Journal: Frontiers in Molecular Neuroscience

Article Title: Improved method for efficient imaging of intracellular Cl − with Cl-Sensor using conventional fluorescence setup

doi: 10.3389/fnmol.2013.00007

Figure Lengend Snippet: Light pass of conventional epifluorescence setup. (A) Excitation and emission spectra of the filter set used in the study (vertical bars) overlapped with emission and excitation spectra for cyan and yellow fluorescent proteins carrying the I152L mutation (CFP and YFP I152L , respectively). The plot is a schematic presentation of spectral bandpasses of the setup designed based on the information provided by the filter set manufacture (Chroma Technology Corp., USA). The absorbance spectra of YFP I152L is reproduced from Galietta et al. . The absorbance spectra of CFP and emission spectra of CFP and YFP were taken from R. Tsien's website ( http://www.tsienlab.ucsd.edu/Documents.htm ). (B) Scheme of the setup. The light source (Xenon arc burner) was connected to a filter wheel including two excitation filters and one neutral density filter (ND filter) placed in front of 430(24) nm filter. The emitted fluorescence was recorded using a digital CCD camera. (C) Schematic presentation of the recorded portion of fluorescence emitted by Cl-Sensor during excitation with a 430(24) nm filter (see Materials and Methods for details). (D) Schematic presentation of the fluorescence signal emitted by Cl-Sensor during excitation with a 500(20) nm filter.

Article Snippet: Image recording was performed using a CoolSNAP HQ Monochrome CCD camera and Metamorph software that includes a multi-dimensional acquisition option (MDA) (Roper scientific sas, Evry, France) (see Figure for scheme of the lightpass).

Techniques: Mutagenesis, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Improved method for efficient imaging of intracellular Cl − with Cl-Sensor using conventional fluorescence setup

doi: 10.3389/fnmol.2013.00007

Figure Lengend Snippet: Stable recording of Cl-Sensor fluorescence using the updated setup. (A) Scheme of the experiment: Cl-Sensor was excited every 20 s using two consecutive 50 ms light pulses through first a 500 nm and then a 430 nm excitation filter. The sequence of excitation was different as compared to Figure . (B) Example of N2a cells transfected with Cl-Sensor and excited at 430 nm (panel b1). CCD camera binning was settled 4 × 4. The image in panel b2 shows a digitally amplified region from b1. Image in b3 shows fluorescence obtained using 500 nm excitation (CCD binning 2 × 2). The arrow indicates an example of a region of interest (ROI) drawn around a cell to measure fluorescence intensity. Scale 20 μm. (C) Left and middle plots illustrate recordings of fluorescence induced using 430 nm and 500 nm excitation pulses. The right plot shows corresponding traces of signal ratio (R 430/500 ). Each color corresponds to the same cell shown in the three plots. The artificial increase of [Cl − ] i was achieved by application of an external solution containing glycine (50 μM) and KCl (100 mM). The black dotted line in the right plot illustrates a mean value of 42 cells recorded in the depictured experiment. (D) Dynamics of R 430/500 change in an experiment similar to the one depictured in (C) but in the presence of Strychnine, a blocker of glycine receptors (0.3 μM) in the control external solution. Notice the slow or inexistent recovery of the R 430/500 value after wash out of glycine and KCl. (E) Comparison of the kinetics of the recovery of R 430/500 (mean ± SEM) in the experiments depictured in panels (C) (right plot, 42 cells) and (D) (38 cells).

Article Snippet: Image recording was performed using a CoolSNAP HQ Monochrome CCD camera and Metamorph software that includes a multi-dimensional acquisition option (MDA) (Roper scientific sas, Evry, France) (see Figure for scheme of the lightpass).

Techniques: Fluorescence, Sequencing, Transfection, Amplification, Control, Comparison