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News Article | May 11, 2017
Site: www.futurity.org

Medical engineers can now take a live look at the inner workings of a small animal with enough resolution to see active organs, flowing blood, circulating melanoma cells, and firing neural networks. The technique, dubbed single-impulse panoramic photoacoustic computed tomography (SIP-PACT), uses both light and ultrasound to peer inside living animals. In Nature Biomedical Engineering, the engineers describe how this hybrid imaging technology can provide a full cross-sectional view of a small animal’s internal functions in real time. Traditional light-based microscopy provides fast, high-resolution images that retain important functional information based on the wavelengths of light (i.e., colors) the tissue absorbs, reflects, or emits. A significant amount of that light is scattered as it travels through tissue, however, so these methods are limited to depths of less than a couple of millimeters. Photoacoustic imaging combines the abilities of multiple imaging techniques into one platform. It uses extremely short laser bursts that safely cause cells or other light absorbers to emit ultrasound waves, which then travel unimpeded back through the tissue to sensors that translate the signal into an image. Using this technique, medical engineers are able to discern delicate features inside the body because different types of molecules absorb light differently. For example, hemoglobin (which defines the color of blood) absorbs more light than the tissue around it, creating a contrast between oxygenated and de-oxygenated blood that makes it possible to take color images of arteries and veins in vivo. “Photoacoustic tomography combines light and sound synergistically for high-resolution imaging of molecular contrast,” says Wang, professor of medical engineering and electrical engineering at California Institute of Technology. Wang conducted this research while the Optical Imaging Laboratory was located at Washington University in St. Louis. He moved the lab to Caltech in January 2017. “Photoacoustic imaging has been highly expected to get real-time whole-body imaging of a small animal with rich functional information,” says Junjie Yao, formerly of the Optical Imaging Laboratory, now of Duke University. “With this advance, researchers can easily watch as drugs are distributed throughout an animal and track how different organs respond,” Yao says, referring to the technique’s ability to track individual molecules as they flow through the blood stream. Ultrasound waves easily travel through tissue, providing a much more in-depth view, but do not have the ability to discern a tissue’s chemical components and therefore do not capture important information that can be conveyed by light-based imaging. Magnetic resonance imaging (MRI) can also see deep into tissue, but requires a strong magnetic field and often takes seconds to minutes to form an image. Limits to the amount of radiation a subject can tolerate makes X-ray imaging and positron emission tomography (PET) impractical for long-term use. Photoacoustic tomography, on the other hand, avoids ionizing radiation altogether and uses only a safe dose of nonionizing energy. As such, it is safe to use on living tissue repeatedly, the engineers say. “It’s basically compressing one second’s worth of summer noon sunlight over a fingernail area into a single nanosecond,” says Yao. “When the laser hits a cell, the energy causes it to heat up a tiny bit and expand instantaneously, creating an ultrasonic wave. It’s like the difference between pushing on something to slowly move it and striking it to cause a vibration.” The result is an imaging technique that can peer up to five centimeters into the typical biological tissue and generate images with sub-millimeter-level resolution, while retaining the functional information provided by traditional optical microscopy. “This penetration range enables functional imaging of whole bodies of small animals. This capability is expected to enable all kinds of biological studies in small animals and to accelerate drug discovery,” Wang says. Wang and his colleagues have been developing photoacoustic tomography for more than 10 years. This latest iteration adds increased speed and panoramic views to the imaging technology’s repertoire. The engineers have built a circular ultrasonic detector and a fast data-acquisition system that can triangulate the origin of an ultrasonic wave from anywhere within the body of a small animal. And with the help of a fast laser, the upgraded device can image the full cross-section of an adult rat 50 times per second, providing detailed movies of its inner workings with 120-micrometer resolution. “The panoramic effect provides information from all directions and all angles, so you do not lose any information from each laser shot,” Yao says. “You can see the dynamics of the body in action—the pumping of the heart, the dilation of arteries, the functioning of various tissues.” The paper describes how the engineers use these abilities to track cancerous melanoma cells as they travel through the blood vessels of a mouse. They also demonstrate the ability to watch the entire brain in real time. “We think that this technology holds great potential for both pre-clinical imaging and clinical translation,” Yao says. The National Institutes of Health supported the work.


News Article | May 17, 2017
Site: www.sciencedaily.com

Engineers at the Optical Imaging Laboratory led by Caltech's Lihong Wang have developed an imaging technology that could help surgeons removing breast cancer lumps confirm that they have cut out the entire tumor -- reducing the need for additional surgeries. About 300,000 new cases of invasive breast cancer are discovered annually. Of these, 60 to 75 percent of patients underwent breast-conserving surgery. Breast-conserving surgeries, or lumpectomies, attempt to remove the entire tumor while retaining as much of the undamaged breast tissue as possible. (In contrast, a mastectomy removes the entire breast.) The extracted tissue is then sent to a lab where it is rendered into thin slices, stained with a dye to highlight key features, and then analyzed. If tumor cells are found on the surface of the tissue sample, it indicates that the surgeon has cut through, not around, the tumor -- meaning that a portion of the tumor remains inside the patient, who will then need a follow-up surgery to have more tissue removed. After a week or two waiting for lab results, 20 to 60 percent of patients find out that they must return for a second surgery to have more tissue removed. But, asks Wang, "what if we could get rid of the waiting? With 3D photoacoustic microscopy, we could analyze the tumor right in the operating room, and know immediately whether more tissue needs to be removed." Wang is a Bren Professor of Medical Engineering and Electrical Engineering in Caltech's Division of Engineering and Applied Science. His lab invented 3D photoacoustic microscopy. Photoacoustic microscopy, or PAM, excites a tissue sample with a low-energy laser, which causes the tissue to vibrate. The system measures the ultrasonic waves emitted by the vibrating tissue. Because nuclei vibrate more strongly than surrounding material, PAM reveals the size of nuclei and the packing density of cells. Cancerous tissue tends to have larger nuclei and more densely packed cells. Indeed, as described by Wang and his team in a paper publishing in the journal Science Advances on May 17, PAM produces images capable of highlighting cancerous features, with no slicing or staining required. Wang conducted this research while the Optical Imaging Laboratory was located at Washington University in St. Louis. He moved the lab to Caltech's Andrew and Peggy Cherng Department of Medical Engineering in January 2017. Although Wang's team has focused primarily on breast cancer tumors, his work has potential applications for any analysis of excised tumors -- from melanoma to pancreatic cancer. In a proof-of-concept scan described in the new paper, PAM analyzed a sample in about three hours. Comparable traditional microscopy takes about seven hours to achieve the same results. However, Wang says that PAM's analysis time could be cut down to 10 minutes or less with the addition of faster laser pulse repetition and parallel imaging. This would make the technology useful for clinical applications. "Because the device never directly touches a patient, there will be fewer regulatory hurdles to overcome before gaining FDA approval for use by surgeons," Wang says. "Potentially, we could make this tool available to surgeons within several years."


News Article | May 17, 2017
Site: www.eurekalert.org

Engineers at the Optical Imaging Laboratory led by Caltech's Lihong Wang have developed an imaging technology that could help surgeons removing breast cancer lumps confirm that they have cut out the entire tumor--reducing the need for additional surgeries. About 300,000 new cases of invasive breast cancer are discovered annually. Of these, 60 to 75 percent of patients underwent breast-conserving surgery. Breast-conserving surgeries, or lumpectomies, attempt to remove the entire tumor while retaining as much of the undamaged breast tissue as possible. (In contrast, a mastectomy removes the entire breast.) The extracted tissue is then sent to a lab where it is rendered into thin slices, stained with a dye to highlight key features, and then analyzed. If tumor cells are found on the surface of the tissue sample, it indicates that the surgeon has cut through, not around, the tumor--meaning that a portion of the tumor remains inside the patient, who will then need a follow-up surgery to have more tissue removed. After a week or two waiting for lab results, 20 to 60 percent of patients find out that they must return for a second surgery to have more tissue removed. But, asks Wang, "what if we could get rid of the waiting? With 3D photoacoustic microscopy, we could analyze the tumor right in the operating room, and know immediately whether more tissue needs to be removed." Wang is a Bren Professor of Medical Engineering and Electrical Engineering in Caltech's Division of Engineering and Applied Science. His lab invented 3D photoacoustic microscopy. Photoacoustic microscopy, or PAM, excites a tissue sample with a low-energy laser, which causes the tissue to vibrate. The system measures the ultrasonic waves emitted by the vibrating tissue. Because nuclei vibrate more strongly than surrounding material, PAM reveals the size of nuclei and the packing density of cells. Cancerous tissue tends to have larger nuclei and more densely packed cells. Indeed, as described by Wang and his team in a paper publishing in the journal Science Advances on May 17, PAM produces images capable of highlighting cancerous features, with no slicing or staining required. Wang conducted this research while the Optical Imaging Laboratory was located at Washington University in St. Louis. He moved the lab to Caltech's Andrew and Peggy Cherng Department of Medical Engineering in January 2017. Although Wang's team has focused primarily on breast cancer tumors, his work has potential applications for any analysis of excised tumors--from melanoma to pancreatic cancer. In a proof-of-concept scan described in the new paper, PAM analyzed a sample in about three hours. Comparable traditional microscopy takes about seven hours to achieve the same results. However, Wang says that PAM's analysis time could be cut down to 10 minutes or less with the addition of faster laser pulse repetition and parallel imaging. This would make the technology useful for clinical applications. "Because the device never directly touches a patient, there will be fewer regulatory hurdles to overcome before gaining FDA approval for use by surgeons," Wang says. "Potentially, we could make this tool available to surgeons within several years." The Science Advances paper is titled "Fast Label-free Multi-layered Histology-like Imaging of Human Breast Cancer by Photoacoustic Microscopy." Among the coauthors are Terence Wong, Ruiying Zhang, Pengfei Hai, Chi Zhang, and Miguel Pleitez, who are current or former members of the Optical Imaging Laboratory, and Rebecca Aft and Deborah Novack, who are clinical collaborators at Washington University. This research was funded by the National Institutes of Health and the Siteman Cancer Center.


Rapozzi V.,University of Udine | Biffi S.,Optical Imaging Laboratory | Garrovo C.,Optical Imaging Laboratory | Xodo L.E.,University of Udine
Cancer Biology and Therapy | Year: 2010

The design of new photosensitizers with enhanced phototoxicity and pharmacokinetic properties remains a central challenge for cancer photodynamic therapy (PDT). In this study, pheophorbide a (Pba) has been pegylated to methoxypolyethylene glycol (mPEG-Pba) to produce a soluble photosensitizer that exhibits a higher tissue distribution than free pba. In vitro studies have shown that mPEG-Pba promotes a fairly strong photosensitizing effect in cancer cells, as previously observed for the unpegylated molecule. mPEG-Pba targets the mitochondria where, following photoactivation, ROS are produced which cause a cellular injury by lipid peroxidation. The effect of pegylation on the photosensitizer biodistribution has been examined in different selected organs of female mice, at different time points after intraperitoneal administration of the drug (50 μmol/Kg body weight). Other than free Pba, which showed a low tissue accumulation, mPEG-Pba has been detected in significant amounts (8 to 16 μg/ml) in liver, spleen, duodenum and kidney and, 3-5 hours after intraperitoneal injection, in moderate amounts (3 to 8 μg/ml) in brain and lung. In vivo optical imaging performed on living female C57/BL6 mice bearing a subcutaneous melanoma mass, showed that injected mPEG-Pba distributes all over the body, with an higher uptake in the tumor respect to free Pba. Our results indicate that although pegylation somewhat decreases the phototoxicity, it significantly increases the drug solubility and tissue distribution and tumor uptake of mPEG-Pba, making the conjugate an interesting photosensitizer for PDT. © 2010 Landes Bioscience.


Agostinis C.,Institute for Maternal and Child Health | Biffi S.,Optical Imaging Laboratory | Garrovo C.,Optical Imaging Laboratory | Durigutto P.,University of Trieste | And 10 more authors.
Blood | Year: 2011

In vitro studies have documented β2 glycoprotein I (β2GPI) binding to endothelial cells (ECs) and trophoblast using antiphospholipid antibodies. The in vivo binding of β2GPI to these cells and the conditions that favor their interaction have not been investigated.We analyzed the in vivo distribution of cyanine 5.5-labeled β2GPI in mice and evaluated the effect of pregnancy and circulating antibodies on its tissue localization. The signal was detected in the liver by whole body scan and ex vivo analysis. The β2GPI failed to bind to the vascular endothelium and reacted only with the ECs of uterine vessels. In pregnant mice the protein was localized on ECs and trophoblast at the embryo implantation sites. Immunized mice showed a similar β2GPI biodistribution to naive mice but the immunized pregnant animals exhibited a significant increase in fetal loss associated with C3 and C9 deposition at the implantation sites. Treatment of mice with LPS after β2GPI-Cy5.5 injection promoted protein localization on gut and brain ECs associated with IgG, C1q, and C9 deposition in immunized mice. These findings indicate that β2GPI binding to EC requires priming with pro-inflammatory factors which is not needed for uterine and placental localization probably dependent on hormonal changes. © 2011 by The American Society of Hematology.


Biffi S.,Optical Imaging Laboratory | Biffi S.,Institute for Maternal and Child Health | Dal Monego S.,Cluster in Biomedicine CBM scrl | Dullin C.,University of Gottingen | And 7 more authors.
PLoS ONE | Year: 2013

Background: Non-invasive in vivo imaging strategies are of high demand for longitudinal monitoring of inflammation during disease progression. In this study we present an imaging approach using near infrared fluorescence (NIRF) imaging in combination with a polyanionic macromolecular conjugate as a dedicated probe, known to target L- and P-selectin and C3/C5 complement factors. Methodology/Principal Findings: We investigated the suitability of dendritic polyglycerol sulfates (dPGS), conjugated with a hydrophilic version of the indocyanine green label with 6 sulfonate groups (6S-ICG) to monitor sites of inflammation using an experimental mouse model of allergic asthma. Accumulation of the NIRF-conjugated dPGS (dPGS-NIRF) in the inflamed lungs was analyzed in and ex vivo in comparison with the free NIRF dye using optical imaging. Commercially available smart probes activated by matrix metalloproteinase's (MMP) and cathepsins were used as a comparative control. The fluorescence intensity ratio between lung areas of asthmatic and healthy mice was four times higher for the dPGS in comparison to the free dye in vivo at four hrs post intravenous administration. No significant difference in fluorescence intensity between healthy and asthmatic mice was observed 24 hrs post injection for dPGS-NIRF. At this time point ex-vivo scans of asthmatic mice confirmed that the fluorescence within the lungs was reduced to approximately 30% of the intensity observed at 4 hrs post injection. Conclusions/Significance: Compared with smart-probes resulting in a high fluorescence level at 24 hrs post injection optical imaging with dPGS-NIRF conjugates is characterized by fast uptake of the probe at inflammatory sites and represents a novel approach to monitor lung inflammation as demonstrated in mice with allergic asthma. © 2013 Biffi et al.


Benincasa M.,University of Trieste | Pelillo C.,University of Trieste | Zorzet S.,University of Trieste | Garrovo C.,Optical Imaging Laboratory | And 3 more authors.
BMC Microbiology | Year: 2010

Background. Bac7 is a proline-rich peptide with a potent in vitro antimicrobial activity against Gram-negative bacteria. Here we investigated its activity in biological fluids and in vivo using a mouse model of S. typhimurium infection. Results. The efficacy of the active 1-35 fragment of Bac7 was assayed in serum and plasma, and its stability in biological fluids analyzed by Western blot and mass spectrometry. The ability of the peptide to protect mice against Salmonella was assayed in a typhoid fever model of infection by determination of survival rates and bacterial load in liver and spleen of infected animals. In addition, the peptide's biodistribution was evaluated by using time-domain optical imaging. Bac7(1-35) retained a substantial in vivo activity showing a very low toxicity. The peptide increased significantly the number of survivors and the mean survival times of treated mice reducing the bacterial load in their organs despite its rapid clearance. Conclusions. Our results provide a first indication for a potential development of Bac7-based drugs in the treatment of salmonellosis and, eventually, other Gram-negative infections. The in vivo activity for this peptide might be substantially enhanced by decreasing its excretion rate or modifying the treatment schedule. © 2010 Benincasa et al; licensee BioMed Central Ltd.


Tosi G.,University of Modena and Reggio Emilia | Bondioli L.,University of Modena and Reggio Emilia | Ruozi B.,University of Modena and Reggio Emilia | Badiali L.,University of Modena and Reggio Emilia | And 7 more authors.
Journal of Neural Transmission | Year: 2011

The presence of the blood-brain barrier (BBB) makes extremely difficult to develop efficacious strategies for targeting contrast agents and delivering drugs inside the Central Nervous System (CNS). To overcome this drawback, several kinds of CNS-targeted nanoparticles (NPs) have been developed. In particular, we proposed poly-lactide-co-glycolide (PLGA) NPs engineered with a simil-opioid glycopeptide (g7), which have already proved to be a promising tool for achieving a successful brain targeting after i.v. administration in rats. In order to obtain CNS-targeted NPs to use for in vivo imaging, we synthesized and administrated in mice PLGA NPs with double coverage: near-infrared (NIR) probe (DY-675) and g 7. The optical imaging clearly showed a brain localization of these novel NPs. Thus, a novel kind of NIR-labeled NPs were obtained, providing a new, in vivo detectable nanotechnology tool. Besides, the confocal and fluorescence microscopy evidences allowed to further confirm the ability of g 7 to promote not only the rat, but also the mouse BBB crossing. © 2010 Springer-Verlag.


Biffi S.,Institute of Maternal and Child Health | Biffi S.,Optical Imaging Laboratory | Bortot B.,Institute of Maternal and Child Health | Carrozzi M.,Institute of Maternal and Child Health | Severini G.M.,Institute of Maternal and Child Health
Diagnostic Molecular Pathology | Year: 2011

In many mitochondrial diseases, different clinical manifestations are related to tissue-specific distribution of mutated mitochondrial DNA (mtDNA). In this study, we describe an assay for the determination of mutated mtDNA copy number in small clinical samples, using standard polymerase chain reaction (PCR) followed by SYBR Green real-time allelic-specific PCR [amplification refractory mutation system-quantitative PCR (ARMS-qPCR)]. To assess the degree of heteroplasmy in a patient harboring 2 cosegregating mtDNA mutations (4415A>G and 9922A>C) starting from picogram amounts of DNA, we first amplified the mutated target sequence by standard PCR, and then analyzed it by real-time ARMS-qPCR. To validate this method, we analyzed by real-time ARMS-qPCR the PCR amplification products derived from different mixtures containing known proportions of mutant and wild-type cloned mtDNA fragments. The correlation coefficient of 0.994 between expected and observed values for the percentage of mutant A4415G confirms that the relative proportion of mutated and wild-type mtDNA was maintained after the first PCR amplification. This method allows the precise quantification of heteroplasmic mutations in DNA samples extracted from hairs, urine, small stomach biopsies, and, more importantly, single-muscle fiber, with a limit of detection close to 0.5%. This nested real-time ARMS-PCR represents a rapid, efficient, and less expensive method for the detection and quantification of heteroplasmic mutant mtDNA, even in very small clinical samples. Copyright © 2011 by Lippincott Williams & Wilkins.


Non-invasive in vivo imaging strategies are of high demand for longitudinal monitoring of inflammation during disease progression. In this study we present an imaging approach using near infrared fluorescence (NIRF) imaging in combination with a polyanionic macromolecular conjugate as a dedicated probe, known to target L- and P-selectin and C3/C5 complement factors.We investigated the suitability of dendritic polyglycerol sulfates (dPGS), conjugated with a hydrophilic version of the indocyanine green label with 6 sulfonate groups (6S-ICG) to monitor sites of inflammation using an experimental mouse model of allergic asthma. Accumulation of the NIRF-conjugated dPGS (dPGS-NIRF) in the inflamed lungs was analyzed in and ex vivo in comparison with the free NIRF dye using optical imaging. Commercially available smart probes activated by matrix metalloproteinases (MMP) and cathepsins were used as a comparative control. The fluorescence intensity ratio between lung areas of asthmatic and healthy mice was four times higher for the dPGS in comparison to the free dye in vivo at four hrs post intravenous administration. No significant difference in fluorescence intensity between healthy and asthmatic mice was observed 24 hrs post injection for dPGS-NIRF. At this time point ex-vivo scans of asthmatic mice confirmed that the fluorescence within the lungs was reduced to approximately 30% of the intensity observed at 4 hrs post injection.Compared with smart-probes resulting in a high fluorescence level at 24 hrs post injection optical imaging with dPGS-NIRF conjugates is characterized by fast uptake of the probe at inflammatory sites and represents a novel approach to monitor lung inflammation as demonstrated in mice with allergic asthma.

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