Uplxapo100xo

STED images of the Abberior STAR 635 and Abberior STAR 580 channels were acquired with an Abberior STEDYCON in the Anschutz Medical Campus Advanced Light Microscopy Core (ALMC) facility, which is installed on an Olympus IX81 microscope stand, using a 100X/1.45NA Olympus UPLXAPO100XO oil immersion objective. Sample regions were first identified by eye in the TRITC epifluorescence channel by selecting parts of the sample with strong punctate labeling in the Abberior STAR 580 channel. Following this, a 5 × 5 tiled overview image was acquired in confocal mode and the regions with even labeling, and the lowest background were selected for STED imaging. ROIs were selected based on the orientation of dendritic branches, due to the nature of the scanning device, which enables faster and more efficient acquisitions, meaning wide rectangles were used about the height of a laterally protruding dendritic branch. Pixel size was 25 nm. Laser power was first established such that a count of ~100 photons per pixel in confocal mode was established, then STED laser power was set to about 3-fold the laser power in confocal mode. Once determined, these laser intensities remained the same across all conditions.

SPT studies were conducted using the same apparatus employed in our previous study,^{33 (link)} with some modifications. A brief explanation including the modifications is provided below. The SPT experiments were conducted at room temperature. We used an inverted fluorescence microscope (IX-73, Olympus Co. Ltd., Japan) with an oil-immersion objective lens of 100×, NA = 1.45, and WD = 0.13 mm (UPLXAPO100XO, Olympus Co. Ltd., Japan) and a confocal scanner unit (CSU-X1, Yokogawa Electric Co. Ltd., Japan) with a zoom lens of 2.0×. The QDs were excited using a solid-state laser with an emission wavelength of 488 nm (OBIS488LS, Coherent CO. Ltd., USA). The laser output power was set to 170 mW. The emissions from the QDs were recorded using an electron-multiplying charge-coupled device (EMCCD) camera (C9100-23B, ImagEM X2, Hamamatsu Photonics Co. Ltd., Japan) at 20 frames per second (exposure time: 0.05 s). In this setup, one pixel of the image corresponded to 0.08 μm; thus, the observed image area corresponded to 40.96 μm × 40.96 μm. The focal plane was set 5 μm above the bottom surface of the column using a piezo-actuator (P-725K, Physik Instrumente GmbH & Co. KG, Germany).

STED images of the Abberior STAR 635 and Abberior STAR 580 channels were acquired with an Abberior STEDYCON in the Anschutz Medical Campus Advanced Light Microscopy Core (ALMC) facility, which is installed on an Olympus IX81 microscope stand, using a 100X/1.45NA Olympus UPLXAPO100XO oil immersion objective. Sample regions were first identified by eye in the TRITC epifluorescence channel by selecting parts of the sample with strong punctate labeling in the Abberior STAR 580 channel. Following this, a 5 × 5 tiled overview image was acquired in confocal mode and the regions with even labeling, and the lowest background were selected for STED imaging. ROIs were selected based on the orientation of dendritic branches, due to the nature of the scanning device, which enables faster and more efficient acquisitions, meaning wide rectangles were used about the height of a laterally protruding dendritic branch. Pixel size was 25 nm. Laser power was first established such that a count of ~100 photons per pixel in confocal mode was established, then STED laser power was set to about 3-fold the laser power in confocal mode. Once determined, these laser intensities remained the same across all conditions.