Age synchronized C. elegans expressing different fluorescently tagged transgenes were mounted on microscope slides with M9 buffer. Samples were rinsed three times with M9 buffer to remove debris and eggs. 1 mM levamisole was used to paralyze worms prior to placing coverslips (0.15 mm, #1 thickness) over samples. Confocal microscopy was performed on an A1R system configured on an automated Nikon TiE inverted microscope using a Nikon 20X air immersion CFI Plan Apochromat VC objective with 0.75 numerical aperture (NA) and 1 mm working distance (WD). An LD laser at 488 nm and DPSS laser at 561 nm were used as light source (LU-N4 laser unit, 15 mW). A hybrid A1 scan head with resonant and galvano scanners were used to adjust laser incidence angles. Images were collected using an A1-DU4G detector unit with 2 GaAsP and 2 PMTs. Acquisition was controlled by NIS Elements imaging software and 3X optical zoom was employed during acquisition. Epifluorescence imaging was performed on an Axio Observer inverted microscope by Zeiss, using a Zeiss 20X Plan-Apochromat air immersion objective (0.8 NA, 0.55 mm WD). Images were acquired using standard filter settings for excitation and emission of fluorescence probes/proteins and recorded on a CCD camera (Zeiss AxioCam MRm). Zeiss Zen software was used to control acquisition. Fiji software was utilized for all image processing and analysis.
Cfi plan apochromat vc
Manufactured by Nikon
The CFI Plan Apochromat VC is an optical lens system designed for microscopy applications. It features apochromatic correction and vibration control technology to provide high-resolution, low-distortion images.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam mrm, by Zeiss (1 mentions)
Axio observer inverted microscope, by Zeiss (1 mentions)
Neo scmos, by Oxford Instruments (1 mentions)
C9100 13, by Hamamatsu Photonics (1 mentions)
Du897, by Oxford Instruments (1 mentions)
Ti e inverted microscope, by Nikon (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Nis elements ar software, by Nikon (1 mentions)
Ultraview vox, by PerkinElmer (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
3 protocols using cfi plan apochromat vc
1
Fluorescent Imaging of C. elegans
2
Spinning-Disc Confocal Microscopy Imaging
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Cells were imaged in complete medium (unless stated otherwise) at an acquisition rate from 5-s to 1-min intervals using a spinning-disc confocal microscope (Ultraview VoX; PerkinElmer) attached to an inverted microscope (IX81; Olympus), equipped with a 100× oil-immersion objective (1.40 NA, UPlanSApo), an EMCCD camera (C9100-13; Hamamatsu Photonics) for image acquisition, and Volocity software (PerkinElmer) to control the acquisition protocol. Fixed samples and live cells were also imaged with a Nikon confocal A1R system and Nikon SIM attached to a Ti-E inverted microscope (Nikon) with Perfect Focus System using a 100× oil immersion objective (1.40 NA, CFI Plan-ApochromatVC). The cameras (Neo sCMOS and DU-897; Andor Technology) were used to acquire images for confocal A1R and SIM systems, respectively, with NIS-Elements AR software (Nikon) to control the acquisition protocol. For z-stack images, cells were imaged at a step size of 0.2–0.5 µm with a total height of 15–20 µm.
3
Serum Starvation and Refeeding Assay
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Serum starvation and refeeding experiment were performed by plating HEK293T cells on 96-well plates and transfecting the cells as described above. After 24 h of transfection, cells were washed with PBS and maintained with serum-free culture medium at 37 °C in a humidified 10% CO2 air for 4–5 h, then the medium was replaced with normal culture medium supplemented with serum, incubated for following 4 or 24 h. For rapamycin treatment, transfected cells were treated first with 100 nM of rapamycin or vehicle (4% ethanol, 4% PEG400, 4% Tween 80, and sterile water) for 2 h before the serum starvation procedure described above. Live-cell imaging for colocalization was performed using a Nikon A1R confocal microscope (Nikon Instruments) mounted in a Nikon Eclipse Ti body, and equipped with CFI Plan Apochromat VC objectives (60×/1.4-NA oil or 40×/0.95-NA air; Nikon) and a Chamlide TC system (maintained 37 °C and 10% or 10% CO2; Live Cell Instrument, Inc). CFP and YFP images (512 × 512 pixels, 72.7 µm2) were taken using 457 and 514-nm laser lines, respectively. Colocalization images were quantified using ImageJ coloc2 plugin.
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