Axioskop 2 fs microscope

The electrophysiological measurements were performed in intact islets essentially using the perforated-patch whole-cell technique in the voltage- or current-clamp modes in δ-cells.
Parallel measurements of [Ca²⁺]_i and membrane potential were performed using an Axioskop 2FS microscope (Zeiss, Oberkochen, Germany) equipped with a 40x/0.8 objective, Lambda DG-4 exciter (Sutter Instruments, USA) and Orca-R2 cooled CCD camera (Hamamatsu, Japan). Images were acquired using an open-source Micromanager software (developed at Ron Vale’s lab, UCSF, San Francisco, USA) and processed using ImageJ. Data analysis was performed in Igor Pro (Wavemetrics).

After all MR images were collected, one representative kidney was imaged with 2-photon confocal microscopy and conventional histology. First, the kidney was sectioned near the center coronal plane, cleared using 4M urea (41 ), and scanned using autofluorescence on a LSM 510 Meta confocal microscope (Carl Zeiss Microscopy, LCC, Thornwood, NY). The laser excitation was two photons at 1000 nm. Emission was detected at 500–550 nm (green channel) and 575–640 nm (magenta channel). Images were acquired at 1.6-μm resolution, at 20-μm depth, and with 14 slices covering 280 μm of tissue using a 20x objective (Zeiss W Plan-Apochromat, NA 1.0). The intact half-sections were then immersed back into 1X (10mM) PBS, embedded in paraffin, and serially sectioned at 5-μm thickness. Sections were stained with Hematoxylin and Eosin (H&E) and Masson’s Trichrome. These slides were scanned using bright field contrast on an Axioskop 2 FS microscope (Carl Zeiss Microscopy, LCC, Thornwood, NY). Images were acquired at 0.65-μm resolution using a 10x objective. Both confocal and optical imaging required tiling to cover the entire kidney. Correction was applied to remove the shading effects and non-uniformities in the tiled images. MR images were manually registered to confocal and optical images to enable comparison.