Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm²) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm⁻¹ used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy.     

Anti-Tumour Efficacy of Capecitabine in a Genetically Engineered Mouse Model of Pancreatic Cancer

Capecitabine (CAP) is a 5-FU pro-drug approved for the treatment of several cancers and it is used in combination with gemcitabine (GEM) in the treatment of patients with pancreatic adenocarcinoma (PDAC). However, limited pre-clinical data of the effects of CAP in PDAC are available to support the use of the GEMCAP combination in clinic. Therefore, we investigated the pharmacokinetics and the efficacy of CAP as a single agent first and then in combination with GEM to assess the utility of the GEMCAP therapy in clinic. Using a model of spontaneous PDAC occurring in Kras^G12D; p53^R172H; Pdx1-Cre (KPC) mice and subcutaneous allografts of a KPC PDAC-derived cell line (K8484), we showed that CAP achieved tumour concentrations (∼25 µM) of 5-FU in both models, as a single agent, and induced survival similar to GEM in KPC mice, suggesting similar efficacy. In vitro studies performed in K8484 cells as well as in human pancreatic cell lines showed an additive effect of the GEMCAP combination however, it increased toxicity in vivo and no benefit of a tolerable GEMCAP combination was identified in the allograft model when compared to GEM alone. Our work provides pre-clinical evidence of 5-FU delivery to tumours and anti-tumour efficacy following oral CAP administration that was similar to effects of GEM. Nevertheless, the GEMCAP combination does not improve the therapeutic index compared to GEM alone. These data suggest that CAP could be considered as an alternative to GEM in future, rationally designed, combination treatment strategies for advanced pancreatic cancer.     

Quantification of Tumor Burden in a Genetically Engineered Mouse Model of Lung Cancer by Micro-CT and Automated Analysis

Genetically engineered mouse models (GEMMs) of lung cancer closely recapitulate the human disease but suffer from the difficulty of evaluating tumor growth by conventional methods. Herein, a novel automated image analysis method for estimating the lung tumor burden from in vivo micro-computed tomography (micro-CT) data is described. The proposed tumor burden metric is the segmented soft tissue volume contained within a chest space region of interest, excluding an estimate of the heart volume. The method was validated by comparison with previously published manual analysis methods and applied in two therapeutic studies in a mutant K-ras GEMM of non–small cell lung carcinoma. Mice were imaged by micro-CT pre-treatment and stratified into four treatment groups: an antibody inhibiting vascular endothelial growth factor (anti-VEGF), chemotherapy, combination of anti-VEGF and chemotherapy, or control antibody. In the first study, post-treatment imaging was performed 4 weeks later. In the second study, mice were scanned serially on a high-throughput scanner every 2 weeks for 8 weeks during treatment. In both studies, the automated tumor burden estimates were well correlated with manual metrics (r value range: 0.83-0.93, P < .0001) and showed a similar, significant reduction in tumor growth in mice treated with anti-VEGF alone or in combination with chemotherapy. Given the fully automated nature of this technique, the proposed analysis method can provide a valuable tool in preclinical drug research for screening and randomizing animals into treatment groups and evaluating treatment efficacy in mouse models of lung cancer in a highly robust and efficient manner.     

Wild-Type Hras Suppresses the Earliest Stages of Tumorigenesis in a Genetically Engineered Mouse Model of Pancreatic Cancer

Oncogenic, activating mutations in KRAS initiate pancreatic cancer. There are, however, two other Ras family members, Nras and Hras, which can be activated in the presence of oncogenic Kras. The role of these wild-type Ras proteins in cancer remains unclear, as their disruption has been shown to enhance or inhibit tumorigenesis depending upon the context. As pancreatic cancer is critically dependent upon Ras signaling, we tested and now report that loss of Hras increases tumor load and reduces survival in an oncogenic Kras-driven pancreatic adenocarcinoma mouse model. These effects were traced to the earliest stages of pancreatic cancer, suggesting that wild-type Hras may suppress tumor initiation. In normal cells, activated Ras can suppress proliferation through p53-dependent mechanisms. We find that the tumor suppressive effects of Hras are nullified in a homozygous mutant p53 background. As such, loss of wild-type Hras fosters the earliest stages of pancreatic cancer in a p53-dependent manner.     

Molecular Flow Quantified beyond the Diffraction Limit by Spatiotemporal Image Correlation of Structured Illumination Microscopy Data

We combine total internal reflection fluorescence structured illumination microscopy with spatiotemporal image correlation spectroscopy to quantify the flow velocities and directionality of filamentous-actin at the T cell immunological synapse. These techniques demonstrate it is possible to image retrograde flow of filamentous-actin at superresolution and provide flow quantification in the form of velocity histograms and flow vector maps. The flow was found to be retrograde and radially directed throughout the periphery of T-cells during synapse formation.     

Molecular Flow Quantified beyond the Diffraction Limit by Spatiotemporal Image Correlation of Structured Illumination Microscopy Data

We combine total internal reflection fluorescence structured illumination microscopy with spatiotemporal image correlation spectroscopy to quantify the flow velocities and directionality of filamentous-actin at the T cell immunological synapse. These techniques demonstrate it is possible to image retrograde flow of filamentous-actin at superresolution and provide flow quantification in the form of velocity histograms and flow vector maps. The flow was found to be retrograde and radially directed throughout the periphery of T-cells during synapse formation.Many biological processes are now being visualized with the use of superresolution fluorescence microscopy techniques. However, localization-based techniques primarily rely on fixed or slow moving samples to permit the collection of structural information. The 10-fold gains in resolution afforded by these superresolution techniques are usually possible through sacrificing the factors that originally made microscopy such a powerful tool: the ability to image live cells. In the case of stimulated emission depletion imaging, the scanning approach associated with this technique may fail to detect faster molecular events when imaging whole cellular regions.Structured illumination microscopy (SIM) is an alternative to these methods (1). It increases the resolution of conventional fluorescence microscopy twofold; it has the advantage of using a wide-field system, providing fast acquisition speeds of whole cells with relatively low laser powers; and it is compatible with standard fluorophores. By using a physical grating to produce interference patterns from a laser, periodic illumination is created. This patterned illumination causes information from higher spatial frequencies to be downmodulated (i.e., shifted) into the optical transfer function (support region) of the lens, resulting in higher-resolution spatial information being captured than is ordinarily obtainable.To quantify the directional motion of intracellular molecules, spatiotemporal image correlation spectroscopy (STICS (2)) was applied. Using spatial image correlation in time, STICS measures the similarity of pixels with those surrounding in lagging frames via a correlation function. The correlation function provides information on both flow velocities and directionality, while discounting static structures through the immobile object filter, achieved by subtracting a moving average of pixel intensities.The formation of an immunological synapse between T cells and antigen-presenting cells is a process requiring many dynamic (3) and subdiffraction-limited clustering events (4–6) to take place. The polymerization of actin is important for the spreading of cells over their target antigen-presenting cells (7), as well as cell mobility and migration (8). Retrograde flow of densely meshed cortical actin is observed at the basal membrane of synapse-forming T cells, where it may have a role in the corralling and clustering of signaling molecules at the plasma membrane (9), as well as at the leading edge of migrating cells (10). Filamentous actin is an extremely dynamic (7), densely packed, and thin (7-nm) structure (11,12).Here, we perform STICS on SIM data acquired on a total internal reflection fluorescence (TIRF) microscope system, which generated an evanescent field of 75-nm depth for excitation. To our knowledge, this is the first demonstration of an image correlation approach to quantify molecular dynamics on subresolution length scales using wide-field microscopy. To demonstrate the technique, we analyze two-dimensional actin flows in CD4⁺ T cells during immunological synapse formation, performed after cross-linking of antigen T cell receptors on a coverslip coated with specific antibodies.Fig. 1 a shows a schematic of the TIRF SIM setup. Excitation light (488 nm) passes through a polarizing module and then a phase-grating block, producing diffracted beams. These are then passed through a diffraction filter module to isolate the −1 and +1 order laser beams. These first-order laser beams are angled through the objective to produce total internal reflection conditions at the glass-water interface. The two evanescent waves interfere at the sample, producing structured illumination. The setup then produces lateral and rotational shifts through three orientations, producing nine raw images containing higher spatial frequencies than can normally be acquired by an objective using standard light microscopy. Fig. 1 b demonstrates the increased resolution obtained from TIRF SIM. Shown are the collected Fourier frequencies compared to those of a conventional microscope (dotted red line). Resolution was also measured using sparse 100-nm diameter fluorescent beads. Fig. 1 c shows a magnified image of these beads from which a line profile was obtained (yellow arrow). The full width at half-maximum of this profile (Fig. 1 d) gives a lateral resolution for the system of 120 nm.Open in a separate windowFigure 1(a) Schematic of the TIRF SIM setup. (b) Demonstration of the doubling of spatial resolution of collected frequencies through a Fourier transform (superimposed red circle demonstrating regular spatial frequency limits). (c) SIM reconstructed image of 100-nm bead (scale bar 0.5 μm). (d) (Plotted line) Bead showing full width at half-maximum of 120 nm.We then applied STICS analysis to quantify actin flow in T cell synapses acquired using TIRF SIM (Fig. 2). Fig. 2 a shows a schematic of the STICS analysis. From the raw data, immobile objects are first filtered by subtracting a moving average of the pixel values. Vector maps were obtained from correlation analysis of the time-series as previously published in Hebert et al. (2) and Brown et al. (13). Fig. 2 b shows a reconstructed TIRF SIM image of a mature T cell immunological synapse, representative of a time-point derived from the time series acquired at 1.28 fps (see Movie S1 in the Supporting Material). From this reconstructed image, two representative regions have been selected. In these regions, pseudo-colored actin flow vectors are overlaid onto the fluorescence intensity image. These range in magnitude from 0.01 μm/min (blue) to 5.61 μm/min (red). It can be observed that all flow vectors are directed radially toward the synapse center. A histogram of this flow is shown in Fig. 2 c. The histogram shows a peak retrograde flow velocity of 1.91 ± 1.27 μm/min. These data are representative of n = 7 T-cell synapses imaged by TIRF SIM.Open in a separate windowFigure 2(a) STICS analysis, performed by isolating mobile from immobile structures through a moving average filter (i) and binning a subset of pixels into blocks of superpixels (ii); the STICS software correlates spatial fluorescence fluctuations through time (iii). The code then outputs vector maps showing directionality and flow velocities. (b) TIRF SIM image of actin flow in a T cell 5 min after contact with a stimulatory coverslip. (Zoomed regions) Retrograde actin flow at the synapse periphery. (c) Histograms showing flow speed statistics of vectors from T-cell synapses (n = 7).     

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.     

Genetically Engineered Lactococcus lactis Protect against House Dust Mite Allergy in a BALB/c Mouse Model

Background

Mucosal vaccine based on lactic acid bacteria is an attractive concept for the prevention and treatment of allergic diseases, but their mechanisms of action in vivo are poorly understood. Therefore, we sought to investigate how recombinant major dust mite allergen Der p2-expressing Lactococcus lactis as a mucosal vaccine induced the immune tolerance against house dust mite allergy in a mouse model.

Methods

Three strains of recombinant L. lactis producing Der p2 in different cell components (extracellular, intracellular and cell wall) were firstly constructed. Their prophylactic potential was evaluated in a Der p2-sensitised mouse model, and immunomodulation properties at the cellular level were determined by measuring cytokine production in vitro.

Results

Der p2 expressed in the different recombinant L. lactis strains was recognized by a polyclonal anti-Der p2 antibody. Oral treatment with the recombinant L. lactis prior sensitization significantly prevented the development of airway inflammation in the Der p2-sensitized mice, as determined by the attenuation of inflammatory cells infiltration in the lung tissues and decrease of Th2 cytokines IL-4 and IL-5 levels in bronchoalveolar lavage. In addition, the serum allergen-specific IgE levels were significantly reduced, and the levels of IL-4 in the spleen and mesenteric lymph nodes cell cultures were also markedly decreased upon allergen stimulation in the mice fed with the recombinant L. lactis strains. These protective effects correlated with a significant up-regulation of regulatory T cells in the mesenteric lymph nodes.

Conclusion

Oral pretreatment with live recombinant L. lactis prevented the development of allergen-induced airway inflammation primarily by the induction of specific mucosal immune tolerance.     

Use of Bioluminescence for Detection of Genetically Engineered Microorganisms Released into the Environment

The persistence and movement of strain JS414 of Xanthomonas campestris pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent X. campestris pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mist- and wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. Our bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Our results demonstrate that transgenic incorporation of the luxCDABE operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.     

PD-0332991, a CDK4/6 Inhibitor,Significantly Prolongs Survival in a Genetically Engineered Mouse Model of Brainstem Glioma

Diffuse intrinsic pontine glioma (DIPG) is an incurable tumor that arises in the brainstem of children. To date there is not a single approved drug to effectively treat these tumors and thus novel therapies are desperately needed. Recent studies suggest that a significant fraction of these tumors contain alterations in cell cycle regulatory genes including amplification of the D-type cyclins and CDK4/6, and less commonly, loss of Ink4a-ARF leading to aberrant cell proliferation. In this study, we evaluated the therapeutic approach of targeting the cyclin-CDK-Retinoblastoma (Rb) pathway in a genetically engineered PDGF-B-driven brainstem glioma (BSG) mouse model. We found that PD-0332991 (PD), a CDK4/6 inhibitor, induces cell-cycle arrest in our PDGF-B; Ink4a-ARF deficient model both in vitro and in vivo. By contrast, the PDGF-B; p53 deficient model was mostly resistant to treatment with PD. We noted that a 7-day treatment course with PD significantly prolonged survival by 12% in the PDGF-B; Ink4a-ARF deficient BSG model. Furthermore, a single dose of 10 Gy radiation therapy (RT) followed by 7 days of treatment with PD increased the survival by 19% in comparison to RT alone. These findings provide the rationale for evaluating PD in children with Ink4a-ARF deficient gliomas.     

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins

Purpose

To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features.

Materials and Methods

Tissue excised from a genetically engineered mouse model of sarcoma was imaged using a subcellular resolution microendoscope after topical application of a fluorescent anatomical contrast agent: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma.

Results

Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach.

Conclusion

The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.     

转基因定量检测用质粒分子标准物质研究进展

近10年来,应用于转基因植物产品定量检测的质粒分子标准物质凭借其易于富集,快捷高效等优点,成为各国检测机构的研究热点。对国内外转基因植物核酸定量检测用的质粒分子标准物质研究进展进行总结和评述,并且对该类标准物质在研制过程中的问题进行分析,同时展望了该类标准物质的发展与应用。     

Detection of Gene Expression in Genetically Engineered Microorganisms and Natural Phytoplankton Populations in the Marine Environment by mRNA Analysis

A simple method that combines guanidinium isothiocyanate RNA extraction and probing with antisense and sense RNA probes is described for analysis of microbial gene expression in planktonic populations. Probing of RNA sample extracts with sense-strand RNA probes was used as a control for nonspecific hybridization or contamination of mRNA with target DNA. This method enabled detection of expression of a plasmid-encoded neomycin phosphotransferase gene (nptII) in as few as 10⁴Vibrio cells per ml in 100 ml of seawater. We have used this method to detect expression of the ribulose-1,5-bisphosphate carboxylase large-subunit gene (rbcL) in Synechococcus cultures and natural phytoplankton populations in the Dry Tortugas, Florida. During a 36-h diel study, rbcL expression of the indigenous phytoplankton was greatest in the day, least at night (1100, 0300, and 0100 h), and variable at dawn or dusk (0700 and 1900 h). These results are the first report of gene expression in natural populations by mRNA isolation and probing. This methodology should be useful for the study of gene expression in microorganisms released into the environment for agricultural or bioremediation purposes and indigenous populations containing highly conserved target gene sequences.     

A High-Throughput In Vitro Drug Screen in a Genetically Engineered Mouse Model of Diffuse Intrinsic Pontine Glioma Identifies BMS-754807 as a Promising Therapeutic Agent

Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system, including a model driven by PDGF-B, H3.3K27M, and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study, an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R, IR, MET, TRKA, TRKB, AURKA, AURKB. In vitro evaluation showed significant cytotoxic effects with an IC₅₀ of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation. Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo, systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC₅₀, suggesting that inadequate drug delivery may limit in vivo efficacy. In summary, an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG, but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice.     

转基因水稻华恢1号检测用质粒标准分子研制

质粒标准分子作为阳性标准品已广泛用于转基因作物及产品的核酸定量检测。但是目前多数质粒标准分子只用于研究,未能成为国家级标准物质。因此,按照国家一级标准物质技术规范,研制了用于检测转基因水稻华恢1号的质粒标准分子,内容包括质粒分子的构建、纯度分析、特异性检测、可替代性研究、均匀性检验、稳定性考察、定值与不确定度评估。结果表明,该质粒分子标准物质半年内稳定,可替代基因组作为华恢1号定量的阳性标准。     

Detection of a Single Genetically Modified Bacterial Cell in Soil by Using Charge Coupled Device-Enhanced Microscopy

Genes encoding bioluminescence from Vibrio harveyi were cloned into Pseudomonas syringae pv. phaseoli-cola, resulting in high levels of bioluminescence. After inoculation of sterile and nonsterile soil slurries with bioluminescent P. syringae, cells could not be identified by conventional light microscopy. However, when we used charge coupled device-enhanced microscopy, bioluminescent single cells were detected easily in dark fields despite masking by soil particulate matter, and in addition, the extent of competition from indigenous soil bacteria could be monitored. The technique which we describe offers great potential for tracking and determining the spatial distribution of genetically marked microorganisms in the environment.     

Microscopic Delineation of Medulloblastoma Margins in a Transgenic Mouse Model Using a Topically Applied VEGFR-1 Probe

The unambiguous demarcation of tumor margins is critical at the final stages in the surgical treatment of brain tumors because patient outcomes have been shown to correlate with the extent of resection. Real-time high-resolution imaging with the aid of a tumor-targeting fluorescent contrast agent has the potential to enable intraoperative differentiation of tumor versus normal tissues with accuracy approaching the current gold standard of histopathology. In this study, a monoclonal antibody targeting the vascular endothelial growth factor receptor 1 (VEGFR-1) was conjugated to fluorophores and evaluated as a tumor contrast agent in a transgenic mouse model of medulloblastoma. The probe was administered topically, and its efficacy as an imaging agent was evaluated in vitro using flow cytometry, as well as ex vivo on fixed and fresh tissues through immunohistochemistry and dual-axis confocal microscopy, respectively. Results show a preferential binding to tumor versus normal tissue, suggesting that a topically applied VEGFR-1 probe can potentially be used with real-time intraoperative optical sectioning microscopy to guide brain tumor resections.     

In Vivo Quantitative Microcomputed Tomographic Analysis of Vasculature and Organs in a Normal and Diseased Mouse Model

Non-bone in vivo micro-CT imaging has many potential applications for preclinical evaluation. Specifically, the in vivo quantification of changes in the vascular network and organ morphology in small animals, associated with the emergence and progression of diseases like bone fracture, inflammation and cancer, would be critical to the development and evaluation of new therapies for the same. However, there are few published papers describing the in vivo vascular imaging in small animals, due to technical challenges, such as low image quality and low vessel contrast in surrounding tissues. These studies have primarily focused on lung, cardiovascular and brain imaging. In vivo vascular imaging of mouse hind limbs has not been reported. We have developed an in vivo CT imaging technique to visualize and quantify vasculature and organ structure in disease models, with the goal of improved quality images. With 1–2 minutes scanning by a high speed in vivo micro-CT scanner (Quantum CT), and injection of a highly efficient contrast agent (Exitron nano 12000), vasculature and organ structure were semi-automatically segmented and quantified via image analysis software (Analyze). Vessels of the head and hind limbs, and organs like the heart, liver, kidneys and spleen were visualized and segmented from density maps. In a mouse model of bone metastasis, neoangiogenesis was observed, and associated changes to vessel morphology were computed, along with associated enlargement of the spleen. The in vivo CT image quality, voxel size down to 20 μm, is sufficient to visualize and quantify mouse vascular morphology. With this technique, in vivo vascular monitoring becomes feasible for the preclinical evaluation of small animal disease models.     

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.