Table of Contents

Volume 1, Issue 1

  July/August/September 2012

  Pages 1 - 85

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 Issue Catalogue (MARC XML)

EDITOR'S CORNER

Open Access Article

Welcome to IntraVital

Roberto Weigert
Page 1
http://dx.doi.org/10.4161/intv.21695
Full Text | PDF




REVIEWS

Open Access Article

Intravital imaging of cell signaling in mice

Laila Ritsma, Bas Ponsioen and Jacco van Rheenen
Pages 2 - 10
http://dx.doi.org/10.4161/intv.20802
Abstract | Full Text | PDF

Cell signaling is mostly studied in in vitro 2D-cell culture models that lack the complex in vivo environment provided by neighboring cells, soluble secreted factors and non-cellular matrix components. Given that many environmental factors control cell signaling, it comes as no surprise that in vitro observations often poorly correlate with in vivo observations. Recent developments in intravital imaging techniques have made it possible to visualize and study cell signaling in individual cells within living animals. Here, we review intravital imaging techniques based on fluorescence microscopy and give examples of how these techniques are being used to study cell signaling.

Open Access Article

Hybrid multiphoton multimodal tomography of in vivo human skin

Karsten König
Pages 11 - 26
http://dx.doi.org/10.4161/intv.21938
Abstract | Full Text | PDF

Clinical multiphoton tomography based on femtosecond near infrared laser pulses for in vivo high-resolution skin imaging has been employed to thousands of volunteers and patients. Two-photon cellular autofluorescence and second harmonic generation of collagen can be detected with single-photon sensitivity and submicron spatial resolution. Also in vivo clinical CARS has been realized to image intratissue lipids and water. Novel developments focus on multimodal hybrid imaging to generate optical tissue biopsies with subcellular resolution, deep-tissue information, and chemical fingerprints. Wide-field imaging tools such as dermoscopes, optical coherence tomographs as well as ultrasound and photoacoustic devices can be integrated. The hybrid tomographs have the potential to trace cosmetics and pharmaceutical components such as sunscreen nanoparticles and anti-aging products in humans. Skin cancer such as malignant melanoma and basal cell carcinoma as well as dermatitis can be detected at an early stage and the efficiency of the treatment can be monitored. These novel hybrid multimodal multiphoton tomographs may become important biopsy-free and label-free imaging tools in personalized medicine, pharmacy, biotechnology as well as cosmetic research.




METHODS ARTICLE

Open Access Article

DISC: A guide to in vivo cytometry

Hélène Moreau and Philippe Bousso
Pages 27 - 31
http://dx.doi.org/10.4161/intv.21896
Abstract | Full Text | PDF

Dynamic in situ cytometry (DISC) is a new approach that combines advantages of both intravital imaging and flow cytometry analysis. The DISC methodology provides the opportunity to link cell behavior and phenotype in vivo and hence to perform cytometry in vivo and in real-time. In addition it enables you to readily display and analyze multiparametric imaging data. Through concrete examples, we present a guide to perform DISC experiments with the help of the custom designed DISCit software.




RESEARCH PAPERS

Open Access Article

Intravital third harmonic generation microscopy of collective melanoma cell invasion: Principles of interface guidance and microvesicle dynamics

Bettina Weigelin, Gert-Jan Bakker and Peter Friedl
Pages 32 - 43
http://dx.doi.org/10.4161/intv.21223
Abstract | Full Text | PDF

Cancer cell invasion is an adaptive process based on cell-intrinsic properties to migrate individually or collectively, and their adaptation to encountered tissue structure acting as barrier or providing guidance. Whereas molecular and physical mechanisms of cancer invasion are well-studied in 3D in vitro models, their topographic relevance, classification and validation toward interstitial tissue organization in vivo remain incomplete. Using combined intravital third and second harmonic generation (THG, SHG), and three-channel fluorescence microscopy in live tumors, we here map B16F10 melanoma invasion into the dermis with up to 600 µm penetration depth and reconstruct both invasion mode and tissue tracks to establish invasion routes and outcome. B16F10 cells preferentially develop adaptive invasion patterns along preformed tracks of complex, multi-interface topography, combining single-cell and collective migration modes, without immediate anatomic tissue remodeling or destruction. The data suggest that the dimensionality (1D, 2D, 3D) of tissue interfaces determines the microanatomy exploited by invading tumor cells, emphasizing non-destructive migration along microchannels coupled to contact guidance as key invasion mechanisms. THG imaging further detected the presence and interstitial dynamics of tumor-associated microparticles with submicron resolution, revealing tumor-imposed conditioning of the microenvironment. These topographic findings establish combined THG, SHG and fluorescence microscopy in intravital tumor biology and provide a template for rational in vitro model development and context-dependent molecular classification of invasion modes and routes.

Open Access Article

Quantitative intravital microscopy of hepatic transport

Clifford M. Babbey, Jennifer C. Ryan, Erin M. Gill, Marwan S. Ghabril, Courtney R. Burch, April Paulman and Kenneth W. Dunn
Pages 44 - 53
http://dx.doi.org/10.4161/intv.21296
Abstract | Full Text | PDF

Intravital microscopy has a long history of application to studies of the liver. Here we demonstrate how intravital multiphoton microscopy may be combined with digital image analysis to quantitatively evaluate the effects of drugs on hepatocyte transport in vivo.

Open Access Article

Intravital multiphoton imaging of rhodamine 123 in the rat liver after intravenous dosing

Xin Liu, Camilla A. Thorling, Lu Jin and Michael S. Roberts
Pages 54 - 59
http://dx.doi.org/10.4161/intv.21450
Abstract | Full Text | PDF

Intravital imaging with multiphoton microscopy was used to investigate the hepatic disposition of rhodamine 123 (RH123) in the exposed liver of anesthetized rats after intravenous dosing. The role played by the biliary canalicular transporter P-glycoprotein (P-gp) on the disposition of RH123 was explored by administering a P-gp inhibitor, cyclosporine A prior to RH123 administration. The fluorescence intensity of RH123 was defined by multiphoton microscopy using a femtosecond laser excitation wavelength of 900 nm whereas the autofluorescence at an excitation wavelength of 740 nm was used to define the morphology of the liver acini. Intravital imaging showed that RH123 was rapidly taken up from the sinusoids into hepatocytes but slowly eliminated from the cells (half-life 2.89 ± 1.37 h). The presence of cyclosporine A did not affect the uptake of RH123 but markedly increased the fluorescence intensity of RH123 in the liver and was associated with a slower elimination of RH123 from the liver (half-life 11.5 ± 4.24 h). In conclusion, the spatial disposition of RH123 in the rat liver and the effects of a transporter inhibitor on its disposition have been monitored over time using intravital imaging.

Open Access Article

Video-rate resonant scanning multiphoton microscopy: An emerging technique for intravital imaging of the tumor microenvironment

Nathaniel D. Kirkpatrick, Euiheon Chung, Daniel C. Cook, Xiaoxing Han, Gabriel Gruionu, Shan Liao, Lance L. Munn, Timothy P. Padera, Dai Fukumura and Rakesh K. Jain
Pages 60 - 68
http://dx.doi.org/10.4161/intv.21557
Abstract | Full Text | PDF

The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates—only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment.

Open Access Article

Nondestructive, serial in vivo imaging of a tissue-flap using a tissue adhesion barrier: Applications for IVM imaging in the mammary fat pad and lymph node

Masakatsu Kotsuma, Natesh Parashurama, Bryan R. Smith, Jonathan Wo, Ken Ito and Sanjiv Sam Gambhir
Pages 69 - 76
http://dx.doi.org/10.4161/intv.21769
Abstract | Full Text | PDF

Intravital Microscopy (IVM) is a powerful tool for imaging of dynamic events in living subjects and benefits from flexibility of various tissue preparation techniques. For example, a “tissue flap” (TF) approach initially affords high spatial resolution and physiological imaging with minimal tissue preparation, but serial TF imaging greatly increases the effects of pathological inflammation, resulting in postoperative adhesions and tissue injury. We took a materials science approach by implanting a commercially available, thin film, biopolymer tissue adhesion barrier (TAB) beneath the TF during serial imaging of the normal and developing breast in transgenic fluorescent mice, and with a fluorescent orthotopic mouse lymphoma model. We applied the TAB post-operatively beneath the TF to isolate the TF from the underlying peritoneum. When re-imaging the TF every 3–4 d, with a new TAB placed each time, we observed reduced hemorrhage, fibrous connective tissue and soft tissue damage. The presence of the TAB enabled sequential imaging of orthopically located EGFP⁺-lymphoma cells and associated vasculature at short intervals. In particular, it enabled visualization and tracking of the same individual fluorescent branches of the mammary gland in both adult and developing mice over time; likewise, it enabled tracking of lymph nodes. We conclude that this simple method affords great potential to serially track rare, microscopic, tissue-wide events in parenchyma or stroma. Potential applications include tracking proliferation and motility of transplanted cancer cells, stem cell-driven tissue growth, and tumor cell-stromal cell interactions at high spatial and temporal resolution.

Open Access Article

Reconstitution of in vivo macrophage-tumor cell pairing and streaming motility on one-dimensional micro-patterned substrates

Ved P. Sharma, Brian T. Beaty, Antonia Patsialou, Huiping Liu, Michael Clarke, Dianne Cox, John S Condeelis and Robert J. Eddy
Pages 77 - 85
http://dx.doi.org/10.4161/intv.22054
Abstract | Full Text | PDF

In mammary tumors, intravital imaging techniques have uncovered an essential role for macrophages during tumor cell invasion and metastasis mediated by an epidermal growth factor (EGF) / colony stimulating factor-1 (CSF-1) paracrine loop. It was previously demonstrated that mammary tumors in mice derived from rat carcinoma cells (MTLn3) exhibited high velocity migration on extracellular matrix (ECM) fibers. These cells form paracrine loop-dependent linear assemblies of alternating host macrophages and tumor cells known as “streams.” Here, we confirm by intravital imaging that similar streams form in close association with ECM fibers in a highly metastatic patient-derived orthotopic mammary tumor (TN1). To understand the in vivo cell motility behaviors observed in streams, an in vitro model of fibrillar tumor ECM utilizing adhesive 1D micropatterned substrates was developed. MTLn3 cells on 1D fibronectin or type I collagen substrates migrated with higher velocity than on 2D substrates and displayed enhanced lamellipodial protrusion and increased motility upon local interaction and pairing with bone marrow-derived macrophages (BMMs). Inhibitors of EGF or CSF-1 signaling disrupted this interaction and reduced tumor cell velocity and protrusion, validating the requirement for an intact paracrine loop. Both TN1 and MTLn3 cells in the presence of BMMs were capable of co-assembling into linear arrays of alternating tumor cells and BMMs that resembled streams in vivo, suggesting the stream assembly is cell autonomous and can be reconstituted on 1D substrates. Our results validate the use of 1D micropatterned substrates as a simple and defined approach to study fibrillar ECM-dependent cell pairing, migration and relay chemotaxis as a complementary tool to intravital imaging.