Optical nanoparticle sensors for quantitative intracellular imaging

Real-time measurements of biological/chemical/physical processes, with no interferences, are an ultimate goal for in vivo intracellular studies. To construct intracellular biosensors that meet such a goal, nanoparticle (NP) platforms seem to be most promising, because of their small size and excellent engineerability. This review describes the development of NP-based opical sensors and their intracellular applications. The sensor designs are classified into two types, based on the sensor structures regarding analyte receptor and signal transducer. Type 1 sensors, with a single component for both receptor and transducer, work by mechanisms similar to those of ‘molecular probes’. Type 2 sensors, with a separate component for receptor and transducer, work by different mechanisms that require the presence of specific NPs. A synergistic increase in optical signal or selectivity has been reported for these second type of NP sensors. With ongoing rapid advances in nanotechnology and instrumentation, these NP systems will soon be capable of sensing at the single-molecule level, at the point of interest within the living cell, and capable of simultaneously detecting multiple analytes and physical parameters. Copyright © 2008 John Wiley & Sons, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61310/1/2_ftp.pd

Ratiometric Singlet Oxygen Nano-optodes and Their Use for Monitoring Photodynamic Therapy Nanoplatforms

Ratiometric photonic explorers for bioanalysis with biologically localized embedding (PEBBLE) nanoprobes have been developed for singlet oxygen, using organically modified silicate (ORMOSIL) nanoparticles as the matrix. A crucial aspect of these ratiometric singlet-oxygen fluorescent probes is their minute size. The ORMOSIL nanoparticles are prepared via a sol-gel–based process and the average diameter of the resultant particles is about 160 nm. These sensors incorporate the singlet-oxygen–sensitive 9,10-dimethyl anthracene as an indicator dye and a singlet-oxygen–insensitive dye, octaethylporphine, as a reference dye for ratiometric fluorescence-based analysis. We have found experimentally that these nanoprobes have much better sensitivity than does the conventional singlet-oxygen–free dye probe, anthracene-9, 10-dipropionic acid disodium salt. The much longer lifetime of singlet oxygen in the ORMOSIL matrix, compared to aqueous solutions, in addition to the relatively high singlet oxygen solubility because of the highly permeable structure and the hydrophobic nature of the outer shell of the ORMOSIL nanoparticles, results in an excellent overall response to singlet oxygen. These nanoprobes have been used to monitor the singlet oxygen produced by “dynamic nanoplatforms” that were developed for photodynamic therapy. The singlet oxygen nanoprobes could potentially be used to quantify the singlet oxygen produced by macrophages.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75523/1/2005-05-18-RA-532.pd

Two‐Photon Fluorescence Imaging Super‐Enhanced by Multishell Nanophotonic Particles, with Application to Subcellular pH

A novel nanophotonic method for enhancing the two‐photon fluorescence signal of a fluorophore is presented. It utilizes the second harmonic (SH) of the exciting light generated by noble metal nanospheres in whose near‐field the dye molecules are placed, to further enhance the dye's fluorescence signal in addition to the usual metal‐enhanced fluorescence phenomenon. This method enables demonstration, for the first time, of two‐photon fluorescence enhancement inside a biological system, namely live cells. A multishell hydrogel nanoparticle containing a silver core, a protective citrate capping, which serves also as an excitation quenching inhibitor spacer, a pH indicator dye shell, and a polyacrylamide cladding are employed. Utilizing this technique, an enhancement of up to 20 times in the two‐photon fluorescence of the indicator dye is observed. Although a significant portion of the enhanced fluorescence signal is due to one‐photon processes accompanying the SH generation of the exciting light, this method preserves all the advantages of infrared‐excited, two‐photon microscopy: enhanced penetration depth, localized excitation, low photobleaching, low autofluorescence, and low cellular damage. The two‐photon fluorescence signal of a fluorophore is enhanced by utilizing the second harmonic of the exciting light generated by noble metal nanospheres in whose near‐field dye molecules are placed. A multishell hydrogel nanoparticle containing a silver core, protective citrate capping, pH indicator dye, and polyacrylamide cladding is utilized for pH sensing and fluorescence imaging in live cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92437/1/2213_ftp.pd

Enhanced Solubility of the Support in an FDM-Based 3D Printed Structure Using Hydrogen Peroxide under Ultrasonication

Fused deposition modeling (FDM), one of the archetypal 3D printing processes, typically requires support structures matched to printed model parts that principally have undercut or overhung features. Thus, the support removal is an essential postprocessing step after the FDM process. Here, we present an efficient and rapid method to remove the support part of an FDM-manufactured product using the phenomenon of oxidative degradation of hydrogen peroxide. This mechanism was significantly effective on polyvinyl alcohol (PVA), which has been widely used as a support material in the FDM process. Compared to water, hydrogen peroxide provided a two times faster dissolution rate of the PVA material. This could be increased another two times by applying ultrasonication to the solvent. In addition to the rapidness, we confirmed that amount of the support residues removed was enhanced, which was essentially caused by the surface roughness of the FDM-fabricated part. Furthermore, we demonstrated that there was no deterioration with respect to the mechanical properties or shape geometries of the obtained 3D printed parts. Taken together, these results are expected to help enhance the productivity of FDM by reducing the postprocessing time and to allow the removal of complicated and fine support structures, thereby improving the design capability of the FDM technique

Endoscopic Thyroidectomy via an Axillo-Breast Approach without Gas Insufflation for Benign Thyroid Nodules and Micropapillary Carcinomas: Preliminary Results

PURPOSE: To examine the feasibility of endoscopic thyroidectomy (ET) via an axillo- breast approach without gas insufflation for large thyroid tumors and micropapillary carcinomas. MATERIALS AND METHODS: The patients in the benign group were separated into groups 1 (n=95, <4 cm in tumor diameter) and 2 (n=37, ≥4 cm in tumor diameter). Also, 57 patients in the micropapillary carcinoma group underwent an endoscopic hemithyroidectomy (HT) (group 3) and were compared with 60 patients who received conventional open HT (group 4). Postoperative functional outcome, local complications, surgical outcomes, and pathological outcomes were compared between the groups. RESULTS: In the benign group, there was no significant difference in mean operating time, hospital stay, or overall perioperative complications between the two groups. In the micropapillary carcinoma group, mean operating time and hospital stay in group 3 were significantly longer than in group 4 (p=0.015 and p≤0.001). The overall perioperative complications did not differ significantly between the groups. The postoperative cosmetic result was better in groups 1-3 (endo group) than in group 4 (open group). CONCLUSION: ET via a gasless axillo-breast approach seems to be a safe procedure even for benign thyroid lesions ≥4 cm and micropapillary carcinomas. Although it has the advantage of better cosmetic results over open thyroidectomy, there is room for improvement in terms of lessening its invasiveness and shortening the operative time.ope

Cell Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages

Cell labeling and tracking are important processes in understanding biologic mechanisms and the therapeutic effect of inoculated cells in vivo. Numerous attempts have been made to label and track inoculated cells in vivo; however, these methods have limitations as a result of their biological effects, including secondary phagocytosis of macrophages and genetic modification. Here, we investigated a new cell labeling and tracking strategy based on metabolic glycoengineering and bioorthogonal click chemistry. We first treated cells with tetra-acetylated N-azidoacetyl-D-mannosamine to generate unnatural sialic acids with azide groups on the surface of the target cells. The azide-labeled cells were then transplanted to mouse liver, and dibenzyl cyclooctyne-conjugated Cy5 (DBCO-Cy5) was intravenously injected into mice to chemically bind with the azide groups on the surface of the target cells in vivo for target cell visualization. Unnatural sialic acids with azide groups could be artificially induced on the surface of target cells by glycoengineering. We then tracked the azide groups on the surface of the cells by DBCO-Cy5 in vivo using bioorthogonal click chemistry. Importantly, labeling efficacy was enhanced and false signals by phagocytosis of macrophages were reduced. This strategy will be highly useful for cell labeling and tracking

Brief Education on Microvasculature and Pit Pattern for Trainees Significantly Improves Estimation of the Invasion Depth of Colorectal Tumors

Objectives. This study was performed to evaluate the effectiveness of education for trainees on the gross findings identified by conventional white-light endoscopy (CWE), the microvascular patterns identified by magnifying narrow-band imaging endoscopy (MNE), and the pit patterns identified by magnifying chromoendoscopy (MCE) in estimation of the invasion depth of colorectal tumors. Methods. A total of 420 endoscopic images of 35 colorectal tumors were used. Five trainees estimated the invasion depth of the tumors by reviewing the CWE images before education. Afterwards, the trainees estimated the invasion depth of the same tumors after brief education on CWE, MNE and MCE images, respectively. Results. The initial diagnostic accuracy for deep submucosal invasion before education and after education on CWE, MNE, and MCE findings was 54.3%, 55.4%, 67.4%, and 76.6%, respectively. The diagnostic accuracy increased significantly after MNE education (P=0.028). The specificity for deep submucosal invasion before education and after education on CWE, MNE, and MCE findings was 47.9%, 45.7%, 65.0%, and 80.7%, respectively. The specificity increased significantly after MNE (P=0.002) and MCE (P=0.005) education. Conclusion. Brief education on microvascular pattern identification by MNE and pit pattern identification by MCE significantly improves trainees’ estimations of the invasion depth of colorectal tumors