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At least 451 records · Page 25Linked to original sources
PubMed2006

Simulation study of ratio calculation formulae of two-colour cDNA microarray data.

In cDNA microarray image processing, there are different methods for calculating the channel ratios. Standard microarray image analysis software, such as the Axon GenePix Pro, calculate the channel ratio from pixels that define a given spot using different methods (i.e. ratio of means, ratio of medians, mean of ratios, median of ratios, and regression ratio). Ratio values calculated using the different methods will then be listed in an output file. Microarray users have to choose one of the available methods at their own discretion, as no guidelines are provided. Therefore, we aim to address one of the most frequently asked questions by the microarray users: which ratio quantity provided by the image analysis software should be used? In this study, we have evaluated the five different ratio calculation approaches using simulation studies. Our results suggest that in most circumstances the ratio of means appears to be the best approach, particularly when the coefficient of variance (CV) of two-channel pixel intensities are small (<0.5) and channel intensities are large. Conversely, the ratio of medians and the median of ratios are more favourable when the CV is large.

Computer Simulation↗
PubMed2004

Single-molecule spectroscopy for nucleic acid analysis: a new approach for disease detection and genomic analysis.

Recently developed single-molecule spectroscopy (SMS) permits the analysis of fluorescent mixtures one molecule at a time. SMS methods provide the means to make rapid measurements on small, complex samples without the need for separations and target amplification enabling a new class of ultrasensitive nucleic acid assays. Here we give a brief overview of the current state of the art of SMS nucleic acid analysis and discuss ongoing work in our laboratory on two-color single-molecule fluorescence detection of specific nucleic acid sequences. In the future, two-color SMS nucleic acid assays will be used for a variety of applications including: gene expression analysis, disease detection and genomics.

DNA↗
PubMed2004

Applications of multi-photon microscopy in cell physiology.

Owing to its many optical and physical advantages for fluorescence excitation, multi-photon microscopy has found a wide range of uses in biology, both in structural and functional studies. In this review we highlight various applications of this technique in different fields of cell physiology and biophysical research. This includes studies on second messenger and ionic signals, on cellular metabolism as well as on genetically engineered probes and indicators. In addition, this techniques has been successfully applied for diffraction-limited photolysis of caged compounds. We also point out some of the problems that were encountered along the still rapidly evolving path of this technique, and draw attention to some of the ongoing developments that will further extend and improve the usefulness of multi-photon excitation, such as fluoresce life-time imaging (FLIM), fluorescence resonance energy transfer (FRET) and entangled photon microscopy approaches.

Animals↗
PubMed2007

Two-versus one photon excitation laser scanning microscopy: critical importance of excitation wavelength.

It is often anticipated that two-photon excitation (TPE) laser scanning microscopy should improve cell survival and tissue penetration relative to conventional one-photon excitation (OPE) confocal scanning laser microscopy (CLSM). However few studies have directly compared live cell imaging using one- vs two-photon laser scanning microscopy. We have used calcein-loaded in situ chondrocytes within cartilage as a model for quantitatively comparing these techniques. TPE reduced photo-bleaching and improved cell viability compared to OPE. Using improved detection sensitivity coupled with increased tissue penetration of the near infra-red TPE laser, it was possible to capture images deeper within the cartilage. However, the advantages of TPE vs OPE were strongly dependent on excitation wavelength. We conclude that optimising TPE conditions is essential for realizing the full benefits of this approach.

Animals↗
PubMed2003

Comparison of wide-field/deconvolution and confocal microscopy in bioengineering. Interest of multi-photon microscopy in the study of articular cartilage.

The increase in lateral and spatial resolutions is one of the major targets of research and development in the field of optical microscopies applied to living tissue. The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates its undeniable advantage on conventional fluorescence microscopy by segregating the planes outside the focussing plane. The methodological and technological advances of the last five years have been fast evolving, especially with regard to the optimisation of CLSM and deconvolution process. The limited analysis in thick tissue have given rise to the development of other techniques, multi-photon excitation microscopy in particular. In this paper, we have applied these techniques on major biological applications in bioengineering (endothelial cell, chondrocyte in 3D-culture, human cartilage) and discussed the technical limitations and perspectives.

Actins↗
PubMed2002

GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice.

To examine the behavior of tumor cells in tumors developing directly from mammary tissue in transgenic models, we have evaluated transgenic mice expressing green fluorescent protein (GFP). Using the mouse mammary virus promoter (MMTV) to directly drive expression of GFP, we find low levels of fluorescence in the mammary and salivary glands of transgenic animals. Using MMTV-Cre or WAP-Cre in combination with the Cre-activatable CAG-CAT-EGFP construct, we find stronger expression of GFP that is still tissue specific. These animals provide a range of expression of GFP that is suitable for analysis of transgenic mammary tumors and metastases in vivo at the single cell level of resolution.

Animals↗
PubMed2003

Clinically validated benchmarking of normalisation techniques for two-colour oligonucleotide spotted microarray slides.

Acquisition of microarray data is prone to systematic errors. A correction, called normalisation, must be applied to the data before further analysis is performed. With many normalisation techniques published and in use, the best way of executing this correction remains an open question. In this study, a variety of single-slide normalisation techniques, and different parameter settings for these techniques, were compared over many replicated microarray experiments. Different normalisation techniques were assessed through the distribution of the standard deviation of replicates from one biological sample across different slides. It is shown that local normalisation outperformed global normalisation, and intensity-based 'LOWESS' outperformed trimmed mean and median normalisation techniques. Overall, the top performing normalisation technique was a print-tip-based LOWESS with zero robust iterations. Lastly, we validated this evaluation methodology by examining the ability to predict oestrogen receptor-positive and -negative breast cancer samples with data that had been normalised using different techniques.

Algorithms↗
PubMed2006

[Quantum dots and their applications in cancer research].

Quantum dots are semiconductor nanocrystals with physical dimensions smaller than the exciton Bohr radius. As their fluorescence emissions are size-tunable, we can acquire any spectrum from ultraviolet (UV) to near-infrared by changing the particles' radiuses. The large Stokes shifts of quantum dots can be used to further improve detection sensitivity. The luminescence intensity is high and stable. Single quantum dots have longer excited state lifetimes, and they appear 10-20 times brighter than organic fluorescent dyes. And they have good biocompatibility because quantum dots with appropriate shells don't interfere with physiological processes, such as growth, development, signaling and motility. With the development of optical labeling and imaging technology, many present conventional biomedical methods have limitations in microcosmic direct real-time researches of bio-molecular interactions and early diagnosis of malignant tumors. The invention of quantum dots and their biomedical applications make them as good markers for tumor cell tracing and targeting in cancer research, such as prostate cancer, mammary cancer, cervical cancer, basal cell carcinoma, liver cancer, and melanoma. The current research is focused on tumor markers imaging and molecular interaction based on tangible carriers such as cells and tissues. The next research orientation would be to tap the potential of this highly sensitive technology to image tumor biomarkers in serum and other body fluids, so as to increase the early diagnosis rate of malignant tumors.

Animals↗
PubMed2006

A new technique for assessing hybrid layer interfacial micromorphology and integrity: two-photon laser microscopy.

PURPOSE: This study describes a two-photon laser fluorescence microscopy technique developed to evaluate the interfacial micromorphology of the hybrid layer in bonded restorations. MATERIALS AND METHODS: Micropermeability of the hybrid layer was characterized by means of simultaneously contrasting a dye-containing adhesive with a differently colored dye placed into the pulp chamber and allowed to diffuse toward the different-colored hybrid layer. A fluorescent red dye (rhodamine B) was incorporated into a commercial dentin bonding agent. Class I preparations (margins in enamel) were made on extracted human third molars. The teeth were restored using conventional methods: bonding agent, composite, finishing, and polishing. An aqueous solution of a yellow/green dye (fluorescein) was then placed into the pulp chamber for 3 h, allowing time to diffuse toward the different-colored bonded interface. The teeth were then embedded, sectioned, and microscopically analyzed using two-photon laser microscopy at 40X magnification. RESULTS: Subsurface fluorescent imaging using this technique enabled interfacial micromorphology to be characterized at submicrometer resolution and provided high-contrast images. The quality of surrounding structures and potential presence of gaps were also precisely assessed. CONCLUSION: Two-photon laser microscopy provided high quality, high-resolution images of the bonded interface and surrounding areas, allowing accurate qualitative and quantitative analysis of the structure and integrity of the hybrid layer.

Bisphenol A-Glycidyl Methacrylate↗
PubMedDate not supplied

Fluorene-based fluorescent probes with high two-photon action cross-sections for biological multiphoton imaging applications.

Two-photon fluorescence microscopy is a powerful tool for the study of dynamic cellular processes and live-cell imaging. Many commercially available fluorescent probes have been used in multiphoton-based imaging studies despite exhibiting relatively low two-photon absorption cross-section values in the tunability range of ultrafast Ti:sapphire lasers commonly used in multiphoton microscopy imaging. Furthermore, available fluorophores may be plagued with low fluorescence quantum yield and/or photoinstability (i.e., photobleaching) on exposure to the high peak power and photon density provided by the ultrafast laser source. To address the demand for better performing dyes, we prepare fluorophores tailored for multiphoton imaging. These fluorophores are based on the fluorene ring system, known to exhibit high fluorescence quantum yield (>0.7) and high photostability. Furthermore, an amine-reactive fluorescent probe for the covalent attachment onto amine-containing biomolecules is also prepared. Epi-fluorescence and two-photon fluorescence microscopy images of H9c2 rat cardiomyoblasts stained with an efficient two-photon absorbing fluorene fluorophore is demonstrated. Additionally, single-photon spectral characteristics of the amine-reactive fluorophore, as well as the two-photon absorption cross sections of its model adduct in solution, and spectral characterization of a bovine serum albumin (BSA) as a model bioconjugate are presented.

Animals↗
PubMed2003

Analysis of histology specimens using lifetime multiphoton microscopy.

Observations of cells or tissues with fluorescence microscopy can provide unique insights into cellular physiology and structure. Such information may reveal the pathological state of a tissue to the physician or information on cytoskeletal dynamics to the research scientist. However, problems of overlapping spectra, low signal, and light scatter impose serious limitations on what can be achieved in practice with fluorescence microscopy. These problems can be addressed in part by the development of new imaging modalities that make maximum use of the information present in the fluorescence signal. We describe the application of a new technology to the study of standard histological pathology specimens: a multiphoton excitation fluorescence microscope that incorporates a novel, photon-counting detector that measures the excited-state lifetimes of fluorescent probes. In initial investigations, we have applied this system to the observation of C. elegans embryos and primate histology specimens, with the objective of identifying potentially diagnostic signatures. Our findings demonstrate that lifetime multiphoton microscopy has considerable potential as a diagnostic tool for pathological investigations.

Animals↗
PubMed2003

Water-soluble quantum dots for multiphoton fluorescence imaging in vivo.

The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

Adipose Tissue↗
PubMed2005

Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy.

BACKGROUND: The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. RESULTS: Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1) the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2) direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. CONCLUSION: Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

Animals↗
PubMed2006

Intracoronary shunt-induced endothelial cell damage in porcine heart.

BACKGROUND: Injury to and dysfunction of the endothelium can compromise the patency of coronary arteries and lead to impaired perfusion of the heart. Such injury can occur after the application of an intravascular shunt and/or a snare to a coronary artery during bypass surgery. In this study, multiphoton microscopy was used to assess the integrity of endothelium in porcine coronary arteries subjected to shunting and snaring during off-pump coronary artery bypass grafting (OPCAB). MATERIALS AND METHODS: In open chest porcine, the left anterior descending artery was manipulated in different regions using snare and shunt to simulate OPCAB. Sections of left anterior descending artery were labeled with fluorescent dyes to evaluate the viability of the endothelium. The structural integrity of the endothelium was evaluated by calcein- and ethidium homodimer-mediated fluorescence. Endothelial functional viability was assessed by measuring cellular esterase activity, calcium mobilization, and endothelial nitric oxide synthase-mediated generation of nitric oxide using fluorescence dyes and multiphoton microscopy. RESULTS: Substantial endothelial damage was observed in shunted region of the coronary arteries. In contrast, endothelium remained structurally viable in regions that were snared, similar to control regions of the coronary arteries that were not manipulated. Esterase activity, calcium mobilization, and nitric oxide generation was greater in the control and snared regions of the coronary arteries in comparison to the shunted region. CONCLUSIONS: The use of intracoronary shunts led to structural damage and attenuation of endothelial function in porcine coronary arteries, whereas snared vessels maintained their viability and integrity, similar to the control sections. The routine use of shunts in OPCABG may lead to endothelial damage and possibly to long-term graft failure.

Anastomosis, Surgical↗
PubMed2006

Applications of combined spectral lifetime microscopy for biology.

Live cell imaging has been greatly advanced by the recent development of new fluorescence microscopy-based methods such as multiphoton laser-scanning microscopy, which can noninvasively image deep into live specimens and generate images of extrinsic and intrinsic signals. Of recent interest has been the development of techniques that can harness properties of fluorescence, other than intensity, such as the emission spectrum and excited state lifetime of a fluorophore. Spectra can be used to discriminate between fluorophores, and lifetime can be used to report on the microenvironment of fluorophores. We describe a novel technique-combined spectral and lifetime imaging-which combines the benefits of multiphoton microscopy, spectral discrimination, and lifetime analysis and allows for the simultaneous collection of all three dimensions of data along with spatial and temporal information.

Animals↗
PubMed2003

An increase in surface area is not required for cell division in early sea urchin development.

Cell division requires an increase in surface area to volume ratio. During early development, surface area can increase, volume can decrease, or surface topography can be optimized to allow for division. While exocytosis is thought to be essential for division [Mol. Biol. Cell 10 (1999), 2735; Proc. Natl. Acad. Sci. USA 99 (2002), 3633], exocytosis doesn't always yield an increase in surface area [Proc. Natl. Acad. Sci. USA 79 (1982), 6712]. We used multiphoton laser scanning microscopy, fluorescence spectroscopy, and electron microscopy to monitor membrane trafficking, surface area, volume, and surface topography during early sea urchin development. Despite extensive membrane trafficking monitored by FM 1-43 fluorescence, we find that the net surface area of the embryo does not change prior to the eight-cell stage. During this period, embryo volume decreases by 15%, and microvilli disappear from interior facing membrane segments. Thus, the first three cell divisions utilize residual membrane liberated by decreasing cytoplasmic volume, and reducing microvilli density on interior facing membranes. Only after the eight-cell stage was a net increase in FM 1-43 fluorescence from the embryo surface detected. Our data suggest that compensatory endocytosis is downregulated after this developmental stage to yield an increase in surface area for cell division.

Animals↗
PubMed2003

Imaging of angiogenesis: from microscope to clinic.

Advances in imaging are transforming our understanding of angiogenesis and the evaluation of drugs that stimulate or inhibit angiogenesis in preclinical models and human disease. Vascular imaging makes it possible to quantify the number and spacing of blood vessels, measure blood flow and vascular permeability, and analyze cellular and molecular abnormalities in blood vessel walls. Microscopic methods ranging from fluorescence, confocal and multiphoton microscopy to electron microscopic imaging are particularly useful for elucidating structural and functional abnormalities of angiogenic blood vessels. Magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), ultrasonography and optical imaging provide noninvasive, functionally relevant images of angiogenesis in animals and humans. An ongoing dilemma is, however, that microscopic methods provide their highest resolution on preserved tissue specimens, whereas clinical methods give images of living tissues deep within the body but at much lower resolution and specificity and generally cannot resolve vessels of the microcirculation. Future challenges include developing new imaging methods that can bridge this resolution gap and specifically identify angiogenic vessels. Another goal is to determine which microscopic techniques are the best benchmarks for interpreting clinical images. The importance of angiogenesis in cancer, chronic inflammatory diseases, age-related macular degeneration and reversal of ischemic heart and limb disease provides incentive for meeting these challenges.

Angiogenesis Inhibitors↗