Subcellular localization of αSyn-GFP in living cells was assessed via confocal microscopy using a ZEISS LSM800 Airyscan microscope (Fig 1C ), equipped with a Plan-Apochromat 63x/1.40 Oil M27 objective, and ZEISS ZEN software control. Micrographs in Figs 2B and 3E were recorded with a Leica SP5 confocal laser scanning microscope, equipped with a Leica HCX PL Apo 63x NA 1.4 oil immersion objective. Cells were counterstained with PI to visualize dead cells and subsequently immobilized on agar slides. Micrographs were processed with the open-source software Fiji [105 (link)]. Gaussian filtering (σ = 1) was applied to reduce image noise, followed by background subtraction (rolling ball radius = 50 pixels). Pictures within an experiment were captured and processed using the same settings.
Hcx pl apo 63x na 1.4 oil immersion objective
Manufactured by Leica
The HCX PL Apo 63x NA 1.4 oil immersion objective is a high-performance objective lens designed for use in microscopy applications. It has a magnification of 63x and a numerical aperture of 1.4, which allows for high-resolution imaging of samples. The objective is optimized for use with oil immersion techniques.
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2 protocols using hcx pl apo 63x na 1.4 oil immersion objective
1
Visualizing αSyn-GFP Subcellular Localization
2
Fluorescence Confocal Microscopy Protocol
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Laser Scanning Confocal Microscopy images were acquired on an inverted Leica TCS SP5 microscope equipped with an HCX PL APO 63X, NA 1.4 oil immersion objective in fluorescence mode. The laser outputs were controlled via the Acousto-Optical Tunable Filter (AOTF) and the two collection windows using the Acousto-Optical Beam Splitter (AOBS) and photomultiplier tubes (PMT) as follows: Fluorescein was excited with an argon laser at 488 nm (12%) and measured with emission settings at 500-550 nm. The helium-neon laser at 633 nm (10%) was only used in transmission mode. Images were collected using the microscope in sequential mode with a line average of 8 and a format of 512*512 pixels or 1024*1024 pixels.
Samples (≈30 μL) were injected in µ-slide (chambered coverslip) with uncoated 8 wells from Ibidi GmbH. Processing of fluorescence confocal acquisitions were performed with the ImageJ freeware.
Samples (≈30 μL) were injected in µ-slide (chambered coverslip) with uncoated 8 wells from Ibidi GmbH. Processing of fluorescence confocal acquisitions were performed with the ImageJ freeware.
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