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Image Search Results
Journal: Frontiers in Synaptic Neuroscience
Article Title: Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light
doi: 10.3389/fnsyn.2019.00016
Figure Lengend Snippet: Procedure for transfection, conversion and imaging. (A) In this schematic, cultured neurons are treated with Cal-Light virus. After expression of the construct (indicated via red fluorescence), photostimulation is applied. Imaging then reveals neurons that were active while receiving the light stimulus (i.e., they co-express GFP and thus appear yellow in merged channels). (B) In this example, CaMPARI virus was injected into S1 cortex; expression of the construct is imaged using an epifluorescence microscope. A large number of neurons express CaMPARI 3 weeks after virus injection. Left, injection site imaged through the cranial window before and after 405 nm photoconversion. Note the reduction of green/red ratio immediately after photoconversion. Right, demonstration of photoconverted neurons in acute brain slices of S1 (parasagittal, 300 μm).
Article Snippet: Neural Basal A media (ThermoFisher Scientific, 10888022) B27 (ThermoFisher Scientific, 17504001) Glutamax (ThermoFisher Scientific, 35050-038) Penicillin-Streptomycin (10,000 U/ml, ThermoFisher Scientific, 15140-122) Poly-L-Lysine (Sigma, P1399 Coverslip coating) Papain (Sigma, P4762) Bovine Serum Albumin (Sigma, A3294) Hibernate A low
Techniques: Transfection, Imaging, Cell Culture, Virus, Expressing, Construct, Fluorescence, Injection, Microscopy
Journal: Frontiers in Synaptic Neuroscience
Article Title: Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light
doi: 10.3389/fnsyn.2019.00016
Figure Lengend Snippet: CaMPARI and Cal-Light expression and functionality in neuronal cell culture. (A) Wide-field epifluorescence images of neuronal cell culture expressing CaMPARI. Top row, pre-photoconversion images showing fluorescence exclusively in the green channel. Bottom row, post-photoconversion images showing mixed green and red fluorescence after ~5 min of total illumination with 395 nm light. (B) Wide-field epifluorescence images of neuronal cell culture expressing Cal-Light. Top row, pre-photoactivation images showing fluorescence in the red (tdTomato) channel. Bottom row, post-photoactivation images showing mixed red and green (GFP) fluorescence after ~8–10 min of total illumination with 470 nm light and after 6 days of expression time. Scale bars, 200 μm and 50 μm in insets. Images were taken on a wide-field epifluorescence microscope (Nikon Ti2) using a 10x air objective (Plan Apo, 0.8 NA, 1,000 μm WD) and a Lumencor Spectra X LED. Fluorescence was imaged through 519/26 nm (GFP) and 642/80 nm (tdTomato) bandpass emission filters.
Article Snippet: Neural Basal A media (ThermoFisher Scientific, 10888022) B27 (ThermoFisher Scientific, 17504001) Glutamax (ThermoFisher Scientific, 35050-038) Penicillin-Streptomycin (10,000 U/ml, ThermoFisher Scientific, 15140-122) Poly-L-Lysine (Sigma, P1399 Coverslip coating) Papain (Sigma, P4762) Bovine Serum Albumin (Sigma, A3294) Hibernate A low
Techniques: Expressing, Cell Culture, Fluorescence, Microscopy
Journal: Frontiers in Synaptic Neuroscience
Article Title: Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light
doi: 10.3389/fnsyn.2019.00016
Figure Lengend Snippet: CaMPARI for all-optical functional connectivity mapping. CaMPARI was expressed in S1 and ChR2-EYFP was expressed in M1 cortex. In acute ex vivo brain slices, violet light (405 nm, 120 mW cm −2 ) was applied to S1 cortex that activates ChR2 and simultaneously drives conversion of post-synaptic CaMPARI-expressing neurons in layer 2/3 and 5. Here, conversion and stimulation light pulses were delivered at 10 Hz for a second, with a step of light for 5 s after the stimulus. There was a 12 s interval between each train, and this was repeated 10 times. Left, one-photon image of CaMPARI green/ChR2-EYFP under 4 × magnification. After violet light illumination, red CaMPARI fluorescence is evident in layers 2/3 and 5. Scale bar, 200 μm. Middle and right panels, post-stimulation/conversion 60 × magnification two-photon images of CaMPARI red/green in layers 2/3 and 5, from the corresponding slices. Scale bar, 50 μm. Exclusively in this Figure, CaMPARI (not CaMPARI2) was expressed.
Article Snippet: Neural Basal A media (ThermoFisher Scientific, 10888022) B27 (ThermoFisher Scientific, 17504001) Glutamax (ThermoFisher Scientific, 35050-038) Penicillin-Streptomycin (10,000 U/ml, ThermoFisher Scientific, 15140-122) Poly-L-Lysine (Sigma, P1399 Coverslip coating) Papain (Sigma, P4762) Bovine Serum Albumin (Sigma, A3294) Hibernate A low
Techniques: Functional Assay, Ex Vivo, Expressing, Fluorescence
Journal: Frontiers in Synaptic Neuroscience
Article Title: Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light
doi: 10.3389/fnsyn.2019.00016
Figure Lengend Snippet: Sparse Cal-Light expression triggered through a cranial window in an awake quietly sitting head-fixed mouse. (A) A large number of neurons express Cal-Light three weeks after virus injection. (A1) Neurons from layer 1 to layer 4 express GFP six days after exposure to 470 nm light while the mouse was head-fixed and awake. A halo of GFP expression marks the extent of spread of the 470 nm light in vivo . (A2) Merged image showing photoactivated, double-labeled neurons (yellow) in layers 2–5. (A3) Magnified view of one strongly photoactivated neuron in layer 3. (B) Example of S1 cortex neurons expressing Cal-Light three weeks after virus injection. (B1,B2) Images are taken slightly off the center of injection to highlight the few intensely GFP expressing neurons in layers 1–4, 6 days after exposure to 470 nm light while the mouse was head-fixed and awake. (B3) Magnified view of one strongly photoactivated neuron (bright yellow) and one less strongly photoactivated neuron (pale yellow) in layer 2. Sections are parasagittal, 300 μm. Scale bar in A 100 μm, in B 200 μm, in insets 50 μm. Slices were imaged on a confocal laser scanning microscope (Nikon A1Rsi+) using a 20x air objective (Plan Apo, 0.8 NA, 1.000 WD) and a 647 nm laser. Fluorescence was imaged through a 700/50 bandpass emission filter.
Article Snippet: Neural Basal A media (ThermoFisher Scientific, 10888022) B27 (ThermoFisher Scientific, 17504001) Glutamax (ThermoFisher Scientific, 35050-038) Penicillin-Streptomycin (10,000 U/ml, ThermoFisher Scientific, 15140-122) Poly-L-Lysine (Sigma, P1399 Coverslip coating) Papain (Sigma, P4762) Bovine Serum Albumin (Sigma, A3294) Hibernate A low
Techniques: Expressing, Virus, Injection, In Vivo, Labeling, Laser-Scanning Microscopy, Fluorescence
Journal: Frontiers in Synaptic Neuroscience
Article Title: Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light
doi: 10.3389/fnsyn.2019.00016
Figure Lengend Snippet: CaMPARI expression: Effects of fixation and use of immunostaining to recover converted neurons. (A) After fixation in 4% PFA, only a few neurons in S1 cortex show some weak red fluorescence signal reflecting the photoconversion of CaMPARI carried out in vivo . (A1) A large number of unconverted green neurons are still visible. (A2) When the CaMPARI 4F6 antibody targeting the converted form of CaMPARI is used, it reveals converted neurons, and (A3) , distinguishes them from neighboring unconverted neurons as seen in the merged image. Bottom row, magnified views of inset regions showing converted vs. non-converted cells. Sections are coronal, 300 μm and were taken directly from an in vitro experiment and placed in PFA overnight. Blood vessels are prominent because the brain was not perfused prior to fixation. Scale bar in A 100 μm, in inset 50 μm.
Article Snippet: Neural Basal A media (ThermoFisher Scientific, 10888022) B27 (ThermoFisher Scientific, 17504001) Glutamax (ThermoFisher Scientific, 35050-038) Penicillin-Streptomycin (10,000 U/ml, ThermoFisher Scientific, 15140-122) Poly-L-Lysine (Sigma, P1399 Coverslip coating) Papain (Sigma, P4762) Bovine Serum Albumin (Sigma, A3294) Hibernate A low
Techniques: Expressing, Immunostaining, Fluorescence, In Vivo, In Vitro