investigation of orthotopically growing primary and secondary bone tumours using intravital microscopy

Here is reported the development of an experimental model using intravital microscopy as a tool to orthotopically investigate malignant bone tumours. Although up to 85% of the most frequently occurring malignant solid tumours, such as lung and prostate carcinomas, metastasize into the bone, and despite the knowledge that a tumour's course may be altered by its surrounding tissue, there is no adequate experimental model available enabling the investigation of orthotopically grown bone tumours in vivo. Intravital microscopy is an internationally accepted experimental method, used in various acute and chronic animal models, that enables qualitative and quantitative analysis of the angiogenesis, microcirculation, growth behaviour, etc. of various benign and malignant tissues. Non-invasive investigations of up to several weeks are possible. Additionally, tissue samples can be taken after termination of the in vivo experiments for further ex vivo investigation (histology, immunohistochemistry, molecular biology, etc.), elucidating the mechanisms that underlie the in vivo observations. Severe combined immunodeficient mice were fitted with a cranial window preparation where the calvaria served as the site for orthotopic implantation of the solid human tumours Saos-2 osteosarcoma (primary) and A 549 lung carcinoma and PC-3 prostate carcinoma (secondary). In all preparations, the take rate was 100%. Histological assessment confirmed the data obtained in vivo, showing typical tumour growth with infiltration of the surrounding osseous and soft tissues. This novel model serves as a valuable tool in understanding the biology of primary and secondary bone tumours in physiological and pathophysiological situations, with implications for the most areas of tumour therapy such as chemotherapy, radiation and antiangiogenesis.     

SOURCES OF CONTAMINATION IN OUTBREAKS OF FOOD POISONING CAUSED BY SALMONELLA BACTERIA FROM MEAT IN THE NETHERLANDS

SUMMARY: In outbreaks of food poisoning from meat, caused by Salmonella organisms, intravital infection and post-mortem contamination of the meat must be distinguished. Man and different species of animals suffering from Salmonella infection may be a source of infection or contamination. In the Netherlands this is especially the case with carriers of Salm. dublin in adult cattle. In the National Salmonella Centre, 41 Salmonella types were isolated from man and animals in the years 1946–1952. In 1949–1952, eighteen outbreaks of food poisoning due to meat products and only one due to fresh meat were examined. In 4 cases an intravital infection of the meat was established; in the remaining cases the source of contamination remained unknown. The causative types were: Salm. dublin, Salm. typhi-murium, Salm. paratyphi B, Salm. oregon, Salm. newport and Salm. bovis-morbificans .     

Going live: A comparative analysis of the suitability of the RFP derivatives RedStar,mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.