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Results for “Microscopy, Fluorescence, Multiphoton”

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PubMed2004

Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2.

We report on a novel and compact reliable laser source capable of short-wavelength two-photon laser scanning fluorescence microscopy based on soliton self-frequency shift effects in photonic crystal fibre. We demonstrate the function of the system by performing two-photon microscopy of smooth muscle cells and cardiac myocytes from the rat pulmonary vein and Chinese hamster ovary cells loaded with the fluorescent calcium indicator fura-2/AM.

Animals↗

PubMed2007

Nonlinear optical microscopy at wavelengths exceeding 1.4 microm using a synchronously pumped femtosecond-pulsed optical parametric oscillator.

A singly resonant femtosecond-pulsed synchronously pumped optical parametric oscillator (SPOPO) is reported that provides a wavelength-tunable alternative to the Cr:forsterite source for nonlinear microscopy applications. Periodically poled lithium niobate within an external resonator was synchronously pumped by a commercial fs-pulsed Nd:YLF laser source to provide fs-pulsed continuously tunable radiation from 1.45 to 1.63 microm. Third harmonic generation (THG) microscopy and three-photon laser scanning fluorescence microscopy (3PLSM) of a range of samples were subsequently performed using this long-wavelength SPOPO source. The ability to tune the excitation wavelength delivered to the sample significantly improved the efficiency of both the 3PLSM and THG imaging experiments.

Animals↗

PubMed2003

A mechanism-based in vitro anticancer drug screening approach for phenolic phytochemicals.

Plant-derived phenolic compounds, including polyphenols (e.g., tannins), flavonoids, and phenolic acids, have been under investigation for their anticancer therapeutic and chemoprevention properties. Recently, certain mechanisms underlying the differential effects of green tea polyphenols (GTPPs) on tumor versus normal cells have been determined. These suggest that GTPPs may simultaneously activate multiple pathways. However, existing screening methods are insufficient for the identification of agents that possess both a cytotoxic effect on tumor cells and a protective effect on normal cells. The current study describes the establishment of an in vitro survival/apoptosis testing system based on detecting these mechanisms by a double-fluorescence method. This system is able to screen potential chemopreventive or therapeutic agents from (but not limited to) plant-derived compounds based on the pathways differentially activated by the agents. Tumor cell death and normal cell survival are detected simultaneously, in a device that co-cultures normal human cells adjacent to human tumor cells.

Antineoplastic Agents↗

PubMed2005

Multi-photon excitation of intrinsic protein fluorescence and its application to pharmaceutical drug screening.

The majority of proteins contain intrinsic fluorophores as natural sensors of molecular structures, dynamics, and interactions. The intrinsic protein fluorescence signal allows for the label-free and, hence, undisturbed and rapid study of protein-ligand interactions. Ultraviolet-based drug screening is hampered by the background, photobleaching, light scattering, inner filter effects, and interfering assay compounds. Such problems can be overcome by means of molecular three-photon excitation (3PE) with infrared femtosecond light pulses since longer excitation wavelengths result in less Raleigh scattering, and the subfemtoliter (confocal-like) 3PE volume minimizes out-of-focus photobleaching, background generation, and inner filter effects. We demonstrate the general feasibility of 3PE for protein spectroscopy and illustrate the technique's excellent potential for high-throughput screening. By using the intrinsic fluorescence intensity of a protein-substrate, we were able to discriminate between ligands of different affinities in binding assays.

Computer-Aided Design↗

PubMed2003

An adjustable-threshold algorithm for the identification of objects in three-dimensional images.

MOTIVATION: To develop a highly accurate, practical and fast automated segmentation algorithm for three-dimensional images containing biological objects. To test the algorithm on images of the Drosophila brain, and identify, count and determine the locations of neurons in the images. RESULTS: A new adjustable-threshold algorithm was developed to efficiently segment fluorescently labeled objects contained within three-dimensional images obtained from laser scanning confocal microscopy, or two-photon microscopy. The result of the test segmentation with Drosophila brain images showed that the algorithm is extremely accurate and provided detailed information about the locations of neurons in the Drosophila brain. Centroids of each object (nucleus of each neuron) were also recorded into an algebraic matrix that describes the locations of the neurons. AVAILABILITY: Interested parties should send their request for the NeuronMapper(TM) program with the segmentation algorithm to artemp@bcm.tmc.edu.

Algorithms↗

PubMed2004

Two-photon imaging of calcium accumulation in rat cerebellar granule cells.

Topical accumulation of calcium ions in neurites and cell bodies of rat cerebellar granule cells was studied by two-photon microscopy in neurons loaded with the Ca-sensitive fluorescent indicator Oregon Green 488 Bapta. High potassium caused a rapid surge of internal calcium ([Ca2+]i) in the cell body, followed by a plateau. In neurites, [Ca2+]i reached a peak and then decreased back to the control level. In contrast, in neurons stimulated by NMDA, [Ca2+]i reached a steady level and remained constant as long as the agonist was present in the bath, either in the cell bodies or in neurites. In the latter, the response to NMDA treatment was smaller and heterogeneous, and [Ca2+]i increased in certain segments of the neurite, but not in others.

Animals↗

PubMed2004

Mechanism of protein binding to spherical polyelectrolyte brushes studied in situ using two-photon excitation fluorescence fluctuation spectroscopy.

We used two-photon excitation fluorescence fluctuation spectroscopy with photon counting histogram (PCH) analysis as a new tool to study the binding of globular proteins to colloidal particles in situ. Whereas fluorescence fluctuations are traditionally evaluated by calculating the autocorrelation function (fluorescence correlation spectroscopy), a complementary PCH analysis has been performed in this study which is advantageous when particle concentrations of a multicomponent system are of interest and the particles can be distinguished through particle brightness differences. The binding of two proteins, staphylococcal nuclease (SNase) and bovine serum albumin (BSA), to spherical polyelectrolyte brushes (SPB) was measured as a function of protein concentration and ionic strength of the solution at pH-values where SNase and BSA are positively and negatively charged, respectively. It has been found that SNase and BSA strongly bind to the SPB regardless of the protein charge. When the ionic strength of the solution is raised to 100 mM, the SPB become resistant to both proteins. These findings provide further evidence for a binding mechanism where the proteins are mainly driven to the SPB by the "counterion evaporation" force, while Coulomb interactions play a minor role. The results of this study characterize the potential of SPB as a new class of carrier particles for proteins whose use in biotechnological applications appears to be rewarding.

Acrylic Resins↗

PubMed2005

Interpreting two-photon imaging data of lymphocyte motility.

Recently, using two-photon imaging it has been found that the movement of B and T cells in lymph nodes can be described by a random walk with persistence of orientation in the range of 2 minutes. We interpret this new class of lymphocyte motility data within a theoretical model. The model considers cell movement to be composed of the movement of subunits of the cell membrane. In this way movement and deformation of the cell are correlated to each other. We find that, indeed, the lymphocyte movement in lymph nodes can best be described as a random walk with persistence of orientation. The assumption of motility induced cell elongation is consistent with the data. Within the framework of our model the two-photon data suggest that T and B cells are in a single velocity state with large stochastic width. The alternative of three different velocity states with frequent changes of their state and small stochastic width is less likely. Two velocity states can be excluded.

Algorithms↗

PubMed2005

Wavelet-based compression of M-FISH images.

Multiplex fluorescence in situ hybridization (M-FISH) is a recently developed technology that enables multi-color chromosome karyotyping for molecular cytogenetic analysis. Each M-FISH image set consists of a number of aligned images of the same chromosome specimen captured at different optical wavelength. This paper presents embedded M-FISH image coding (EMIC), where the foreground objects/chromosomes and the background objects/images are coded separately. We first apply critically sampled integer wavelet transforms to both the foreground and the background. We then use object-based bit-plane coding to compress each object and generate separate embedded bitstreams that allow continuous lossy-to-lossless compression of the foreground and the background. For efficient arithmetic coding of bit planes, we propose a method of designing an optimal context model that specifically exploits the statistical characteristics of M-FISH images in the wavelet domain. Our experiments show that EMIC achieves nearly twice as much compression as Lempel-Ziv-Welch coding. EMIC also performs much better than JPEG-LS and JPEG-2000 for lossless coding. The lossy performance of EMIC is significantly better than that of coding each M-FISH image with JPEG-2000.

Algorithms↗

PubMed2005

Quantitative analysis of the factors that affect the determination of colocalization coefficients in dual-color confocal images.

Colocalization analysis is a subject of great interest that allows us to determine those locations at which two or more types of molecules or structures are found simultaneously. In confocal dual-color images, in order to consider a given position as colocalized, the following two conditions must be satisfied: 1) the colors emitted by the molecules labeled with different fluorophores must occupy the same pixel in the image and 2) the intensities of each component of the image must be within a certain range. Since it is not straightforward to assess these two conditions without ambiguity, this can lead to either considering false colocalizations as positive, or, conversely, leaving out positive locations. In fact, at present, there is not a general procedure to determine the area of colocalization that contains those pixels that can be considered as really colocalized (i.e., the pixels for which the above conditions are fulfilled). As a result, it is the user who must decide on the appropriate selection which introduces personal bias. These issues and the guides for a computational procedure independent of the user that allows us to quantify the degree of colocalization in a dual-color system are discussed.

Algorithms↗

PubMed2006

Robust estimation of bioaffinity assay fluorescence signals.

In this paper, the challenging problem of robust mean-signal estimation of a single-step microparticle bioaffinity assay is investigated. For this purpose, a density estimation-based robust algorithm (DER) was developed. The DER algorithm was comparatively evaluated with four other parameter estimation methods (mean value, median filtering, least square estimation, Welsch robust m-estimator). Two important questions were raised and investigated: 1) Which of the five methods can robustly estimate the mean bioaffinity signal? and 2) How many microparticles need to be measured in order to obtain an accurate estimate of the mean signal value? To answer the questions, bootstrap and coefficient of variation (CV) analyses were performed. In the CV analysis, the DER algorithm gave the best results: The CV ranged from 0.8% to 4.9% when the number of microparticles used for the mean signal estimation varied from 800 to 30. In the bootstrap analysis of the standard error, the DER algorithm had the smallest variance. As a conclusion, it can be underlined that: 1) of all methods tested, the DER algorithm gave the most consistent and reproducible results according to the bootstrap and CV analysis; 2) using the DER algorithm accurate estimates could be calculated based on 80-100 particles, corresponding to a typical assay measurement time of 1 min; and 3) the investigated bioaffinity signals contained a large number of outliers (observations that severely deviate from the majority of data) and therefore robust techniques were necessary for the mean signal estimation tasks.

Algorithms↗

PubMed2005

Maximum-likelihood techniques for joint segmentation-classification of multispectral chromosome images.

Traditional chromosome imaging has been limited to grayscale images, but recently a 5-fluorophore combinatorial labeling technique (M-FISH) was developed wherein each class of chromosomes binds with a different combination of fluorophores. This results in a multispectral image, where each class of chromosomes has distinct spectral components. In this paper, we develop new methods for automatic chromosome identification by exploiting the multispectral information in M-FISH chromosome images and by jointly performing chromosome segmentation and classification. We (1) develop a maximum-likelihood hypothesis test that uses multispectral information, together with conventional criteria, to select the best segmentation possibility; (2) use this likelihood function to combine chromosome segmentation and classification into a robust chromosome identification system; and (3) show that the proposed likelihood function can also be used as a reliable indicator of errors in segmentation, errors in classification, and chromosome anomalies, which can be indicators of radiation damage, cancer, and a wide variety of inherited diseases. We show that the proposed multispectral joint segmentation-classification method outperforms past grayscale segmentation methods when decomposing touching chromosomes. We also show that it outperforms past M-FISH classification techniques that do not use segmentation information.

Algorithms↗

PubMed2006

Optimized temporal response in multichannel two-photon fluorescence lifetime microscopy using a photonic crystal fibre.

The integration of fibre optics into an imaging system for the convenient delivery and collection of light has resulted in many hybrid forms of novel biomedical optical instrumentation. Although it is extremely robust and cost effective, fibre integration requires special consideration in a time-domain fluorescence lifetime imaging schema where multipath propagation in the fibre causes significant spread in photon transit times. In this study, we investigated the effect of the length of a multimode collection fibre on the temporal performance of a multichannel fluorescence lifetime microscope and demonstrated the effectiveness of a photonic crystal fibre as a means of optimizing the collection and delivery of emitted fluorescence in terms of temporal resolution. The findings are pertinent to all studies that employ a multimode optical fibre to collect and deliver an emitted fluorescence signal from a sample to a remote detector for measurement of the characteristic fluorescence lifetime.

Animals↗

PubMed2006

Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: differentiation of agonists and antagonists.

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit beta-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin-mediated clathrin-coated pits, whereas, beta-arrestin-2 conjugated green fluorescence protein (beta-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

Arrestins↗

PubMed2002

Miniaturization of fluorescence microscopes using fibre optics.

In both medical research and diagnostics characterization of biological tissue on the cellular level relies on high-resolution optical microscopy. In most cases, however, tissue is excised for microscopic investigation, in part because conventional microscopes are bulky instruments. Imaging of cells in the intact living organism has been difficult. Over the last decade several groups have developed miniature confocal microscopes that use fibre optics to deliver light to the specimen and to measure either reflected or excited fluorescence light. In addition, two-photon excitation recently has been employed in a small portable 'fibrescope'. A potential clinical application of these microscope probes is their endoscopic use for optical biopsy of inner organs or guidance of conventional biopsy. As a mobile research tool they may permit imaging of neuronal activity in the brain of awake, behaving animals. Here, we review technological approaches to build miniaturized fluorescence microscopes and discuss their potential applications.

Animals↗

PubMed2002

Functional imaging of single synapses in brain slices.

The strength of synaptic connections in the brain is not fixed, but can be modulated by numerous mechanisms. Traditionally, electrophysiology has been used to characterize connections between neurons. Electrophysiology typically reports the activity of populations of synapses, while most mechanisms of plasticity are thought to operate at the level of single synapses. Recently, two-photon laser scanning microscopy has enabled us to perform optical quantal analysis of individual synapses in intact brain tissue. Here we introduce the basic principle of the two-photon microscope and discuss its main differences compared to the confocal microscope. Using calcium imaging in dendritic spines as an example, we explain the advantages of simultaneous dual-dye imaging for quantitative calcium measurements and address two common problems, dye saturation and background fluorescence subtraction.

Animals↗

PubMed2005

Cooling blocks rat hippocampal neurotransmission by a presynaptic mechanism: observations using 2-photon microscopy.

Over the past decade there has been great interest in the therapeutic potential of brain cooling for epilepsy, stroke, asphyxia and other neurological diseases. However, there is still no consensus regarding the neurophysiological effect(s) of brain cooling. We employed standard physiological techniques and 2-photon microscopy to directly examine the effect of temperature on evoked neurotransmitter release in rat hippocampal slices. We observed a monotonic decline in extracellular synaptic potentials and their initial slope over the temperature range 33-20 degrees C, when the slices were cooled to a new set point in less than 5 s. Imaging the fluorescent synaptic marker FM1-43 with 2-photon microscopy showed that the same cooling protocol dramatically reduced transmitter release between 33 and 20 degrees C. Cooling also reduced the terminal FM1-43 destaining that was induced by direct depolarization with elevated K+, indicating that axonal conduction block cannot account for our observations. The temperature dependence of FM1-43 destaining correlated well with the effect of temperature on field potential slope, compatible with a presynaptic explanation for our electrophysiological observations. Optical measurement of FM1-43 dissociation from cell membranes was not affected by temperature, and rapid cooling of slices loaded with FM1-43 did not increase their fluorescence. Our experiments provide visible evidence that a major neurophysiological effect of cooling in the mammalian brain is a reduction in the efficacy of neurotransmitter release. This presynaptic effect may account for some of the therapeutic benefits of cooling in epilepsy and possibly stroke.

Animals↗

PubMed2005

A new quantitative (two-photon extracellular polar-tracer imaging-based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets.

We have developed an imaging approach to estimate the diameter of exocytic vesicles that are smaller than the resolution of an optical microscope and present within intact tissue. This approach is based on two-photon excitation imaging of polar tracers in the extracellular medium, is designated TEPIQ (two-photon extracellular polar-tracer imaging-based quantification), and has three variants. TEPIQ analysis of DeltaV measures vesicle volume with a fluid-phase tracer, sulforhodamine B (SRB). TEPIQ analysis of DeltaS determines vesicle surface area with a polar membrane tracer, FM1-43. TEPIQ analysis of DeltaV/DeltaS estimates vesicle diameter from the SRB/FM1-43 fluorescence ratio. TEPIQ analysis is insensitive to microscope settings because the same setup is used for calibration and actual experiments. We tested the validity of TEPIQ with glucose-induced exocytosis from beta-cells within pancreatic islets. The three TEPIQ variants yielded estimates for the mean diameter of exocytic vesicles of between 340 and 390 nm, consistent with the size of insulin granules. TEPIQ analysis relies on the combination of two-photon excitation imaging, the narrow intercellular spaces of intact tissue, and the presence of diffusible polar tracers in the extracellular medium. It allows quantitative imaging of exocytosis within secretory organs, yielding estimates of vesicle diameter with nanometer resolution.

Anatomy, Cross-Sectional↗