Multicolor Fluorescent Intravital Live Microscopy (FILM) for Surgical Tumor Resection in a Mouse Xenograft Model

Background

Complete surgical resection of neoplasia remains one of the most efficient tumor therapies. However, malignant cell clusters are often left behind during surgery due to the inability to visualize and differentiate them against host tissue. Here we establish the feasibility of multicolor fluorescent intravital live microscopy (FILM) where multiple cellular and/or unique tissue compartments are stained simultaneously and imaged in real time.

Methodology/Principal Findings

Theoretical simulations of imaging probe localization were carried out for three agents with specificity for cancer cells, stromal host response, or vascular perfusion. This transport analysis gave insight into the probe pharmacokinetics and tissue distribution, facilitating the experimental design and allowing predictions to be made about the localization of the probes in other animal models and in the clinic. The imaging probes were administered systemically at optimal time points based on the simulations, and the multicolor FILM images obtained in vivo were then compared to conventional pathological sections. Our data show the feasibility of real time in vivo pathology at cellular resolution and molecular specificity with excellent agreement between intravital and traditional in vitro immunohistochemistry.

Conclusions/Significance

Multicolor FILM is an accurate method for identifying malignant tissue and cells in vivo. The imaging probes distributed in a manner similar to predictions based on transport principles, and these models can be used to design future probes and experiments. FILM can provide critical real time feedback and should be a useful tool for more effective and complete cancer resection.     

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.     

Targeted nanoparticles for imaging incipient pancreatic ductal adenocarcinoma

Background

Pancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, typically presenting with metastasis at the time of diagnosis and exhibiting profound resistance to existing therapies. The development of molecular markers and imaging probes for incipient PDAC would enable earlier detection and guide the development of interventive therapies. Here we sought to identify novel molecular markers and to test their potential as targeted imaging agents.

Methods and Findings

Here, a phage display approach was used in a mouse model of PDAC to screen for peptides that specifically bind to cell surface antigens on PDAC cells. These screens yielded a motif that distinguishes PDAC cells from normal pancreatic duct cells in vitro, which, upon proteomics analysis, identified plectin-1 as a novel biomarker of PDAC. To assess their utility for in vivo imaging, the plectin-1 targeted peptides (PTP) were conjugated to magnetofluorescent nanoparticles. In conjunction with intravital confocal microscopy and MRI, these nanoparticles enabled detection of small PDAC and precursor lesions in engineered mouse models.

Conclusions

Our approach exploited a well-defined model of PDAC, enabling rapid identification and validation of PTP. The developed specific imaging probe, along with the discovery of plectin-1 as a novel biomarker, may have clinical utility in the diagnosis and management of PDAC in humans.     

Use of Echocardiography Reveals Reestablishment of Ventricular Pumping Efficiency and Partial Ventricular Wall Motion Recovery upon Ventricular Cryoinjury in the Zebrafish

Aims

While zebrafish embryos are amenable to in vivo imaging, allowing the study of morphogenetic processes during development, intravital imaging of adults is hampered by their small size and loss of transparency. The use of adult zebrafish as a vertebrate model of cardiac disease and regeneration is increasing at high speed. It is therefore of great importance to establish appropriate and robust methods to measure cardiac function parameters.

Methods and Results

Here we describe the use of 2D-echocardiography to study the fractional volume shortening and segmental wall motion of the ventricle. Our data show that 2D-echocardiography can be used to evaluate cardiac injury and also to study recovery of cardiac function. Interestingly, our results show that while global systolic function recovered following cardiac cryoinjury, ventricular wall motion was only partially restored.

Conclusion

Cryoinjury leads to long-lasting impairment of cardiac contraction, partially mimicking the consequences of myocardial infarction in humans. Functional assessment of heart regeneration by echocardiography allows a deeper understanding of the mechanisms of cardiac regeneration and has the advantage of being easily transferable to other cardiovascular zebrafish disease models.     

Intravital imaging of neutrophil recruitment in intestinal ischemia-reperfusion injury

Background

Neutrophils are known to be key players in innate immunity. Activated neutrophils induce local inflammation, which results in pathophysiologic changes during intestinal ischemia-reperfusion injury (IRI). However, most studies have been based on static assessments, and few have examined real-time intravital neutrophil recruitment. We herein report a method for imaging and evaluating dynamic changes in the neutrophil recruitment in intestinal IRI using two-photon laser scanning microscopy (TPLSM).

Life cycle assessment of electricity transmission and distribution—part 2: transformers and substation equipment

Purpose  

The purpose of this paper is to characterize the environmental impacts of equipment used in power transmission and distribution. This study is divided in two parts, each addressing different main components of the electrical grid system. This part is concerned with the impacts of transformers and substation equipment while in part 1 a similar analysis is presented for power lines and cables.     

Dynamic <Emphasis Type="Italic">in vivo</Emphasis> imaging and cell tracking using a histone fluorescent protein fusion in mice

Background  

Advances in optical imaging modalities and the continued evolution of genetically-encoded fluorescent proteins are coming together to facilitate the study of cell behavior at high resolution in living organisms. As a result, imaging using autofluorescent protein reporters is gaining popularity in mouse transgenic and targeted mutagenesis applications.     

Motmot, an open-source toolkit for realtime video acquisition and analysis

Background  

Video cameras sense passively from a distance, offer a rich information stream, and provide intuitively meaningful raw data. Camera-based imaging has thus proven critical for many advances in neuroscience and biology, with applications ranging from cellular imaging of fluorescent dyes to tracking of whole-animal behavior at ecologically relevant spatial scales.     

Data-guide for brain deformation in surgery: comparison of linear and nonlinear models

Background  

Pre-operative imaging devices generate high-resolution images but intra-operative imaging devices generate low-resolution images. To use high-resolution pre-operative images during surgery, they must be deformed to reflect intra-operative geometry of brain.     

Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee

Background  

Optical imaging is an attractive non-invasive way to evaluate the expression of a transferred DNA, mainly thanks to its lower cost and ease of realization. In this study optical imaging was evaluated for monitoring and quantification of the mouse knee joint and tibial cranial muscle electrotransfer of a luciferase encoding plasmid. Optical imaging was applied to study the kinetics of luciferase expression in both tissues.     

Edge detection in microscopy images using curvelets

Background  

Despite significant progress in imaging technologies, the efficient detection of edges and elongated features in images of intracellular and multicellular structures acquired using light or electron microscopy is a challenging and time consuming task in many laboratories.     

Evaluation of the diagnostic performance of infrared imaging of the breast: a preliminary study

Background  

The study was conducted to investigate the diagnostic performance of infrared (IR) imaging of the breast using an interpretive model derived from a scoring system.     

A new approach to dual-color two-photon microscopy with fluorescent proteins

Background  

Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs) is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA) efficiency.     

Electrical impedance tomography imaging using a priori ultrasound data

Background  

Different imaging systems (e.g. electrical, magnetic, and ultrasound) rely on a wide variety of physical properties, and the datasets obtained from such systems provide only partial information about the unknown true state. One approach is to choose complementary imaging systems, and to combine the information to achieve a better representation.     

Quantitative Analysis of Peripheral Tissue Perfusion Using Spatiotemporal Molecular Dynamics

Background

Accurate measurement of peripheral tissue perfusion is challenging but necessary to diagnose peripheral vascular insufficiency. Because near infrared (NIR) radiation can penetrate relatively deep into tissue, significant attention has been given to intravital NIR fluorescence imaging.

Methodology/Principal Findings

We developed a new optical imaging-based strategy for quantitative measurement of peripheral tissue perfusion by time-series analysis of local pharmacokinetics of the NIR fluorophore, indocyanine green (ICG). Time-series NIR fluorescence images were obtained after injecting ICG intravenously in a murine hindlimb ischemia model. Mathematical modeling and computational simulations were used for translating time-series ICG images into quantitative pixel perfusion rates and a perfusion map. We could successfully predict the prognosis of ischemic hindlimbs based on the perfusion profiles obtained immediately after surgery, which were dependent on the preexisting collaterals. This method also reflected increases in perfusion and improvements in prognosis of ischemic hindlimbs induced by treatment with vascular endothelial growth factor and COMP-angiopoietin-1.

Conclusions/Significance

We propose that this novel NIR-imaging-based strategy is a powerful tool for biomedical studies related to the evaluation of therapeutic interventions directed at stimulating angiogenesis.     

<Emphasis Type="Italic">In vivo</Emphasis> molecular imaging of experimental joint inflammation by combined <Superscript>18</Superscript>F-FDG positron emission tomography and computed tomography

Introduction  

The purpose of this work was to establish and validate combined small animal positron emission tomography - computed tomography (PET/CT) as a new in vivo imaging method for visualisation and quantification of joint inflammation.     

Inside Cover: Three‐dimensional intravital imaging in bone research (J. Biophotonics 12/2019)

This review is aimed at interpreting development and advantages of intravital imaging as an emerging invaluable methodology and summarizing related representative discoveries in bone physiologies and pathologies. It also indicates current limitations, further refinement, and extended application of intravital imaging in bone research. Further details can be found in the article by Yuhao Liu, Quan Yuan, and Shiwen Zhanget ( e201960075 ).

     

Image fusion for dynamic contrast enhanced magnetic resonance imaging

Background  

Multivariate imaging techniques such as dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) have been shown to provide valuable information for medical diagnosis. Even though these techniques provide new information, integrating and evaluating the much wider range of information is a challenging task for the human observer. This task may be assisted with the use of image fusion algorithms.     

A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D

Background  

To exploit the flood of data from advances in high throughput imaging of optically sectioned nuclei, image analysis methods need to correctly detect thousands of nuclei, ideally in real time. Variability in nuclear appearance and undersampled volumetric data make this a challenge.     

The usefulness of magnetic resonance imaging of the hand and wrist in very early rheumatoid arthritis

Introduction  

Magnetic resonance imaging (MRI) was used to study the hand and wrist in very early rheumatoid arthritis (RA), and the results were compared with early and established disease.