Measurements were performed using Na+-binding benzofuran isophthalate AM indicator (SBFI) (Invitrogen). SBFI was chosen as it is sensitive to [Na+]i in the range 0 to 50 mM [Na+]i (Kd = 11.3 mM). The cover slip with primary astrocytes was washed with Hepes Krebs-Ringer buffer (KCl 4 mM, NaCl 136 mM, Hepes 20 mM, Na2HPO4*2H20 0.56 mM, KH2PO4 0.59 mM, D-glucose 5.6 mM, MgCl2*6H2O 0.9 mM, NaHCO3 10 mM, CaCl2*2H2O 1.4 mM) and loaded for 40 min at RT with SBFI 14.7 µM, Pluronic F-127 0.2% (Invitrogen). The cover slip was then mounted into a closed POCmini-2 chamber (PeCon GmbH). Perfusion flow during measurements was estimated to be ∼35 µl/s. Recordings were performed at RT in an upright epifluorescence microscope (Axioscope 2 FS Plus, Carl Zeiss) using a 63×/1.4 NA oil-immersion objective lens, polychrome IV monochromator (Till Photonics) and a cooled CCD camera (ORCA-ERG, Hamamatsu). Fluorescence intensity at 510/30 nm was recorded from alternating 340/15 nm and 380/15 nm excitations with a frame rate 0.2 Hz. Transfected cells were identified by mCherry fluorescence, excited at 543/15 nm and detected with 590 nm long pass filter.
Orca erg
Manufactured by Hamamatsu Photonics
Sourced in Japan
The ORCA-ERG is a scientific instrument designed for high-performance electroretinography (ERG) measurements. It features a back-illuminated sCMOS image sensor that provides high quantum efficiency, low readout noise, and fast frame rates. The ORCA-ERG is capable of recording the electrical responses of the retina to light stimuli, which is a widely used technique in vision research and ophthalmology.
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4 protocols using orca erg
1
Measurement of Intracellular Sodium Dynamics in Astrocytes
2
Photothermal Effect on Collagen/γ-PGA Hydrogel
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To investigate the photothermal effect on collagen/γ-PGA hydrogel, ICG (10–100 µg) as an NIR absorbing dye was added to the collagen/γ-PGA solutions (1 mL) and the solutions were then incubated at 37°C for 30 min to formulate hydrogel. Next, an NIR laser (760 nm laser diode, Photodigm, Richardson, TX, USA) was used to illuminate the collagen/γ-PGA/ICG hydrogel (0.47 W/cm2, 3 min). For NIR fluorescence imaging, collagen/γ-PGA/ICG hydrogel was placed in a chamber sealed from light and connected to a cold charge-coupled device camera (Orca ERG; Hamamatsu Photonics, Hamamatsu City, Japan). NIR images of ICG in the collagen/γ-PGA/ICG hydrogel were acquired using a 760-nm LED light as the excitation light source and an 845/55 emission filter. To confirm the temperature increase in collagen/γ-PGA/ICG hydrogel after irradiation with an NIR laser (0.63 W/cm2, 5 min), the change in temperature of the collagen/γ-PGA/ICG hydrogel in vivo was measured using a thermal imaging camera (FLIR TG165, FLIR Instruments, Wilsonville, OR, USA).
3
In Vivo Lung Imaging with ICG
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For the labeling of cells with ICG, control and shRAP80‐2 HeLa cells suspended in cell culture medium were incubated with ICG (1 mg/mL) at 37°C for 3 h. Unlabeled ICG was removed by washing the cell mixture with PBS (GIBCO Invitrogen, Grand Island, NY, USA). After washing, the ICG labeled cells were re‐suspended into PBS and a fixed number of cells (1 × 106 cells/100 μL) were injected via tail vein. For near‐infrared (NIR) fluorescence imaging, lungs were dissected at 1, 2 and 7 days after injection. The lungs were placed in a chamber sealed against light and connected to the cold charge‐coupled device (CCD) camera (Orca ERG; Hamamatsu Photonics, Hamamatsu City, Japan). NIR images of lungs were acquired using a 760‐nm LED light as the excitation light source and an 845/55 emission filter.
4
Inverse Multifractal Analysis of Tissue Refractive Index
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The spatial distribution of tissue refractive index (RI) was recorded by a differential interference contrast (DIC) microscope (Olympus IX-81, United States). At a magnification of 60×, these DIC images were recorded by a CCD camera (ORCA-ERG, Hamamatsu, 1344 × 1024 pixel dimension 6.45 μm). The elastic scattering spectra from the multiple sites of the biopsied tissue sections were recorded by the angle resolved spectral light scattering measurements (Fig. 1). In brief, light emitted from a Xe-lamp (HPX-2000, Ocean Optics, United States) was collimated by a combination of lenses and illuminated the tissue sample at the center of a goniometric arrangement (spot size ∼1-mm-diameter). The collimated scattered light from the sample was focused into a collecting fiber probe coupled to a spectrometer (USB4000FL, Ocean Optics) for wavelength resolved signal detection. The recordings of spectra were performed (360 to 800 nm) with a spectral resolution of 2.05 nm, where the angular range was kept at 10 deg to 150 deg with an interval of 10 deg. For the inverse multifractal study, the spectra were recorded at backscattering angle θ ¼ 150 deg (Fig. 3).
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