High-resolution Perilimbal Imaging Protocol

Samples were imaged and processed as described before [13 (link),14 (link)]. Briefly, full-thickness perilimbal scans were acquired with a confocal microscope designed for high-speed ribbon-scanning and large scale image stitching (RS-G4, Caliber I.D., Andover, MA, USA). The system was fitted with a scanning stage (SCANplus IM 120 × 80, #00-24-579-0000; Märzhäuser Wetzlar GmbH & Co. KG, Wetzlar, Germany) and an Olympus 25×, 1.05 NA water immersion objective (XLPLN25XWMP2; Olympus). Volumetric scans were acquired with a voxel size of 0.365 × 0.365 × 2.43 μm. A scan-zoom of 1.5 was used during acquisition to achieve the desired resolution. Images were acquired over a single channel with an excitation wavelength of 561 nm and an emission filter of 630/60. Laser percentage, high voltage (HV), and offset were held constant throughout the volume at 7, 85, and 15, respectively.

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Loewen R.T., Waxman S., Wang C., Atta S., Chen S., Watkins S.C., Watson A.M, & Loewen N.A. (2020). 3D-Reconstruction of the human conventional outflow system by ribbon scanning confocal microscopy. PLoS ONE, 15(5), e0232833.

Publication 2020

XLPLN25XWMP2

Confocal microscope Held Immersion Scan

Corresponding Organization : Central South University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Volumetric scans acquired with a voxel size of 0.365 × 0.365 × 2.43 μm

control variables

Laser percentage held constant at 7
High voltage (HV) held constant at 85
Offset held constant at 15
Excitation wavelength of 561 nm
Emission filter of 630/60
Scan-zoom of 1.5 used during acquisition

controls

Positive control: None mentioned
Negative control: None mentioned

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