1Department of Neuroscience, Medical University of South Carolina
A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.
Published December 12, 2012. Keywords: Neuroscience, Molecular Biology, Cellular Biology, Anatomy, Physiology, Two-photon imaging, non-rodent, cortical maps, functional architecture, orientation pinwheel singularity, optical imaging, calcium-sensitive dye, bulk loading, single-cell electroporation
1Institute of Neurobiology, Heinrich Heine University Düsseldorf
We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.
Published October 8, 2014. Keywords: Neuroscience, Neurosciences, two-photon microscopy, patch-clamp, UV-flash photolysis, mouse, hippocampus, caged compounds, glutamate, brain slice, dendrite, sodium signals
1Institute of Bioengineering and Swiss Institute of Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne, 2Department of Cell and Developmental Biology and Knight Cancer Institute, Oregon Health & Science University
The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.
Published April 22, 2014. Keywords: Bioengineering, Intravital imaging, epifluorescence, two-photon imaging, Tumor matrix, Matrix remodeling
1Department of Physics, University of Wisconsin - Milwaukee, 2Department of Biological Sciences, University of Wisconsin - Milwaukee
By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of Förster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.
Published January 19, 2011. Keywords: Cellular Biology, Forster (Fluorescence) Resonance Energy Transfer (FRET), protein-protein interactions, protein complex, in vivo determinations, spectral resolution, two-photon microscopy, G protein-coupled receptor (GPCR), sterile 2 alpha-factor protein (Ste2p)
1Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, 2Department of Neuroscience, Brown University - National Institutes of Health Graduate Partnership Program, 3Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, 4Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown
Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.
Published January 5, 2013. Keywords: Neuroscience, Medicine, Anatomy, Neurobiology, Surgery, Cerebral Cortex, Frontal Cortex, Stereotaxic Techniques, Molecular Imaging, Neuronal Plasticity, Neurosciences, In Vivo Imaging, Two-photon Microscopy, Experience-dependent Gene Expression, Arc-GFP Mice, Cranial Window, in situ hybridization, immunohistochemistry, animal model
JoVE Clinical and Translational Medicine
1Developmental Biology Institute of Marseille, Aix Marseille University, 2European Research Center for Medical Imaging, Campus de la Timone, 3Vesalius Research Center, KU Leuven Campus Gasthuisberg
We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.
Published April 21, 2014. Keywords: Medicine, Glioblastoma multiforme, intravital two-photon imaging, animal model, chronic cranial window, brain tumors, neuro-oncology.
1Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz
Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.
Published May 12, 2014. Keywords: Neuroscience, dendritic spine, mouse cortex, in vivo, two-photon microscopy, thinned-skull, imaging
1Friedrich Miescher Institute for Biomedical Research, 2Max Planck Institute of Neurobiology, 3Department of Biosystems Science and Engineering, ETH Zurich
Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.
Published February 20, 2014. Keywords: Behavior, Two-photon imaging, Virtual Reality, mouse behavior, adeno-associated virus, genetically encoded calcium indicators
JoVE Clinical and Translational Medicine
1Neuroscience Center, University of Helsinki
Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.
Published April 6, 2014. Keywords: Medicine, Trauma, Nervous System, animal models, Brain trauma, in vivo multiphoton microscopy, dendrite, astrocyte, microglia, second harmonic generation.
1Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine
In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.
Published October 1, 2014. Keywords: Bioengineering, fluorescence, single particle tracking, laser scanning microscope, two-photon, vesicle transport, live-cell imaging, optics