ER was labeled with D1ER and mitochondria stained with TMRM (50 nM). Cells were imaged on a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) equipped with 100× objective lens (Plan-Fluor x100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems) and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric corporation). The D1ER and TMRM were alternately excited with 488 and 561 nm laser lines, respectively, and emissions were acquired at 530 and 600 nm using a charged CCD camera (CoolSNAP-HQ, Photometrics, Tucson, AZ, USA). Z-stacks of both channels in 0.2 µm increments were recorded. VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. Images were blindly deconvoluted with NIS-elements v5.1 (Nikon, Vienna, Austria). The colocalization was determined on a single-cell level using ImageJ and the plugin coloc2. The Pearson coefficient was calculated.
Plan fluor x100 1.45 oil
Manufactured by Zeiss
Sourced in Germany
The Plan-Fluor x100/1.45 Oil is a high-performance microscope objective lens manufactured by Zeiss. It features a magnification of 100x and a numerical aperture of 1.45, designed for use with oil immersion. The lens is optimized for fluorescence microscopy applications.
Automatically generated - may contain errors
4 protocols using plan fluor x100 1.45 oil
1
Imaging ER and Mitochondria Colocalization
2
Multi-Channel Confocal Microscopy Imaging
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If not stated otherwise, all experiments were performed with a Zeiss array confocal laser scanning microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) by using 100x objective lens (Plan-Fluor x100/1.45 Oil, Zeiss, Germany). This was equipped with a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, an AOTF-based laser merge module for the 405, 445, 473, 488, 514, and 561 nm laser lines (Visitron Systems), and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric Corporation). Data acquisition and control of the fluorescence microscope were performed using Visiview 4.2.01 (Visitron, Puchheim, Germany).
3
Imaging of ER-Mitochondria Colocalization
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D1ER-overexpressing HeLa cells were stained for 10 min with 200 nM MitoTracker® Red CMXRos and imaged directly. High-resolution images of cells were recorded by using a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) equipped with 100x objective lens (Plan-Fluor x100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems) and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric corporation). The D1ER and Mitotracker® Red CMXRos were alternately excited with 488 and 561 nm laser lines, respectively, and emissions were acquired at 353 and 600 nm using a charged CCD camera (CoolSNAP-HQ, Photometrics, Tucson, AZ, USA). Z-stacks of both channels in 0.2 μm increments were recorded. The software VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. Images were blind deconvoluted with NIS-elements (Nikon, Austria). The colocalization was determined on a single cell level using ImageJ and the plugin coloc2. The Pearson coefficient and the Costes thresholded Manders coefficient were calculated.
4
Visualizing Mitochondrial-ER Interactions in PAEC Cells
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D1ER-containing adenovirus infected PAEC cells were stained for 10 min with 200 nM MitoTracker® Red CMXRos and imaged directly. High resolution images of cells were recorded by using a confocal spinning disk microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) equipped with 100x objective lens (Plan-Fluor x100/1.45 Oil, Zeiss), a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, AOTF-based laser merge module for laser line 405, 445, 473, 488, 561, and 561 nm (Visitron Systems) and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric corporation). The D1ER and Mitotracker ® Red CMXRos were alternately excited with 488 and 561 nm laser lines, respectively, and emissions were acquired at 353 and 600 nm using a charged CCD camera (CoolSNAP-HQ, Photometrics, Tucson, AZ, USA). Z-stacks of both channels in 0.2 µm increments were recorded. The software VisiView acquisition software (Universal Imaging, Visitron Systems) was used to acquire the imaging data. Images were blind deconvoluted with NIS-elements (Nikon, Austria). The colocalization was determined on a single cell level using ImageJ and the plugin coloc2. The Pearson coefficient and the Costes tresholded Manders 1 or 2 coefficients were calculated. The MitoTracker® Red CMXRos channel was assigned to channel 1 and D1ER to channel 2.
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