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Laser capture microscopy

Jaebum Chung, Hangwen Lu, Xiaoze Ou, Haojiang Zhou, Changhuei Yang
Fourier ptychographic (FP) microscopy is a coherent imaging method that can synthesize an image with a higher bandwidth using multiple low-bandwidth images captured at different spatial frequency regions. The method's demand for multiple images drives the need for a brighter illumination scheme and a high-frame-rate camera for a faster acquisition. We report the use of a guided laser beam as an illumination source for an FP microscope. It uses a mirror array and a 2-dimensional scanning Galvo mirror system to provide a sample with plane-wave illuminations at diverse incidence angles...
November 1, 2016: Biomedical Optics Express
Dingrong Yi, Linghua Kong, Ranjith K Kankala, Zi Wang
There is an unmet need for researchers in life sciences and clinical pathology to obtain untainted target cells with very high accuracy, which are suitable for subsequent genome and protein analysis. In this paper, an electrostatic capture laser microdissection technology (ECM) is proposed and explained. Following microscopic identification and separation of target cells from the surrounding tissues using laser cutting, the ECM uses electrostatic forces to capture target cells. Validation experiments indicate that ECM can capture a wide assortment of contamination-free homogeneous samples, ranging from very tiny pieces of a few micrometers in diameter to large pieces with a surface area of over 40,000 µm2...
November 28, 2016: Microscopy and Microanalysis
Philip Pearce, Paul Brownbill, Jiří Janáček, Marie Jirkovská, Lucie Kubínová, Igor L Chernyavsky, Oliver E Jensen
During pregnancy, oxygen diffuses from maternal to fetal blood through villous trees in the placenta. In this paper, we simulate blood flow and oxygen transfer in feto-placental capillaries by converting three-dimensional representations of villous and capillary surfaces, reconstructed from confocal laser scanning microscopy, to finite-element meshes, and calculating values of vascular flow resistance and total oxygen transfer. The relationship between the total oxygen transfer rate and the pressure drop through the capillary is shown to be captured across a wide range of pressure drops by physical scaling laws and an upper bound on the oxygen transfer rate...
2016: PloS One
Rita Valenzuela, Maria A Costa-Besada, Javier Iglesias-Gonzalez, Emma Perez-Costas, Begoña Villar-Cheda, Pablo Garrido-Gil, Miguel Melendez-Ferro, Ramon Soto-Otero, Jose L Lanciego, Daniel Henrion, Rafael Franco, Jose L Labandeira-Garcia
The renin-angiotensin system (RAS) was initially considered as a circulating humoral system controlling blood pressure, being kidney the key control organ. In addition to the 'classical' humoral RAS, a second level in RAS, local or tissular RAS, has been identified in a variety of tissues, in which local RAS play a key role in degenerative and aging-related diseases. The local brain RAS plays a major role in brain function and neurodegeneration. It is normally assumed that the effects are mediated by the cell-surface-specific G-protein-coupled angiotensin type 1 and 2 receptors (AT1 and AT2)...
October 20, 2016: Cell Death & Disease
Kenji Hanabusa, Takuya Ueda, Shingo Takata, Masahiro Suzuki
Fluorescein-, benzothiazole-, quinoline-, stilbene-, and carbazole-containing fluorescent gelators have been synthesized by connecting gelation-driving segments, including l-isoleucine, l-valine, l-phenylalanine, l-leucine residue, cyclo(l-asparaginyl-l-phenylalanyl), and trans-(1R,2R)-diaminocyclohexane. The emission behaviors of the gelators were investigated, and their gelation abilities studied against 15 solvents. The minimum gel concentration, variable-temperature spectroscopy, transmission electron microscopy, scanning electron microscopy, fluorescence microscopy (FM), and confocal laser scanning microscopy (CLSM) were used to characterize gelation...
November 14, 2016: Chemistry: a European Journal
Ana Rita S Pereira, Teresa F Mendes, Augusto Ministro, Mariana Teixeira, Mariana Filipe, Jorge M Santos, Rita N Bárcia, J Goyri-O'Neill, Fausto Pinto, Pedro E Cruz, Helder J Cruz, Susana Constantino Rosa Santos
BACKGROUND: Mesenchymal stem cells derived from human umbilical cord tissue, termed UCX®, have the potential to promote a full range of events leading to tissue regeneration and homeostasis. The main goal of this work was to investigate UCX® action in experimentally induced hindlimb ischemia (HLI). METHODS: UCX®, obtained by using a proprietary technology developed by ECBio (Amadora, Portugal), were delivered via intramuscular injection to C57BL/6 females after unilateral HLI induction...
September 29, 2016: Stem Cell Research & Therapy
Toshiaki Takahashi, Florian Friedmacher, Julia Zimmer, Prem Puri
PURPOSE: Congenital diaphragmatic hernia (CDH) is presumed to originate from defects in the primordial diaphragmatic mesenchyme, mainly comprising of muscle connective tissue (MCT). Thus, normal diaphragmatic morphogenesis depends on the structural integrity of the underlying MCT. Developmental mutations that inhibit normal formation of diaphragmatic MCT have been shown to result in CDH. Desmin (DES) is a major filament protein in the MCT, which is essential for the tensile strength of the developing diaphragm muscle...
September 20, 2016: Pediatric Surgery International
Holly L Lutz, Nicholas J Marra, Felix Grewe, Jenny S Carlson, Vaidas Palinauskas, Gediminas Valkiūnas, Michael J Stanhope
Acquiring genomic material from avian malaria parasites for genome sequencing has proven problematic due to the nucleation of avian erythrocytes, which produces a large ratio of host to parasite DNA (∼1 million to 1 bp). We tested the ability of laser capture microdissection microscopy to isolate parasite cells from individual avian erythrocytes for four avian Plasmodium species, and subsequently applied whole genome amplification and Illumina sequencing methods to Plasmodium relictum (lineage pSGS1) to produce sequence reads of the P...
September 21, 2016: Parasitology Research
Evelyn Ploetz, Eitan Lerner, Florence Husada, Martin Roelfs, SangYoon Chung, Johannes Hohlbein, Shimon Weiss, Thorben Cordes
Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE)...
September 19, 2016: Scientific Reports
Agnieszka K Dąbrowska, Christian Adlhart, Fabrizio Spano, Gelu-Marius Rotaru, Siegfried Derler, Lina Zhai, Nicholas D Spencer, René M Rossi
The skin properties, structure, and performance can be influenced by many internal and external factors, such as age, gender, lifestyle, skin diseases, and a hydration level that can vary in relation to the environment. The aim of this work was to demonstrate the multifaceted influence of water on human skin through a combination of in vivo confocal Raman spectroscopy and images of volar-forearm skin captured with the laser scanning confocal microscopy. By means of this pilot study, the authors have both qualitatively and quantitatively studied the influence of changing the depth-dependent hydration level of the stratum corneum (SC) on the real contact area, surface roughness, and the dimensions of the primary lines and presented a new method for characterizing the contact area for different states of the skin...
2016: Biointerphases
Yung-Luen Shih, Jason Chou, Ming-Yang Yeh, Hsiao-Min Chou, Hsiu-Chen Chou, Hsu-Feng Lu, Hung-Sheng Shang, Fu-Shin Chueh, Yung-Lin Chu, Shu-Ching Hsueh, Jing-Gung Chung
Casticin, a polymethoxyflavone, has been demonstrated to possess anticancer activities, yet no study has shown in detail that casticin induces DNA damage in lung cancer cells. The purpose of this study was to investigate the possible molecular mechanisms of casticin which induce DNA damage and nuclear condensation in murine melanoma cancer B16F10 cells. In this study, by examining and capturing images using phase contrast microscopy, we found that casticin induced cell morphological changes. Moreover, it decreased the total number of viable cells which was measured by flow cytometry...
October 2016: Oncology Reports
Sorina Suarasan, Monica Focsan, Monica Potara, Olga Soritau, Adrian Florea, Dana Maniu, Simion Astilean
In this work, we developed a new pH- and temperature-responsive nanochemotherapeutic system based on Doxorubicin (DOX) noncovalently bound to biosynthesized gelatin-coated gold nanoparticles (DOX-AuNPs@gelatin). The real-time release profile of DOX was evaluated at different pH values (7.4, 5.3, and 4.6) and temperatures (22-45 °C) in aqueous solutions, and its therapeutic performance was examined in vitro against MCF-7 breast cancer cells. TEM, dark-field scattering, and wide-field fluorescence microscopy indicated the effective uptake of nanochemotherapeutics with the subsequent release and progressive accumulation of DOX in cell nuclei...
September 7, 2016: ACS Applied Materials & Interfaces
Toshiaki Takahashi, Florian Friedmacher, Julia Zimmer, Prem Puri
Introduction Diaphragmatic morphogenesis depends on proper formation of muscle connective tissue (MCT) and underlying extracellular matrix (ECM). Fibrillin-1 is an essential ECM protein and crucial for the structural integrity of MCT in the developing diaphragm. Recently, mutations in the fibrillin-1 gene (FBN1) have been identified in cases of congenital diaphragmatic hernia (CDH), thus suggesting that alterations in FBN1 gene expression may lead to diaphragmatic defects. We designed this study to investigate the hypothesis that the diaphragmatic expression of fibrillin-1 is decreased in the MCT of nitrofen-induced CDH...
August 14, 2016: European Journal of Pediatric Surgery
Dongzhi Yang, Liangzhu Feng, Casey A Dougherty, Kathryn E Luker, Daiqin Chen, Meagan A Cauble, Mark M Banaszak Holl, Gary D Luker, Brian D Ross, Zhuang Liu, Hao Hong
Angiogenesis, i.e. the formation of neovasculatures, is a critical process during cancer initiation, progression, and metastasis. Targeting of angiogenic markers on the tumor vasculature can result in more efficient delivery of nanomaterials into tumor since no extravasation is required. Herein we demonstrated efficient targeting of breast cancer metastasis in an experimental murine model with nano-graphene oxide (GO), which was conjugated to a monoclonal antibody (mAb) against follicle-stimulating hormone receptor (FSHR)...
October 2016: Biomaterials
Susanne Nichterwitz, Geng Chen, Julio Aguila Benitez, Marlene Yilmaz, Helena Storvall, Ming Cao, Rickard Sandberg, Qiaolin Deng, Eva Hedlund
Laser capture microscopy (LCM) coupled with global transcriptome profiling could enable precise analyses of cell populations without the need for tissue dissociation, but has so far required relatively large numbers of cells. Here we report a robust and highly efficient strategy for LCM coupled with full-length mRNA-sequencing (LCM-seq) developed for single-cell transcriptomics. Fixed cells are subjected to direct lysis without RNA extraction, which both simplifies the experimental procedures as well as lowers technical noise...
2016: Nature Communications
Ben J Glasgow
A conventional fluorescence microscope was previously constructed for simultaneous imaging of two colors to gain subdiffraction localization. The system is predicated on color separation of overlapping Airy discs, construction of matrices of Cartesian coordinates to determine locations as well as centers of the point spread functions of fluorophores. Quantum dots that are separated by as little as 10 nm were resolved in the x-y coordinates. Inter-fluorophore distances that vary by 10 nm could also be distinguished...
February 2016: Proceedings of SPIE
Liheng Bian, Jinli Suo, Jaebum Chung, Xiaoze Ou, Changhuei Yang, Feng Chen, Qionghai Dai
Fourier ptychographic microscopy (FPM) is a novel computational coherent imaging technique for high space-bandwidth product imaging. Mathematically, Fourier ptychographic (FP) reconstruction can be implemented as a phase retrieval optimization process, in which we only obtain low resolution intensity images corresponding to the sub-bands of the sample's high resolution (HR) spatial spectrum, and aim to retrieve the complex HR spectrum. In real setups, the measurements always suffer from various degenerations such as Gaussian noise, Poisson noise, speckle noise and pupil location error, which would largely degrade the reconstruction...
2016: Scientific Reports
Loïc Binan, Javier Mazzaferri, Karine Choquet, Louis-Etienne Lorenzo, Yu Chang Wang, El Bachir Affar, Yves De Koninck, Jiannis Ragoussis, Claudia L Kleinman, Santiago Costantino
The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications...
2016: Nature Communications
Emily M Kudalkar, Trisha N Davis, Charles L Asbury
Here we present our standard protocol for studying the binding of kinetochore proteins to microtubules as a paradigm for designing single-molecule total internal reflection fluorescence (TIRF) microscopy experiments. Several aspects of this protocol require empirical optimization, including the method for anchoring the polymer or substrate to the coverslip, the type and amount of blocking protein to prevent nonspecific protein adsorption to the glass, the appropriate protein concentration, the laser power, and the duration of imaging...
2016: Cold Spring Harbor Protocols
Ali Kamen, Shanhui Sun, Shaohua Wan, Stefan Kluckner, Terrence Chen, Alexander M Gigler, Elfriede Simon, Maximilian Fleischer, Mehreen Javed, Samira Daali, Alhadi Igressa, Patra Charalampaki
Diagnosis of tumor and definition of tumor borders intraoperatively using fast histopathology is often not sufficiently informative primarily due to tissue architecture alteration during sample preparation step. Confocal laser microscopy (CLE) provides microscopic information of tissue in real-time on cellular and subcellular levels, where tissue characterization is possible. One major challenge is to categorize these images reliably during the surgery as quickly as possible. To address this, we propose an automated tissue differentiation algorithm based on the machine learning concept...
2016: BioMed Research International
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