All samples were imaged using an Olympus FV3000RS (Olympus) laser scanning confocal microscope under the control of Olympus Fluoview FV3000 software. Fluorescent images were acquired using a 10X air objective (Olympus, UPlan Super Apochromat, NA 0.4, WD 3.1 mm) or 30X oil objective (Olympus, UPlan Super Apochromat, NA 1.05, WD 0.80 mm); and laser diode (405, 488, and 561 nm) laser lines. Imaging settings were consistent between samples for all conditions within the same experiment. Image stacks were converted to TIFF format and max z-projections were reconstructed in ImageJ.
Uplan super apochromat
Manufactured by Olympus
The UPlan Super Apochromat is an optical lens system designed for microscopy applications. It is crafted with high-quality materials and advanced manufacturing techniques to provide excellent optical performance. The core function of this product is to deliver superior image quality and resolution through its precise optical design and superior color correction capabilities.
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2 protocols using uplan super apochromat
1
Confocal Microscopy Imaging Protocol
2
Fluorescent Cell Targeting for Patch Recordings
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To target fluorescent cells for patch recording, two photon imaging was used (Olympus FV1200MPE BASIC (BX-61WI) microscope, 25×, 1.05 NA water-immersion objective (XLPL25XWMP, Olympus), an ultrafast pulsed laser (Mai Tai DeepSee HP, Spectra-Physics) tuned to 910 nm, and the imaging software Fluoview 4.1 (Olympus)). To acquire an image stack, RGCs were filled during electrophysiological recordings with Alexa hydrazide 488 or 594 (100 µM, Invitrogen), and were imaged following the recording, either using the two-photon or the single-photon (confocal) configuration of the two-photon microscope. Tissue in which fluorescent proteins were expressed was often fixed and immunostained (see above), and subsequently imaged on a confocal microscope (Olympus FV3000, UPlan Super Apochromat objectives, 30xS, 1.05 NA, or 60x2S, 1.3 NA) in the Leduc Imaging Facility, Brown University. Confocal and two-photon stacks were processed in Fiji (https://imagej.net/software/fiji ), and collapsed using either maximum intensity or maximum standard deviation projections.
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