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PubMedDate not supplied

Mapping piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation microscopy.

Taking advantage of the electric field-enhanced second-harmonic generation effect in bulk gallium nitride (GaN) and indium gallium nitride (InGaN) quantum wells, we demonstrated the piezoelectric field distribution mapping in bulk GaN and InGaN multiple-quantum-well (MQW) samples using scanning second-harmonic generation (SHG) microscopy. Scanning SHG microscopy and the accompanying third-harmonic generation (THG) microscopy of the bulk GaN sample were demonstrated using a femtosecond Cr:forsterite laser at a wavelength of 1230 nm. Taking advantage of the off-resonant electric field-enhanced SHG effect and the bandtail state-resonance THG effect, the second- and third-harmonic generation microscopic images obtained revealed the piezoelectric field and bandtail state distributions in a GaN sample. Combined with 720 nm wavelength excited two-photon fluorescence microscopy in the same sample, the increased defect density around the defect area was found to suppress bandedge photoluminescence, to increase yellow luminescence, to increase bandtail state density, and to decrease residue piezoelectric field intensity. Scanning SHG microscopy of the InGaN MQW sample was resonant excited with 800 nm femtosecond pulses from a Ti:sapphire laser in order to suppress SHG contribution from the bulk GaN substrate. Taking advantage of the strong piezoelectric field inside the InGaN quantum well, the wavelength resonant effect, and the electric field-enhanced SHG effect of InGaN quantum wells, resonant scanning SHG microscopy revealed the piezoelectric field distribution inside the wells. Combined with accompanying three-photon fluorescence microscopy from the bulk GaN substrate underneath the quantum wells, the direct correspondence between the piezoelectric field strength inside the quantum well and the substrate quality can be obtained. According to our study, the GaN substrate area with bright bandedge luminescence corresponds to the area with strong SHG signals indicating a higher stained-induced piezoelectric field. These scanning harmonic generation microscopies exhibit superior images of the piezoelectric field and defect state distributions in GaN and InGaN MQWs not available before. Combining with scanning multiphoton fluorescence microscopy, these techniques open new ways for the physical property study of this important material system and can provide interesting details that are not readily available by other microscopic techniques.

Journal Article↗

PubMed2004

Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

Erythrocytes↗

PubMed2003

Multiphoton endoscopy.

Despite widespread use of multiphoton fluorescence microscopy, development of endoscopes for nonlinear optical imaging has been stymied by the degradation of ultrashort excitation pulses that occurs within optical fiber as a result of the combined effects of group-velocity dispersion and self-phase modulation. We introduce microendoscopes (350-1000 microm in diameter) based on gradient-index microlenses that effectively eliminate self-phase modulation within the endoscope. Laser-scanning multiphoton fluorescence endoscopy exhibits micrometer-scale resolution. We used multiphoton endoscopes to image fluorescently labeled neurons and dendrites.

Animals↗

PubMed2006

Visualization of the post-Golgi trafficking of multiphoton photoactivated transferrin receptors.

Newly synthesized membrane proteins are sorted in the trans-Golgi network (TGN) on the basis of sorting signals carried in their cytoplasmic domains and delivered to their final destinations in the secretory and endocytic pathways. Although previous studies have suggested the involvement of early endosomes in the biosynthetic pathway of transmembrane proteins, the precise trafficking routes followed by the newly synthesized plasma membrane proteins, such as transferrin receptors (TfRs), after exit from the TGN remain unclear. In this report, first, we demonstrated the advantages of photoactivating PA-GFP, a variant of the Aequorea victoria green fluorescent protein (GFP), with multiphoton laser light rather than single-photon laser light, in terms of photoactivation efficiency and spatial resolution. We then applied the multiphoton photoactivation technique to selectively photoactivate the TfR tagged with PA-GFP (PA-GFP-TfR) at the TGN, and monitored the movement of the photoactivated PA-GFP-TfR in live cells. We observed that the PA-GFP-TfR photoactivated at the TGN are transported to the Tfn(+)EEA1(+) endosomal compartments after exiting the TGN. These data support the notion that early endosomes can serve as a sorting station for not only internalized plasma membrane proteins in the endocytic pathway but also newly synthesized membrane proteins in the post-Golgi secretory pathway.

Autoantigens↗

PubMed2004

Multiphoton polarization and generalized polarization microscopy reveal oleic-acid-induced structural changes in intercellular lipid layers of the skin.

We have demonstrated that both multiphoton polarization and generalized polarization (GP) microscopy may be combined to characterize the structural changes of intercellular lipids in skin. Both polarization and GP (at 440- and 490-nm emission) images obtained by analysis of Laurdan fluorescence suggest that the treatment of oleic acid results in a skin surface with a more random packing of lipid molecules, which allows easier water penetration. Our results show that combined polarization and GP microscopy can be used to characterize the physical and chemical changes in biological structures.

Culture Techniques↗

PubMed2005

Improved depth resolution in video-rate line-scanning multiphoton microscopy using temporal focusing.

By introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain video-rate images with depth resolution similar to point-by-point scanning multiphoton microscopy while mechanically scanning in only one dimension. This is achieved by temporal focusing of the illumination pulse: The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration (and highest peak intensity) at the focal plane before stretching again beyond it. This method is applied to produce, in a simple and scalable setup, video-rate two-photon excitation fluorescence images of Drosophila egg chambers with nearly 100,000 effective pixels and 1.5 microm depth resolution.

Animals↗

PubMed2005

Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy.

The successful study of dendritic signaling and computation requires the ability to simultaneously monitor neuronal activity at multiple cellular sites. While the difficulties of accessing dendritic submicron structures with conventional micropipette approaches are generally overcome by optical recording techniques, their spatio-temporal resolution has limited such studies to few sites or slow signals. Here we present a novel approach to functional imaging, termed random-access multiphoton (RAMP) microscopy, which combines multiphoton excitation with an inertia-free scanning mechanism. RAMP microscopy employs two-dimensional acousto-optic deflection to rapidly position a focused near-infrared ultrafast laser beam between dwell periods at multiple user-selected sites. Because neuronal structures are generally sparse, activity located throughout various compartments, including thin dendritic branches and spines, can be mapped at high frame rates while maintaining the signal-to-noise ratio of conventional scanning microscopy. Moreover, RAMP microscopy maintains the excellent structural imaging capability of multiphoton excitation, i.e., intrinsic optical sectioning and high lateral resolution from within highly light-scattering brain tissue. RAMP microscopy thus comprises a versatile tool for investigating correlations of dendritic structure and function with significantly enhanced experimental throughput.

Action Potentials↗

PubMed2006

Fourier-transform coherent anti-Stokes Raman scattering microscopy.

We report a novel Fourier-transform-based implementation of coherent anti-Stokes Raman scattering (CARS) microscopy. The method employs a single femtosecond laser source and a Michelson interferometer to create two pulse replicas that are fed into a scanning multiphoton microscope. By varying the time delay between the pulses, we time-resolve the CARS signal, permitting easy removal of the nonresonant background while providing high resolution, spectrally resolved images of CARS modes over the laser bandwidth (approximately 1500 cm(-1)). We demonstrate the method by imaging polystyrene beads in solvent.

Algorithms↗

PubMed2004

Cytomics--new technologies: towards a human cytome project.

BACKGROUND: Molecular cell systems research (cytomics) aims at the understanding of the molecular architecture and functionality of cell systems (cytomes) by single-cell analysis in combination with exhaustive bioinformatic knowledge extraction. In this way, loss of information as a consequence of molecular averaging by cell or tissue homogenisation is avoided. PROGRESS: The cytomics concept has been significantly advanced by a multitude of current developments. Amongst them are confocal and laser scanning microscopy, multiphoton fluorescence excitation, spectral imaging, fluorescence resonance energy transfer (FRET), fast imaging in flow, optical stretching in flow, and miniaturised flow and image cytometry within laboratories on a chip or laser microdissection, as well as the use of bead arrays. In addition, biomolecular analysis techniques like tyramide signal amplification, single-cell polymerase chain reaction (PCR), and the labelling of biomolecules by quantum dots, magnetic nanobeads, or aptamers open new horizons of sensitivity and molecular specificity at the single-cell level. Data sieving or data mining of the vast amounts of collected multiparameter data for exhaustive multilevel bioinformatic knowledge extraction avoids the inadvertent loss of information from unknown molecular relations being inaccessible to an a priori hypothesis. CHALLENGE: It seems important to address the challenge of a human cytome project using hypothesis-driven molecular information collection from disease associated cell systems, supplemented by systematic and exhaustive knowledge extraction. This will allow the description of the molecular setup of normal and abnormal cell systems within a relational knowledge system, permitting the standardised discrimination of abnormal cell states in disease. As one of the consequences, individualised predictions of further disease course in patients (predictive medicine by cytomics) by characteristic discriminatory data patterns will permit individualised therapies, identification of new pharmaceutical targets, and establishment of a standardised framework of relevant molecular alterations in disease. This special issue of Cytometry, on new technologies in cytomics, focuses on prominent examples of this presently fast-moving scientific field, and represents one of the preconditions for the formulation of a human cytome project.

Cell Biology↗

PubMed2004

Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation.

We compare conventional infrared laser based three-photon excitation with a visible laser based two-photon excitation scheme for imaging the ultraviolet fluorophore serotonin in solution and in live cells. To obtain a signal level of 1000 photons per second per mM serotonin solution, we need a back aperture power of 5 mW at 550 nm (for two-photon excitation) and 33 mW at 740 nm (for three-photon excitation). The detectivity of serotonin (defined as the concentration of serotonin that yields a signal equivalent to three times the standard deviation of the signal obtained from the buffer alone) is 12 microM for two-photon, and 220 microM for three-photon excitation. Surprisingly, for live cell imaging of vesicular serotonin in serotonergic cells, three-photon excitation appears to provide better image contrast than two-photon excitation. The origin of this is traced to the concentration-dependent shift of the serotonin emission spectrum.

Animals↗

PubMed2005

From micro to nano: recent advances in high-resolution microscopy.

Improving the spatial resolution of optical microscopes is important for a vast number of applications in the life sciences. Optical microscopy allows intact samples and living cells to be studied in their natural environment, tasks that are not possible with other microscopy methods (e.g. electron microscopy). Major advances in the past two decades have significantly improved microscope resolution. By using interference and structured light methods microscope resolution has been improved to approximately 100 nm, and with non-linear methods a ten times improvement has been demonstrated to a current resolution limit of approximately 30 nm. These methods bring together old theoretical concepts such as interference with novel non-linear methods that improve spatial resolution beyond the limits that were previously assumed to be unreachable.

Image Enhancement↗

PubMed2005

An extended vision for dynamic high-resolution intravital immune imaging.

The past few years have seen the application of confocal and especially two-photon microscopy to the dynamic high-resolution imaging of lymphocytes and antigen presenting cells within organs such as lymph nodes and thymus. After summarizing some of the published results obtained to date using these methods, we describe our view of how this technology will develop and be applied in the near future. This includes its extension to a wide variety of non-lymphoid tissues, to the tracking of functional responses in addition to migratory behavior, to the analysis of molecular events previously studied only in vitro, to dissection of the interplay between hematopoietic and stromal elements, to visualization of a wider array of cell types including neutrophils, macrophages, NK cells, NKT cells and others, and to the interaction of the host with infectious agents. Reaching these goals will depend on a combination of new tools for genetic manipulations, novel fluorescent reporters, enhanced instrumentation, and better surgical techniques for the extended imaging of live animals. The end result will be a new level of understanding of how orchestrated cell movement and interaction contribute to the physiological and pathological activities of the immune system.

Animals↗

PubMed2004

Multiphoton microscopy guides neurotrophin modification with poly(ethylene glycol) to enhance interstitial diffusion.

Brain-derived neurotrophic factor (BDNF) is a promising therapeutic agent for the treatment of neurodegenerative diseases. However, the limited distribution of this molecule after administration into the brain tissue considerably hampers its efficacy. Here, we show how multiphoton microscopy of fluorescently tagged BDNF in brain-tissue slices provides a useful and rapid screening method for examining the diffusion of large molecules in tissues, and for studying the effects of chemical modifications-for example, conjugating with polyethylene glycol (PEG)-on the diffusion constant. This single variable, obtained by monitoring short-term diffusion in real time, can be effectively used for rational drug design. In this study on fluorescently tagged BDNF and BDNF-PEG, we identify slow diffusion as a major contributing factor to the limited penetration of BDNF, and demonstrate how chemical modification can be used to overcome this barrier.

Animals↗

PubMed2006

Unraveling the relationship between macula densa cell volume and luminal solute concentration/osmolality.

At the macula densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both macula densa and cortical thick ascending limb (cTAL) cells occurs via furosemide-sensitive sodium-chloride-potassium cotransport. In macula densa, apical entry of sodium chloride leads to changes in cell volume, although there are conflicting data regarding the directional change in macula densa cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in macula densa cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and macula densa cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80 mmol/l at constant osmolality led to cell swelling in macula densa and cTAL cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80 mmol/l with concomitant increases in osmolality caused sustained decreases in macula densa cell volume but transient increases in cTAL cell volume. Increases in luminal osmolality with urea also resulted in macula densa cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is macula densa cell shrinkage, which may play a role in the macula densa cell signaling process.

Animals↗

PubMed2005

Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion.

We tracked pathogenic myelin basic protein-specific CD4+ effector T cells in early central nervous system (CNS) lesions of experimental autoimmune encephalomyelitis (EAE) by combining two-photon imaging and fluorescence video microscopy. We made two key observations: (a) the majority of the cells (65%) moved fast (maximal speed 25 microm/min) and apparently nondirected through the compact tissue; and (b) a second group of effector T cells (35%) appeared tethered to a fixed point. Polarization of T cell receptor and adhesion molecules (lymphocyte function-associated antigen 1) towards this fixed point suggests the formation of immune synapses. Nonpathogenic, ovalbumin-specific T cells were not tethered in the CNS and did not form synapse-like contacts, but moved through the tissue. After intrathecal injection of antigen, 40% of ovalbumin-specific T cells became tethered. Conversely, injection of anti-major histocompatibility complex class II antibodies profoundly reduced the number of stationary pathogenic T cells within the CNS (to 15%). We propose that rapid penetration of the CNS parenchyma by numerous autoimmune effector T cells along with multiple autoantigen-presentation events are responsible for the fulminate development of clinical EAE.

Animals↗

PubMed2005

A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids.

Synthetic vectors based on reducible polycations consisting of histidine and polylysine residues (HIS RPCs) were evaluated for their ability to deliver nucleic acids. Initial experiments showed that RPC-based vectors with at least 70% histidine content mediated efficient levels of gene transfer without requirement for the endosomolytic agent chloroquine. Significant gene transfer was observed in a range of cell types achieving up to a 5-fold increase in the percentage of transfected cells compared to 25 kDa PEI, a gold standard synthetic vector. In contrast to 25 kDa PEI, HIS RPCs also mediated efficient transfer of other nucleic acids, including mRNA encoding green fluorescent protein in PC-3 cells and siRNA directed against the neurotrophin receptor p75(NTR) in post-mitotic cultures of rat dorsal root ganglion cell neurons. Experiments to elevate intracellular glutathione and linear profiling of cell images captured by multiphoton fluorescent microscopy highlighted that parameters such as the molecular weight and rate of cleavage of HIS RPCs were important factors in determining transfection activity. Altogether, these results demonstrate that HIS RPCs represent a novel and versatile type of vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids. This should enable different or a combination of therapeutic strategies to be evaluated using a single type of polycation-based vector.

Animals↗

PubMed2004

Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes.

Oxidative stress-induced mitochondrial dysfunction has been shown to play a crucial role in the pathogenesis of a wide range of diseases. Protecting mitochondrial function, therefore, is vital for cells to survive during these disease processes. In this study, we demonstrate that melatonin, a chief secretory product of the pineal gland, readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptotic events and death in rat brain astrocytes (RBA-1). The early protection provided by melatonin in mitochondria of intact living cells was investigated by the application of time-lapse conventional, confocal, and multiphoton fluorescent imaging microscopy coupled with noninvasive mitochondria-targeted fluorescent probes. In particular, we observed that melatonin effectively prevented exogenously applied H2O2-induced mitochondrial swelling in rat brain astrocytes at an early time point (within 10 min) and subsequently reduced apoptotic cell death (150 min later). Other early apoptotic events such as plasma membrane exposure of phosphatidyl serine and the positive YOPRO-1 staining of the early apoptotic nucleus were also prevented by melatonin. A mechanistic study at the mitochondrial level related to the early protection provided by melatonin revealed that the indole molecule significantly reduced mitochondrial reactive oxygen species (ROS) formation induced by H2O2 stress. Melatonin also prevented mitochondrial ROS generation caused by other organic hydroperoxides including tert-butyl hydroperoxide and cumene hydroperoxide. This antioxidative effect of melatonin is more potent than that of vitamin E. Via its ability to reduce mitochondrial ROS generation, melatonin prevented H2O2-induced mitochondrial calcium overload, mitochondrial membrane potential depolarization, and the opening of the mitochondrial permeability transition (MPT) pore. As a result, melatonin blocked MPT-dependent cytochrome c release, the downstream activation of caspase 3, the condensation and karyorrhexis of the nucleus and apoptotic fragmentation of nuclear DNA. Thus, the powerful mitochondrial protection provided by melatonin reinforces its therapeutic potential to combat a variety of oxidative stress-induced mitochondrial dysfunctions as well as mitochondria-mediated apoptosis in various diseases.

Animals↗

PubMedDate not supplied

Second harmonic imaging of intrinsic signals in muscle fibers in situ.

We use second harmonic generation (SHG) imaging to study and quantify a strong intrinsic SHG signal in skeletal muscle fiber preparations and single isolated myofibrils. The intrinsic signal follows the striation pattern of the muscle cells and is positioned at the sarcomeric location of the myosin filaments. Interestingly, the signal is enhanced at the region where the myosin heads are located on the myosin filaments. As the intrinsic signal reflects the subcellular structure in an accurate way, SHG can be used for noninvasive high resolution structural imaging without exogenous labels in living muscle cells. This may be very important for detecting changes in myofibrillar organization occurring under pathophysiological conditions, e.g., in cardiac and skeletal myopathies. Due to the strong dependency of SHG on orientation and symmetries of the tissue, it may allow the study of dynamic interactions between the contractile proteins actin and myosin during force production and muscle shortening. Furthermore, SHG imaging can be combined with other nonlinear microscopical techniques, such as laser scanning multiphoton fluorescence microscopy, to simultaneously measure other dynamic cellular processes, representing a complementary method and extending the range of nonlinear microscopical methods.

Animals↗