Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 16 de 16
Filter
Add more filters










Publication year range
1.
Microorganisms ; 9(8)2021 Aug 16.
Article in English | MEDLINE | ID: mdl-34442824

ABSTRACT

Strains of the food-borne pathogen Listeria (L.) monocytogenes have diverse virulence potential. This study focused on the virulence of three outbreak strains: the CC1 strain PF49 (serovar 4b) from a cheese-associated outbreak in Switzerland, the clinical CC2 strain F80594 (serovar 4b), and strain G6006 (CC3, serovar 1/2a), responsible for a large gastroenteritis outbreak in the USA due to chocolate milk. We analysed the genomes and characterized the virulence in vitro and in vivo. Whole-genome sequencing revealed a high conservation of the major virulence genes. Minor deviations of the gene contents were found in the autolysins Ami, Auto, and IspC. Moreover, different ActA variants were present. Strain PF49 and F80594 showed prolonged survival in the liver of infected mice. Invasion and intracellular proliferation were similar for all strains, but the CC1 and CC2 strains showed increased spreading in intestinal epithelial Caco2 cells compared to strain G6006. Overall, this study revealed long-term survival of serovar 4b strains F80594 and PF49 in the liver of mice. Future work will be needed to determine the genes and molecular mechanism behind the long-term survival of L. monocytogenes strains in organs.

2.
Biomed Opt Express ; 8(7): 3329-3342, 2017 Jul 01.
Article in English | MEDLINE | ID: mdl-28717570

ABSTRACT

We demonstrate a 60 mg light video-endomicroscope with a cylindrical shape of the rigid tip of only 1.6 mm diameter and 6.7 mm length. A novel implementation method of the illumination unit in the endomicroscope is presented. It allows for the illumination of the biological sample with fiber-coupled LED light at 455 nm and the imaging of the red-shifted fluorescence light above 500 nm in epi-direction. A large numerical aperture of 0.7 leads to a sub-cellular resolution and yields to high-contrast images within a field of view of 160 µm. A miniaturized chip-on-the-tip CMOS image sensor with more than 150,000 pixels captures the multicolor images at 30 fps. Considering size, plug-and-play capability, optical performance, flexibility and weight, we hence present a probe which sets a new benchmark in the field of epifluorescence endomicroscopes. Several ex-vivo and in-vivo experiments in rodents and humans suggest future application in biomedical fields, especially in the neuroscience community, as well as in medical applications targeting optical biopsies or the detection of cellular anomalies.

3.
Acta Neurochir (Wien) ; 156(8): 1427-40, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24792966

ABSTRACT

BACKGROUND: In gliomas molecular biomarkers are increasingly gaining diagnostic, prognostic and predictive significance. Determination of biomarker status after biopsy is important as not all patients are eligible for open tumor resection. We developed and validated prospectively (6/10-12/11) a protocol allowing for both reliable determination of multiple biomarkers and representative histological diagnoses from small-sized biopsies. METHODS: All molecular stereotactic biopsies were performed according to a detailed workflow. The selection of specimens best suited for molecular analyses was intra-operatively guided by the attending neuropathologist. Postoperative screening was done by methylation specific PCR using two distinct cryopreserved specimens to test for reproducibility of the findings and to rule out contamination. The DNA of a single best-suited specimen (1 mm(3)) was subjected to detailed molecular analysis (MGMT promoter methylation, IDH1/2 mutational status, LOH 1p and/or 19q). RESULTS: 159 consecutively enrolled untreated gliomas were analyzed (94 glioblastomas, 2 gliosarcomas, 24 anaplastic astrocytomas, 10 oligo-tumors grade II/III, 20 grade II astrocytomas and 9 pilocytic astrocytomas). Transient morbidity was 2 %. Overall, the drop-out rate due to tissue contamination was 0.4 %. Median time from biopsy to histological and molecular genetic analyses was 3 and 5 days, respectively. Distributions of the respective biomarker status for tumor subgroups were consistent with the literature. The final histological diagnosis was changed/modified in 5/159 patients according to molecular findings. Treatment after molecular biopsy was highly personalized. CONCLUSIONS: Molecular stereotactic biopsy is feasible and safe, can be implemented in daily clinical practice, improves diagnostic precision and enables personalized treatment.


Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Stereotaxic Techniques , Adult , Age Factors , Biomarkers, Tumor , Biopsy , Brain Neoplasms/genetics , Brain Neoplasms/surgery , DNA Methylation , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Female , Glioma/genetics , Glioma/surgery , Humans , Male , Middle Aged , Mutation , Precision Medicine , Prognosis , Promoter Regions, Genetic , Reproducibility of Results , Tumor Suppressor Proteins/genetics , Young Adult
4.
Photodiagnosis Photodyn Ther ; 11(3): 307-18, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24699453

ABSTRACT

BACKGROUND: Intraoperative blood vessel detection based on intraluminal indocyanin-green (ICG) would allow to minimize the risk of blood vessel perforation during stereotactic brain tumor biopsy. For a fiber-based approach compatible with clinical conditions, the maximum tolerable excitation light power was derived from simulations of the thermal heat load on the tissue. METHODS: Using the simulation software LITCIT, the temperature distribution in human brain tissue was calculated as a function of time for realistic single-fiber probes (0.72mm active diameter, numerical aperture 0.35, optional focusing to 0.29mm diameter) and for the optimum ICG excitation wavelength of 785nm. The asymptotic maximum temperature in the simulated tissue region was derived for different radiant fluxes at the distal fiber end. Worst case values were assumed for all other parameters. In addition to homogeneous (normal and tumor) brain tissue with homogeneous blood perfusion, models with localized extra blood vessels incorporated ahead of the distal fiber end were investigated. RESULTS: If one demands that destruction of normal brain tissue must be excluded by limiting the tissue heating to 42°C, then the radiant flux at the distal fiber end must be limited to 33mW with and 43mW without focusing. When considering extra blood vessels of 0.1mm diameter incorporated into homogeneously perfused brain tissue, the tolerable radiant flux is reduced to 22mW with and 32mW without focusing. The threshold value according to legal laser safety regulations for human skin tissue is 28.5mW. CONCLUSIONS: For the envisaged modality of blood vessel detection, light power limits for an application-relevant fiber configuration were determined and found to be roughly consistent with present legal regulations for skin tissue.


Subject(s)
Body Temperature/physiology , Brain/physiology , Microscopy, Fluorescence/methods , Models, Neurological , Stereotaxic Techniques , Surgery, Computer-Assisted/methods , Animals , Body Temperature/radiation effects , Brain/radiation effects , Brain/surgery , Computer Simulation , Humans , Indocyanine Green , Light , Lighting/methods , Radiation Dosage
5.
Photodiagnosis Photodyn Ther ; 10(4): 552-60, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24284111

ABSTRACT

BACKGROUND: New treatment strategies for malignant gliomas are indispensible, due to the poor prognosis for patients. Fluorescence diagnosis (FD) and photodynamic therapy (PDT) are currently under intensive investigation and seem to improve the prognosis. Especially for deep seated malignant brain lesions and in order to optimize therapy new diagnostic tools are needed. METHODS: In a syngeneic subcutaneous glioma mouse model we investigated the time dependent hypericin (HYP) uptake in malignant tumor tissue by microendoscopically fluorescence measurements. The HYP fluorescence in tumor was also detected by fluorescence microscopy (FM) and was compared to endoscopic data. RESULTS: Both methods, microendoscopy and FM, demonstrated time dependent HYP uptake in subcutaneously implanted mouse glioma. Maximum of HYP uptake was achieved after 6h, measured with both methods. FM reached a 10-fold increase in fluorescence intensity compared to the autofluorescence. Measured by microendoscopy a 2.2-fold HYP fluorescence intensity compared to the autofluorescence was detected. Microendoscopy enables visualization of small vessels even in healthy brain tissue by intravascular HYP fluorescence. CONCLUSION: The new developed microendoscope enables not only fluorescence based discrimination of tumor and healthy tissue, but also semiquantitative measurements of fluorescence intensities in vivo. Individual repetitive fluorescence diagnosis will become possible by this method and opens up new possibilities for determining optimal settings of light applications for PDT.


Subject(s)
Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Disease Models, Animal , Endoscopy/methods , Glioma/metabolism , Glioma/pathology , Spectrometry, Fluorescence/methods , Animals , Anthracenes , Cell Line, Tumor , Metabolic Clearance Rate , Mice , Perylene/analogs & derivatives , Perylene/pharmacokinetics , Photosensitizing Agents/pharmacokinetics , Reproducibility of Results , Sensitivity and Specificity , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Tissue Distribution
6.
Opt Express ; 20(24): 26117-26, 2012 Nov 19.
Article in English | MEDLINE | ID: mdl-23187467

ABSTRACT

Proper treatment of deep seated brain tumors requires correct histological diagnosis which unambiguously necessitates biopsy sampling. Stereotactically guided sampling of biopsies is widely used but bears the danger of incorrect sampling locations and damage to intracerebral blood vessels. Here, we present a minimally invasive contact endoscopic probe that can be inserted into the tissue inside a standard biopsy needle and allows for fluorescence detection of both tumorous tissue and intracerebral blood vessels. Outer diameter of our contact probe is smaller than 1.5 mm, field-of-view in the range of several hundred microns; the optical design allows for simultaneous detection and visualization of tissue autofluorescence and selective fluorescence signals from deep seated brain tumors and vasculature as shown on in vivo animal models. We demonstrate the tumor detection capability during stereotactic needle insertion in a clinical pilot trial. Using our probe, we expect stereotactic interventions to become safer and more precise and the technology might ultimately be used also for various other kinds of applications.


Subject(s)
Biopsy, Needle/instrumentation , Brain Neoplasms/diagnosis , Brain/pathology , Endoscopes , Endoscopy/methods , Neurosurgical Procedures/instrumentation , Stereotaxic Techniques/instrumentation , Animals , Brain/surgery , Brain Neoplasms/surgery , Equipment Design , Mice , Neoplasms, Experimental
7.
Article in English | MEDLINE | ID: mdl-22180739

ABSTRACT

How are visual scenes encoded in local neural networks of visual cortex? In rodents, visual cortex lacks a columnar organization so that processing of diverse features from a spot in visual space could be performed locally by populations of neighboring neurons. To examine how complex visual scenes are represented by local microcircuits in mouse visual cortex we measured visually evoked responses of layer 2/3 neuronal populations using 3D two-photon calcium imaging. Both natural and artificial movie scenes (10 seconds duration) evoked distributed and sparsely organized responses in local populations of 70-150 neurons within the sampled volumes. About 50% of neurons showed calcium transients during visual scene presentation, of which about half displayed reliable temporal activation patterns. The majority of the reliably responding neurons were activated primarily by one of the four visual scenes applied. Consequently, single-neurons performed poorly in decoding, which visual scene had been presented. In contrast, high levels of decoding performance (>80%) were reached when considering population responses, requiring about 80 randomly picked cells or 20 reliable responders. Furthermore, reliable responding neurons tended to have neighbors sharing the same stimulus preference. Because of this local redundancy, it was beneficial for efficient scene decoding to read out activity from spatially distributed rather than locally clustered neurons. Our results suggest a population code in layer 2/3 of visual cortex, where the visual environment is dynamically represented in the activation of distinct functional sub-networks.

8.
Cold Spring Harb Protoc ; 2011(11): 1340-9, 2011 Nov 01.
Article in English | MEDLINE | ID: mdl-22046041

ABSTRACT

Neural tissue is organized in three-dimensional (3D) networks of neuronal and glial cell populations. To understand the functional organization of these networks, it is desirable to achieve 3D activity measurements from large cell populations in intact tissue with high temporal resolution. Repeated acquisition of image stacks with standard laser-scanning microscopes is too slow. This protocol describes fast 3D calcium imaging in the living brain using mechanical laser scanning with standard galvanometric mirrors and a piezoelectric focusing element. The purpose of 3D laser scanning is to create a 3D line scan that samples relatively homogenously from a particular observation volume. The spatial resolution of this approach is low, except along the line. However, the main goal is not to resolve subcellular structures, but rather to hit as many cell bodies as possible within the volume. In this manner, local network activity can be inferred from the somatic calcium signals of a significant fraction of the cell population. With a sinusoidal swinging microscope objective as a constraint, 3D scan trajectories are generated that sample fluorescence signals from the majority of cells within a cuboidal volume. Measurements with 10-Hz temporal resolution can be achieved for population calcium signals from several hundreds of identified neurons and glial cells within cuboids with side lengths of ∼250 µm. An example cellular 3D orientation map of the rat visual cortex is presented. This 3D laser-scanning technique enables direct observation of in vivo neural network dynamics in cell populations of substantial size.


Subject(s)
Brain/cytology , Brain/physiology , Imaging, Three-Dimensional/methods , Microscopy, Confocal/methods , Nerve Net/cytology , Nerve Net/physiology , Animals , Rats
9.
Article in English | MEDLINE | ID: mdl-20461230

ABSTRACT

Fluorescent calcium (Ca(2+)) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca(2+) sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca(2+) transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca(2+) transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca(2+) dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 - in combination with various optical techniques - thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.

10.
Opt Express ; 17(8): 6421-35, 2009 Apr 13.
Article in English | MEDLINE | ID: mdl-19365467

ABSTRACT

Nonlinear microscopy techniques crucially rely on efficient signal detection. Here, we present a ring of large-core optical fibers for epi-collection of fluorescence photons that are not transmitted through the objective and thus normally wasted. Theoretical treatments indicated that such a supplementary fiber-optic light collection system (SUFICS) can provide an up to 4-fold signal gain. In typical in vivo imaging experiments, the fiber-ring channel was brighter than the objective channel down to 800 microm depth, thus providing a gain >2. Moreover, SUFICS reduced noise levels in calcium imaging experiments by about 23%. We recommend SUFICS as a generally applicable, effective add-on to nonlinear microscopes for enhancing fluorescence signals.


Subject(s)
Fiber Optic Technology/instrumentation , Image Enhancement/instrumentation , Microscopy, Fluorescence/instrumentation , Spectrometry, Fluorescence/instrumentation , Computer-Aided Design , Equipment Design , Equipment Failure Analysis , Light , Nonlinear Dynamics , Reproducibility of Results , Scattering, Radiation , Sensitivity and Specificity
11.
Proc Natl Acad Sci U S A ; 106(9): 3496-501, 2009 Mar 03.
Article in English | MEDLINE | ID: mdl-19211787

ABSTRACT

Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent indicator dyes or the calcium-sensor protein G-CaMP2, we discovered spontaneous calcium waves that filled approximately ellipsoidal domains of Bergmann glia processes. Waves spread in 3 dimensions at a speed of 4-11 microm/s to a diameter of approximately 50 microm, slowed during expansion, and were reversibly blocked by P2 receptor antagonists. Consistent with the hypothesis that ATP acts as a diffusible trigger of calcium release waves, local ejection of ATP triggered P2 receptor-mediated waves that were refractory to repeated activation. Transglial waves represent a means for purinergic signals to act with local specificity to modulate activity or energetics in local neural circuits.


Subject(s)
Calcium/metabolism , Cerebellum/metabolism , Adenosine Triphosphate/metabolism , Animals , Cerebellum/drug effects , Mice , Purinergic P2 Receptor Antagonists , Rats , Receptors, Purinergic P2/metabolism
12.
Physiology (Bethesda) ; 22: 358-65, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18073408

ABSTRACT

Spatiotemporal activity patterns in local neural networks are fundamental to brain function. Network activity can now be measured in vivo using two-photon imaging of cell populations that are labeled with fluorescent calcium indicators. In this review, we discuss basic aspects of in vivo calcium imaging and highlight recent developments that will help to uncover operating principles of neural circuits.


Subject(s)
Calcium Signaling , Microscopy, Fluorescence, Multiphoton/methods , Nerve Net/metabolism , Neuroglia/metabolism , Neurons/metabolism , Action Potentials , Animals , Artifacts , Fluorescent Dyes/chemistry , Humans , Imaging, Three-Dimensional , Models, Neurological , Neuropil/chemistry , Reproducibility of Results , Signal Processing, Computer-Assisted , Time Factors
13.
J Neurophysiol ; 98(6): 3770-9, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17898141

ABSTRACT

Imaging technologies are well suited to study neuronal dendrites, which are key elements for synaptic integration in the CNS. Dendrites are, however, frequently oriented perpendicular to tissue surfaces, impeding in vivo imaging approaches. Here we introduce novel laser-scanning modes for two-photon microscopy that enable in vivo imaging of spatiotemporal activity patterns in dendrites. First, we developed a method to image planes arbitrarily oriented in 3D, which proved particularly beneficial for calcium imaging of parallel fibers and Purkinje cell dendrites in rat cerebellar cortex. Second, we applied free linescans -- either through multiple dendrites or along a single vertically oriented dendrite -- to reveal fast dendritic calcium dynamics in neocortical pyramidal neurons. Finally, we invented a ribbon-type 3D scanning method for imaging user-defined convoluted planes enabling simultaneous measurements of calcium signals along multiple apical dendrites. These novel scanning modes will facilitate optical probing of dendritic function in vivo.


Subject(s)
Dendrites/physiology , Neurons/physiology , Animals , Calcium Signaling/physiology , Cerebellum/cytology , Cerebellum/physiology , Data Interpretation, Statistical , Image Processing, Computer-Assisted , Microscopy, Confocal , Neocortex/cytology , Neocortex/physiology , Purkinje Cells/physiology , Pyramidal Cells/physiology , Rats , Rats, Wistar
14.
Nat Methods ; 4(1): 73-9, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17143280

ABSTRACT

Spatiotemporal activity patterns in three-dimensionally organized cellular networks are fundamental to the function of the nervous system. Despite advances in functional imaging of cell populations, a method to resolve local network activity in three dimensions has been lacking. Here we introduce a three-dimensional (3D) line-scan technology for two-photon microscopy that permits fast fluorescence measurements from several hundred cells distributed in 3D space. We combined sinusoidal vibration of the microscope objective at 10 Hz with 'smart' movements of galvanometric x-y scanners to repeatedly scan the laser focus along a closed 3D trajectory. More than 90% of cell somata were sampled by the scan line within volumes of 250 microm side length. Using bulk-loading of calcium indicator, we applied this method to reveal spatiotemporal activity patterns in neuronal and astrocytic networks in the rat neocortex in vivo. Two-photon population imaging using 3D scanning opens the field for comprehensive studies of local network dynamics in intact tissue.


Subject(s)
Astrocytes/physiology , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Microscopy, Fluorescence, Multiphoton/methods , Nerve Net/physiology , Neurons/physiology , Animals , Lasers , Models, Neurological , Neocortex/cytology , Neocortex/physiology , Neural Networks, Computer , Nonlinear Dynamics , Rats , Sensitivity and Specificity
15.
Opt Lett ; 29(21): 2521-3, 2004 Nov 01.
Article in English | MEDLINE | ID: mdl-15584281

ABSTRACT

We present a miniature, flexible two-photon microscope consisting of a fused coherent optical fiber bundle with 30,000 cores and a gradient-index lens objective. The laser focus of a standard two-photon laser-scanning microscope was scanned over the entrance surface of the fiber bundle, resulting in sequential coupling into individual cores. Fluorescent light was detected through the fiber bundle. Micrometer-sized fluorescent beads and pollen grains were readily resolved. In addition, fluorescently labeled blood vessels were imaged through the fiber bundle in rat brain in vivo.


Subject(s)
Endoscopes , Equipment Failure Analysis , Fiber Optic Technology/instrumentation , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence, Multiphoton/instrumentation , Tomography, Optical Coherence/instrumentation , Animals , Microscopy, Confocal/methods , Microscopy, Fluorescence, Multiphoton/methods , Miniaturization , Neocortex/cytology , Optical Fibers , Phantoms, Imaging , Pollen/ultrastructure , Rats , Reproducibility of Results , Sensitivity and Specificity
16.
Opt Lett ; 29(11): 1285-7, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-15209273

ABSTRACT

We demonstrate propagation of femtosecond pulses in the 800-nm range through a hollow-core photonic crystal fiber with preserved temporal and spectral profiles for pulse energies up to 4.6 nJ. Without the use of a prechirping unit, 170-fs pulses were transmitted essentially undistorted at 812 nm, near the zero-dispersion wavelength. Because of the air guidance of pulses, intensity-dependent nonlinear effects were minimal, with only 15% pulse broadening occurring at 350-mW average output power. This fiber thus is excellently suited for applications that require single-mode delivery of high-energy ultrashort pulses to the fiber output face such as, for example, miniaturized multiphoton microscopes.

SELECTION OF CITATIONS
SEARCH DETAIL
...