Live and fixed kidney imaging was performed using a Nikon Ti2-E microscope equipped with a CSU-W1 spinning disk (Yokogawa), a white light LED, laser illumination (100 mW 405, 488, and 561 nm lasers and a 75 mW 640 nm laser), a Prime 95B back-illuminated sCMOS camera (Photometrics), motorized stage, 4x/0.2 NA, 10x/0.25 NA and 20x/0.5 NA lenses (Nikon), and a stagetop environmental enclosure (OkoLabs). In dispase treatment experiments, a 150 μm stack (21 frames, 7.5 μm step size) of the kidney cortex was collected at each stage position every 5 minutes. Z-stacks of fixed and stained dispase-treated kidneys or cut caps (and controls) were similarly collected using 5 μm step size (100–200 μm, 21–41 frames) using either the 10x or 20x lens. In order to image the cortical kidney surface, caps were oriented cortex-side down against the glass surface of 35 mm dishes while uncut controls were oriented cortex-side down in the bottom of 2 mm-diameter PDMS wells, created as described above. The soft material model was imaged by submerging it in a transparent glass water bath and filming through an amber trans-illuminator filter (IO Rodeo) during manual manipulations under blue 470 nm LED illumination (ThorLabs M470L2-C1) using an iPhone 8 video camera.
M470l2 c1
Manufactured by Thorlabs
The M470L2-C1 is a 470 nm LED from Thorlabs. It provides an optical output power of 1 W.
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3 protocols using m470l2 c1
1
Live Kidney Imaging Using Spinning Disk
2
Optogenetic Activation of Pyramidal Cells
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For acute slice experiments, we used a cesium-based internal solution containing (in mM) 117 cs-methanesulfonate, 15 CsCl, 10 TEA-Cl, 10 HEPES, 10 phosphocreatine, 8 NaCl, 4 Mg2-ATP, 1 MgCl2, 0.5 Na2-GTP, and 0.2 EGTA for experiments measuring inhibitory currents. Inhibitory currents were isolated pharmacologically by including 10μM NBQX and 50μm D-AP5 in the ACSF. Virus expressing cells were visually identified and targeted by mRuby expression. To optically activate Cry2Olig expressing pyramidal cells, slices were illuminated with 470 nm LED light (ThorLabs M470L2-C1) continuously or via 20s pulses through the 40x dipping objective located directly over the recorded cell. With an illumination area of 33.18mm2 the tissue was exposed to an irradiance of 0.17 mW/mm2. For electrical stimulation a homemade nichrome stimulating electrode was placed ~200 μm from the patched cell and pulsed at 0.1 Hz at 100mA (A-M Systems 2100 Isolated pulse stimulator). The stimulation electrode was placed in the stratum radiatum.
3
Transcranial Flavoprotein Fluorescence Imaging
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A binocular microscope (MVX10, Olympus) equipped with a 0.63 x, 0.15 NA objective (MV PLAPO, Olympus), an Olympus U-M49002XL filter cube (excitation filter: 470/40 nm, dichroic mirror: 495 nm high pass, emission filter: 525/50 nm) and a CMOS camera (Zyla 4.2 Plus, Andor) was used to transcranially record flavoprotein fluorescence signals (Takahashi et al., 2006) . The skull surface was covered with 0.9% NaCl solution to increase bone transparency and excited with a 470 nm LED (M470L2-C1, Thorlabs) using a light intensity of 4.2 mW below the objective. Images (128 3 128 pixels) were recorded at 10 Hz sampling rate with custom-written acquisition software (LabVIEW 2014, National Instruments).
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