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PubMed2006

Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts.

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Arabidopsis↗
PubMed1999

The Radio Afterglow and the Host Galaxy of the X-Ray-rich GRB 981226.

We report the discovery of a radio transient VLA 232937.2-235553, coincident with the proposed X-ray afterglow for the gamma-ray burst GRB 981226. This gamma-ray burst (GRB) has the highest ratio of X-ray to gamma-ray fluence of all the GRBs detected by BeppoSAX so far, and yet no corresponding optical transient was detected. The radio light curve of VLA 232937.2-235553 is qualitatively similar to that of several other radio afterglows. At the subarcsecond position provided by the radio detection, optical imaging reveals an extended R=24.9 mag object, which we identify as the host galaxy of GRB 981226. Afterglow models that invoke a jetlike geometry for the outflow or that require an ambient medium with a radial density dependence, such as that produced by a wind from a massive star, are both consistent with the radio data. Furthermore, we show that the observed properties of the radio afterglow can explain the absence of an optical transient without the need for large extinction local to the GRB.

Journal Article↗
PubMed1992

[An image analysis system for optic disc in glaucoma].

An image analysis system for glaucomatous optic disc was developed. The key for optic disc measurement was to trace accurately the boundaries of the optic cup and disc. The 2-dimension measurement modality adopted was the heuristic boundary tracing method, and the 3-dimension measurement was performed by observation of the photographs, arbitrary selection of points, and automatic plotting and calculation. In order to measure accurately the optic disc in glaucoma follow-up, the authors made use of the cross correlation line by line scanning method, after proper correction of the photographs, to detect any positional shift in two photographs. By clinical verification, the 95% confidence criterion of normal optic cup boundary shifting was 1.7 pixels. The optic disc diameter of the disc images was over 200 pixels, and any shift over 2 pixels (1%) would have clinical significance, which was a high degree of precision.

Glaucoma↗
PubMed2004

ZP8, a neuronal zinc sensor with improved dynamic range; imaging zinc in hippocampal slices with two-photon microscopy.

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(2+) sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn(2+) complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca(2+) and Mg(2+). In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM). The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn(2+) within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn(2+) stores.

Animals↗
PubMed2004

Circulatory basis of fMRI signals: relationship between changes in the hemodynamic parameters and BOLD signal intensity.

Blood oxygenation level-dependent functional magnetic resonance imaging (BOLD-fMRI) is widely used as a tool for functional brain mapping. During brain activation, increases in the regional blood flow lead to an increase in blood oxygenation and a decrease in paramagnetic deoxygenated hemoglobin (deoxy-Hb), causing an increase in the MR signal intensity at the site of brain activation. However, not a few studies using fMRI have failed to detect activation of areas that ought to have been activated. We assigned BOLD-positive (an increase in the signal intensity), BOLD-negative (a decrease in the signal intensity), and BOLD-silent (no change) brain activation to respective circulatory conditions through a description of fMRI signals as a function of the concentration of oxygenated Hb (oxy-Hb) and deoxy-Hb obtained with near-infrared optical imaging (NIOI). Using this model, we explain the sensory motor paradox in terms of BOLD-positive, BOLD-negative, and BOLD-silent brain activation.

Adult↗
PubMed2005

Brain tissue phantoms for optical near infrared imaging.

In this study, solid, stable, and cost-effective optical phantoms of scalp-skull, white matter and grey matter are developed by inverse method. To begin with, to obtain a range of optical parameters, absorption and reduced scattering coefficients (mu(a) and mu(s)', respectively), 20 homogeneous phantoms were made of paraffin wax by using optically contrast black and highly scattering white colouring materials in different combination. By comparing the measured reflectance values for each phantom got from the four channel reflectometer with that obtained from steady-state diffusion equation, the values of mu(a) and mu(s)' were determined. Next, phantoms which exhibit specific optical properties of scalp-skull, white and grey matter are developed iteratively by comparing actual reflectance measurements got by adjusting the colour concentration with the predicted reflectance values from the diffusion equation. This is done as follows: to obtain mu(a) of 0.04 mm(-1) for scalp-skull, 9.5 mg of black dye per 100 ml of wax added since more attenuation of light occurs in bone tissue. To obtain a mu(s)' 6.0 mm(-1) for white matter in brain tissue, 190 mg of white dye per 100 ml of wax was used to facilitate more scatter of light. The colour concentrations of phantoms were then adjusted to obtain the predetermined values of optical parameters for scalp-skull, grey and white matter.

Animals↗
PubMed1999

Correlated measurements of free and total intracellular calcium concentration in central nervous system neurons.

Transient changes in the intracellular concentration of free calcium ([Ca2+])i) act as a trigger or modulator for a large number of important neuronal processes. Such transients can originate from voltage- or ligand-gated fluxes of Ca2+ into the cytoplasm from the extracellular space, or by ligand- or Ca2+(-)gated release from intracellular stores. Characterizing the sources and spatio-temporal patterns of [Ca2+]i transients is critical for understanding the role of different neuronal compartments in dendritic integration and synaptic plasticity. Optical imaging of fluorescent indicators sensitive to free Ca2+ is especially suited to studying such phenomena because this approach offers simultaneous monitoring of large regions of the dendritic tree in individual living central nervous system neurons. In contrast, energy-dispersive X-ray (EDX) microanalysis provides quantitative information on the amount and location of intracellular total, i.e., free plus bound, calcium (Ca) within specific subcellular dendritic compartments as a function of the activity state of the neuron. When optical measurements of [Ca2+]i transients and parallel EDX measurements of Ca content are used in tandem, and correlated simultaneously with electrophysiological measurements of neuronal activity, the combined information provides a relatively general picture of spatio-temporal neuronal total Ca fluctuations. To illustrate the kinds of information available with this approach, we review here results from our ongoing work aimed at evaluating the role of various Ca uptake, release, sequestration, and extrusion mechanisms in the generation and termination of [Ca2+]i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. Our observations support the long-standing speculation that the dendritic endoplasmic reticulum acts not only as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses, but also as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca2+ influx.

Animals↗
PubMed2001

Poly[N-(2-hydroxypropyl)methacrylamide] polymers diffuse in brain extracellular space with same tortuosity as small molecules.

Integrative optical imaging was used to show that long-chain synthetic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) polymers in a range of molecular weights from 7.8 to 1057 kDa were able to diffuse through the extracellular space in rat neocortical slices. Tortuosity (square root of ratio of diffusion coefficient in aqueous medium to that in brain) measured with such polymers averaged 1.57, a value similar to that obtained previously with tetramethylammonium, a small cation. When PHPMA was conjugated with bovine serum albumin (BSA) to make a bulky polymer with molecular weight 176 kDa, the tortuosity rose to 2.27, a value similar to that obtained previously with BSA alone and with 70-kDa dextran. The method of image analysis was justified with diffusion models involving spherical and nonspherical initial distributions of the molecules.

Animals↗
PubMed2005

Imaging in neurooncology.

Imaging in patients with brain tumors aims toward the determination of the localization, extend, type, and malignancy of the tumor. Imaging is being used for primary diagnosis, planning of treatment including placement of stereotaxic biopsy, resection, radiation, guided application of experimental therapeutics, and delineation of tumor from functionally important neuronal tissue. After treatment, imaging is being used to quantify the treatment response and the extent of residual tumor. At follow-up, imaging helps to determine tumor progression and to differentiate recurrent tumor growth from treatment-induced tissue changes, such as radiation necrosis. A variety of complementary imaging methods are currently being used to obtain all the information necessary to achieve the above mentioned goals. Computed tomography and magnetic resonance imaging (MRI) reveal mostly anatomical information on the tumor, whereas magnetic resonance spectroscopy and positron emission tomography (PET) give important information on the metabolic state and molecular events within the tumor. Functional MRI and functional PET, in combination with electrophysiological methods like transcranial magnetic stimulation, are being used to delineate functionally important neuronal tissue, which has to be preserved from treatment-induced damage, as well as to gather information on tumor-induced brain plasticity. In addition, optical imaging devices have been implemented in the past few years for the development of new therapeutics, especially in experimental glioma models. In summary, imaging in patients with brain tumors plays a central role in the management of the disease and in the development of improved imaging-guided therapies.

Animals↗
PubMed1994

Pixel perception and voxel vision constructs for a new paradigm of dental care.

The introduction to dentistry of a multimedia computed approach with instant images, optical impressions, electronic periodontal probing and occlusal contact measurements, electronic storage and communications, has the potential to improve the quality of care, significantly reduce downtime, simplify infection control, expedite treatment, and take the dentist off the "treadmill" of the multi-operatory practice.

Computer-Aided Design↗
PubMed1999

Analysis of optical signals evoked by peripheral nerve stimulation in rat somatosensory cortex: dynamic changes in hemoglobin concentration and oxygenation.

The origins of reflected light changes associated with neuronal activity (optical signals) were investigated in rat somatosensory cortex with optical imaging, microspectrophotometry, and laser-Doppler flowmetry, and dynamic changes in local hemoglobin concentration and oxygenation were focused on. Functional activation was carried out by 2-second, 5-Hz electrical stimulation of the hind limb under chloralose anesthesia. These measurements were performed at the contralateral parietal cortex through a thinned skull. Regional cortical blood flow (rCBF) started to rise 1.5 seconds after the stimulus onset, peaked at 3.5 seconds (26.7% +/- 9.7% increase over baseline), and returned to near baseline by 10 seconds. Optical signal responses at 577, 586, and 805 nm showed a monophasic increase in absorbance coincident with the increase in rCBF; however, the signal responses at 605 and 760 nm were biphasic (an early increase and late decrease in absorbance) and microanatomically heterogeneous. The spectral changes of absorbance indicated that the concentrations of both total hemoglobin and oxyhemoglobin increased together with rCBF; deoxyhemoglobin, increased slightly but distinctly (P = 0.016 at 1.0 seconds, P = 0.00038 at 1.5 seconds) just before rCBF increases, then decreased. The authors conclude that activity-related optical signals are greatly associated with a moment-to-moment adjustment of rCBF and metabolism to neuronal activity.

Animals↗
PubMed2005

Applications of optical coherence tomography in dermatology.

Histology represents the gold standard for morphological investigation of the skin, though biopsy may alter the original morphology, is non-repeatable on the same site and always requires an iatrogenic trauma. In the past decade, advances in optics, fibre as well as laser technology have enabled the development of a novel non-invasive optical biomedical imaging technique, optical coherence tomography (OCT). The latter is based on a classic optical measurement method known as low-coherence interferometry that enables non-invasive, high resolution, two- or three-dimensional, cross-sectional imaging of microstructural morphology in biological tissue in situ. Using conventional OCT with a lateral resolution of 10-15 microm, the stratum corneum of glabrous skin (palmoplantar), the epidermis and the upper dermis can usually be identified, as well as skin appendages and blood vessels. For example, non-invasive monitoring of cutaneous inflammation, hyperkeratotic conditions and photoadaptive processes is possible by means of OCT. Furthermore, the development of high-output broadband light sources, e.g. femtosecond Ti:sapphire laser, might soon enable ultrahigh image resolutions of about 1 microm in order to investigate skin tissue on the cellular level, which could potentially allow the differentiation between benign and malignant tissues. Beyond a high resolution morphology in OCT images, tissue characterization by additional local physical parameters, such as the scattering coefficient and refractive index may be of great value, in particular in cosmetics and the pharmaceutical industry. Functional OCT imaging based on spectroscopy, tissue birefringence, elastography and Doppler flow reveals further information on tissue properties and represents an important progress of OCT technique in the field of dermatology. Therefore, the advanced versions of OCT technique might not only lead to significant new insights in skin physiology and pathology, but also in diagnosis and therapeutic control of cutaneous disorders with respect to non-invasive diagnosis of conditions and monitoring of disease activity in addition to treatment effects over time.

Humans↗
PubMedDate not supplied

Repetitive, noninvasive imaging of cyclooxygenase-2 gene expression in living mice.

The cyclooxygenase-2 (COX-2) gene plays a role in a wide variety of normal physiologic pathways and is a major target of pharmacologic intervention in a large number of pathophysiologic contexts, including pain, fever, inflammation, and cancer. Expression of the COX-2 gene is induced in a wide range of cells, in response to an ever-increasing number of stimuli. The regulation of the COX-2 gene has been the subject of extensive study, using traditional transfection techniques with reporter gene constructs. Regulation of the COX-2 gene in living animals, however, requires sacrifice of the animal and in situ hybridization and/or immunohistochemical studies. We have utilized in vivo optical imaging technology with a cooled charged coupled device camera to image the expression of the firefly luciferase gene in tumor xenografts that are stably transfected with a chimeric gene containing the first kilobase of the murine COX-2 promoter. Induction of luciferase gene expression following systemic lipopolysaccharide/endotoxin administration can be robustly demonstrated; both a dose-response relationship and a time course for luciferase expression from the COX-2 promoter can be noninvasively analyzed in the tumor xenografts. These data suggest expression from the COX-2 promoter will be easily analyzed in transgenic mice, in knock-in mice, and in somatic cell and gene transfer experiments.

Animals↗
PubMedDate not supplied

[Combination of new electrophysiological and imaging techniques in the study of primary visual cortex function].

INTRODUCTION: This work summarizes current research focused on explaining orientation selectivity of primary visual cortex (V1), and describes the electrophysiological and imaging techniques than are being used. DEVELOPMENT: The study of orientation selectivity in V1 is key to understanding the cortical mechanisms implicated in the processing of sensory information, but this enterprise has proved more challenging than previously thought and there is no consensus about the best model to explain V1 neurons activity. Ongoing research is focused on determining the importance of the different inputs that a cortical cell receives (thalamic and lateral cortical inputs), and their link to cortical architecture. To achieve that, current research is combining optical imaging techniques with intracellular recordings of V1 neurons. Recent findings have found differences in the synaptic integration performed by neurons located in the iso orientation domains vs orientation centers of the functional V1 map. CONCLUSIONS: Data describing synaptic activity combined with the cortical functional structure are yielding new clues about V1 computation, suggesting that there is more than one mechanism capable of generating orientation selectivity.

Electrophysiology↗
PubMedDate not supplied

Chemical shift selective magnetic resonance imaging of the optic nerve in patients with acute optic neuritis.

Optic neuritis is often the first manifestation of multiple sclerosis (MS). Sixteen patients with acute optic neuritis and one patient with benign intracranial hypertension (BIH) were investigated by magnetic resonance imaging, using a chemical shift selective double spin echo sequence. In 3 of the 16 patients, abnormalities were seen. In one patient with bilateral symptoms, signal hyperintensity and swelling of the right side of the chiasm were found. In another patient the optic nerve was found diffusely enlarged with only a marginally increased signal in the second echo. In the third patient an area of signal hyperintensity and swelling was seen in the left optic nerve. In the patient with BIH the subarachnoid space which surrounds the optic nerves was enlarged. Even using this refined pulse sequence, avoiding the major artefact in imaging the optic nerve, the chemical shift artefact, lesions were only shown in 3/16 (19%) of the patients with optic neuritis. Nevertheless, the presented chemical shift selective double spin echo sequence may be of great value for detection of retrobulbar lesions.

Acute Disease↗
PubMed2006

Imaging membrane potential in dendrites and axons of single neurons.

This review focuses on the use of imaging techniques to record electrical signaling in the fine processes of neurons such as dendrites and axons. Voltage imaging began with the use and development of externally applied voltage-sensitive dyes. With the introduction of internally applied dyes and advances in detection technology, it is now possible to record supra-threshold action potential responses, as well as sub-threshold synaptic potentials, in fine neuronal processes including dendritic spines. The development of genetically coded sensors, as well as variants of laser scanning microscopy such as second harmonic generation, offers promise for further advances in this field. Through the use and further development of these methods, optical imaging of membrane potential will continue to be a valuable tool for investigators wishing to explore the electrical events underlying single neuronal computation.

Action Potentials↗
PubMed2006

Investigating neural-hemodynamic coupling and the hemodynamic response function in the awake rat.

An understanding of the relationship between changes in neural activity and the accompanying hemodynamic response is crucial for accurate interpretation of functional brain imaging data and in particular the blood oxygen level-dependent (BOLD) fMRI signal. Much physiological research investigating this topic uses anesthetized animal preparations, and yet, the effects of anesthesia upon the neural and hemodynamic responses measured in such studies are not well understood. In this study, we electrically stimulated the whisker pad of both awake and urethane anesthetized rats at frequencies of 1-40 Hz. Evoked field potential responses were recorded using electrodes implanted into the contralateral barrel cortex. Changes in hemoglobin oxygenation and concentration were measured using optical imaging spectroscopy, and cerebral blood flow changes were measured using laser Doppler flowmetry. A linear neural-hemodynamic coupling relationship was found in the awake but not the anesthetized animal preparation. Over the range of stimulation conditions studied, hemodynamic response magnitude increased monotonically with summed neural activity in awake, but not in anesthetized, animals. Additionally, the temporal structure of the hemodynamic response function was different in awake compared to anesthetized animals. The responses in each case were well approximated by gamma variates, but these were different in terms of mean latency (approximately 2 s awake; 4 s anesthetized) and width (approximately 0.6 s awake; 2.5 s anesthetized). These findings have important implications for research into the intrinsic signals that underpin BOLD fMRI and for biophysical models of cortical hemodynamics and neural-hemodynamic coupling.

Anesthetics↗
PubMed2003

A novel method of imaging lysosomes in living human mammary epithelial cells.

Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.

Acetylglucosamine↗