Mito-PAGFP–based mitochondrial fusion assay was performed using a confocal microscope (LSM 510 META; Carl Zeiss) equipped with Plan-Apochromat 100×/1.4D oil DIC M27 objective lens (Carl Zeiss) as described previously (Karbowski et al., 2004 (link); Karbowski et al., 2006 (link)). In brief, after acquisition of a preactivation image, an ∼5-µm-diameter circular region of interest was photoactivated by brief irradiation with 351/364-nm light (using Coherent Enterprise Ion Laser 80.0mW), followed by time-lapse imaging using 488-nm excitation light and 488-nm Argon Ion Laser (25.0 mW set at 0.3%). 15 postactivation images were collected with interval between images set to ∼2 min. To avoid z-section shift, focus was maintained using the “Multi-time Macro” and the autofocusing system (utilizing linescans to detect the reflection off the coverglass). Images were acquired and analyzed using ZEN 2009 image acquisition software (Carl Zeiss).
Zen 2009 image acquisition software
Manufactured by Zeiss
Zen 2009 is an image acquisition software developed by Zeiss. It is designed to capture and manage digital images from Zeiss microscopy systems. The software provides core functionalities for image acquisition, processing, and storage.
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4 protocols using zen 2009 image acquisition software
1
Mito-PAGFP Mitochondrial Fusion Assay
2
Confocal Microscopy Imaging Protocol
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Fluorescent images were collected using an LSM 710 laser scanning confocal microscope and Zen 2009 image acquisition software (Zeiss). Individual channels were collected sequentially. Laser lines for excitation were 405 nm, 488 nm, and 561 nm. Emissions were collected between 440 and 480, 505 and 550, and 580 and 625 nm for blue, green and red fluorescence respectively. For all images, global enhancement of brightness and contrast was performed using Zen Lite 2011 image analysis software (Zeiss). For direct quantitative comparisons (where stated), all images were acquired and processed under identical conditions.
3
Confocal Microscopy Imaging Protocol
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Fluorescent images were collected using a LSM 710 laser scanning confocal microscope and Zen 2009 image acquisition software (Zeiss). Individual channels were collected sequentially. Laser lines for excitation were 405 nm, 488 nm, 561 nm and 633 nm. Emissions were collected between 440–480, 505–550, 580–625 and 650–700 nm for blue, green, red and far-red fluorescence respectively. For all images, global enhancement of brightness and contrast was performed using Zen Lite 2011 image analysis software (Zeiss). For direct quantitative comparisons (where stated), all images were acquired and processed under identical conditions.
4
Retinal Histology Imaging Protocol
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To examine retinal histology fluorescent images were collected using a LSM 710 laser scanning confocal microscope and Zen 2009 image acquisition software (Zeiss). Individual channels were collected sequentially. Laser lines for excitation were 405 nm, 488 nm and 561 nm. Emissions were collected between 440–480, 505–550, and 580–630 nm for blue, green and red fluorescence respectively. For all images, global enhancement of brightness and contrast was performed using Zen Lite 2011 image analysis software (Zeiss). Images of retina flatmounts represent maximum intensity projections generated from confocal slices images collected every 2.5 μm, spanning from the ganglion cell layer to the inner plexiform layer. For direct quantitative comparisons all images were acquired and processed under identical conditions.
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