Confocal fluorescence images and videos were acquired using an inverted Zeiss LSM 710 confocal microscope with a 60X Plan-Apochromat objective. Epifluorescence and bright-field images were acquired using an upright Nikon Eclipse Ni-E fluorescence microscope equipped with a Ds-Ri2 camera and CFI Plan Apochromat Lambda objectives. For PAS histology images, Z stacks were acquired and all-in-focus images were created using the NIS Elements Extended Depth of Focus plugin. All images were processed using ImageJ. All image modifications were performed on entire images (no masking was used) and were performed identically between genotypes.
Cfi plan apochromat lambda
Manufactured by Nikon
Sourced in Japan
The CFI Plan Apochromat Lambda is a high-performance microscope objective lens designed by Nikon. It features a plan-apochromatic optical design and utilizes extra-low dispersion glass to provide superior chromatic aberration correction across a wide range of wavelengths.
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Lab products found in correlation
Eclipse ti e, by Nikon (8 mentions)
Volocity software, by PerkinElmer (7 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Eclipse ni e fluorescence microscope, by Nikon (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Ultraview spinning disc confocal scanner unit, by PerkinElmer (1 mentions)
Nis element software, by Nikon (1 mentions)
Zyla 4.2 plus, by Oxford Instruments (1 mentions)
Di01 t405 488 568 647, by IDEX Corporation (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Ds ri2, by Nikon (1 mentions)
Eclipse ti2 e inverted microscope, by Tokai Hit (1 mentions)
C hgfi, by Nikon (1 mentions)
Hoechst 33342, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 chicken anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Velocity software, by PerkinElmer (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Eclipse ti u inverted microscope, by Oxford Instruments (1 mentions)
Observer d1 inverted microscope, by Zeiss (1 mentions)
18 protocols using cfi plan apochromat lambda
1
Multimodal Imaging Protocols for Microscopy
2
Live-cell Imaging of Pax7-ZsGreen Cells
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Pax7-ZsGreen cells were isolated by FACS and plated for live-cell imaging into 0.1% gelatin-coated 24-well glass-bottom dishes (NC9988706; Mattek; Thermo Fisher Scientific; Waltham, MA; 12,000 cells/well) containing MGM with 20% CS-FBS. Cells were treated with MGM containing vehicle (0.03% ethanol in PBS) or E2 (final concentration 100 pM) at the time of plating and again 18 h after plating. Time-lapse imaging was performed from 18 h to 72 h after plating with a Nikon Eclipse Ti-inverted fluorescence microscope equipped with an automated stage (Prior), and a custom chamber to maintain a constant 37°C temperature, high humidity, and 5% CO2. Multiple positions were analyzed per group with images acquired every 10 min using phase contrast. Images were collected using a ×20 CFI Plan Apochromat Lambda (NA = 0.75) objective (Nikon). For each condition, at least 100 individual cells were tracked. Following imaging, data were exported as individual TIFFs for each position and time point. ImageJ software package was used to concatenate TIFF images from each location and manually measure time to first division of each cell. Cell size at 18 h after plating was measured following pixel-based classification and cell segmentation with ilastik (version 1.3.3) and CellProfiler (version 4.0.5), respectively.
3
Quantification of Autophagy Markers
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To quantify accumulation of LGG-1–positive structures, we generated a single copy insertion reporter of GFP::LGG-1 by CRISPR-Cas9. The number of GFP::LGG-1 puncta was scored at the larvae l (L1) stage. Fluorescent images in 40–50 z series (0.5 µm/section) were captured using a 60× objective (CFI Plan Apochromat Lambda; NA 1.45; Nikon) on an inverted fluorescence microscope (Eclipse Ti-E; Nikon) with an UltraView spinning-disc confocal scanner unit (PerkinElmer Inc.) with 488 (emission filter 525 [W50]) laser. Serial optical sections were analyzed, and the number of GFP::LGG-1 puncta was quantified by Volocity software (PerkinElmer Inc.). At least 20 animals were quantified in each strain.
4
Yeast Cell Microscopy Protocol
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Yeast cells were grown in appropriate media (SCD media for Figs. 2 F , 6 F , S1 F, and S3 C; YPL media for Fig. S3 A) to log phase, concentrated, and immobilized on microscope slides. Fluorescent images were captured at 25° C using a 100× objective (CFI Plan Apochromat Lambda, NA 1.45; Nikon) with immersion oil (type NF) on an inverted fluorescence microscope (Eclipse Ti-E; Nikon) with a spinning-disk confocal scanner unit (UltraView; PerkinElmer) with 488 (emission filter 525 [W50]) and 561 (dual-band emission filter 445 [W60] and 615 [W70]) lasers. Z-stack images with 0.5-µm increments were acquired with Volocity software (PerkinElmer) and processed with Volocity and ImageJ software.
5
Live-Cell Fluorescence Microscopy Protocol
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Fluorescent live-cell microscopy (Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 3 , Figure 4 , Figure 5 , Figure 6 D, and S2 ) was performed at room temperature on a Yokogawa CSU-WI imaging system, which was equipped with a Nikon Eclipse Ti2 inverted microscope, a 100 × 1.45 NA CFI Plan Apochromat Lambda objective lens (Nikon); 405-, 488-, and 561-nm laser lines; and a photometrics Prime BSI camera. Due to fluorescence of Tet, imaging of fluorescently tagged proteins was performed on cells that had been washed into fresh Tet-free media for 20 min prior to imaging. 1,6-hexanediol-treated cells were also imaged on this system (Fig. 5 ). These cells were grown and induced with Tet in YE4S as described, washed for 20 min into fresh YE4S, then incubated another 10 min in the presence or absence of 5% (w/v) 1,6-hexanediol dissolved in media.
6
Live Cell Imaging of Transfected Cells
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Cells were cultured on glass bottom dishes (801001; NEST). 24 h after transfection of plasmids, cells were cultured in HBSS medium and imaged at 37°C and 5% CO2 using a 100× objective (CFI Plan Apochromat Lambda, NA 1.45; Nikon) with immersion oil on an inverted fluorescence microscope (Eclipse Ti-E; Nikon) with a spinning-disk confocal scanner unit (UltraView; PerkinElmer). Images were taken every 5 s for 1 h. The images were analyzed with Volocity software (PerkinElmer).
7
Multimodal Fluorescence Imaging Techniques
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TIRF and epifluorescence imaging was performed on an inverted microscope (Nikon Ti Eclipse) with a 488 nm excitation laser and a 525/50 nm emission filter. For SDC imaging, an inverted Nikon TI microscope equipped with a confocal scanner unit (CSU–W1 disk; Yokogawa), which contains a four–band dichroic beamsplitter (Di01–T405/488/568/647; Semrock) and a 525/50 nm emission filter, was used. Both microscopes were operated by Nikon NIS element software. All images were collected by a 100×/1.45 NA oil objective (CFI Plan Apochromat Lambda; Nikon) with sCMOS cameras (Zyla 4.2 plus, Andor).
8
Confocal and Brightfield Microscopy Protocol
9
Live Imaging of Phagosomes in C. elegans
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Embryos at the precomma or comma stage, or adult worms at 24 or 36 h after L4/adult molt, were mounted on agar pads in M9 buffer without (embryos) or with 5-mM levamisole (adult worms; levamisole prevents animals from moving but does not affect the germline or the gonad). Fluorescent images were captured using a 100× objective (CFI Plan Apochromat Lambda; NA 1.45; Nikon) with immersion oil (type NF) on an inverted fluorescence microscope (Eclipse Ti-E; Nikon) with a spinning-disk confocal scanner unit (UltraView; PerkinElmer) with 488 (emission filter 525 [W50]) and 561 (dual-band emission filter 445 [W60] and 615 [W70]) lasers. Images in 20–25 z series (1.0 µm/section) were captured every 1 or 2 min for 1–2 h (embryos) or 2–6 h (adult worms) at 20° C and were viewed and analyzed using Volocity software (PerkinElmer). The numbers of phagosomes that were followed and quantified are indicated in the figures and figure legends. In general, at least 10 phagosomes from three independent animals were followed and quantified in each strain.
10
Photographic Imaging of Insect Aedeagus and Habitus
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Habitus images were taken using a Canon 5DSR digital camera, equipped with a lens EF 75–300 mm f/4–5.6 linking a Nikon CFI Plan Apochromat Lambda 4× or 2× objective lens. Illumination was by flash, and each photo was taken by a macro slide system.
Aedeagus images were taken using a Nikon D610 digital camera, linking a Zeiss V microscope, with 5× and 10× objective lens. A cable shutter release was used to prevent the camera from shaking. The number of images taken was depending on the size of the aedeagus.
To get full depth of focus, all images were stacked with HELICON FOCUS 6 (http://www.heliconsoft.com/heliconsoft-products/helicon-focus/ ) and the resulting output, edited with Adobe Photoshop CC (https://www.photoshop.com/ ).
Aedeagus images were taken using a Nikon D610 digital camera, linking a Zeiss V microscope, with 5× and 10× objective lens. A cable shutter release was used to prevent the camera from shaking. The number of images taken was depending on the size of the aedeagus.
To get full depth of focus, all images were stacked with HELICON FOCUS 6 (
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