Fluorescence quantification for AtRGS1-YFP internalization was performed as described by Urano et al. (2012) (link) and Fu et al. (2014) (link). Sterilized, stratified seeds were germinated in 6-well plates containing 2 ml 1/2 × MS liquid medium (pH adjusted to 5.75 with 5 N KOH) at 23°C under darkness. Seedlings (7-day-old) were treated with 0 or 3% D -glucose (w/v) for 30 min. Hypocotyl epidermal cells located 2–4 mm below the cotyledon were imaged (Z stacks obtained) using a Zeiss LSM710 confocal laser scanning microscope equipped with a 20× Plan-NeoFluor (N.A. = 0.5) objective and a 40× C-Apochromat (N.A. = 1.20) water immersion objective. YFP fluorescence was excited by a 514 nm argon laser and detected at 526–569 nm by a photomultiplier detector. At least 10 sets of images from 5 seedlings were obtained for internalization quantification analysis by ImageJ software.
Plan neofluor
Manufactured by Zeiss
The Plan-Neofluor is an optical lens designed for microscopy applications. It provides a flat field of view and high numerical aperture for improved image quality and clarity. The lens is optimized for fluorescence microscopy techniques.
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Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
C apochromat, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Stemi sv11, by Zeiss (1 mentions)
Plan neofluar, by Zeiss (1 mentions)
A plan, by Zeiss (1 mentions)
Axioskop 2 mot plus, by Zeiss (1 mentions)
Fluorodishes, by World Precision Instruments (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Axiovert 100, by Zeiss (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
6 protocols using plan neofluor
1
Fluorescence Quantification of AtRGS1-YFP
2
3D Optic Lobe Imaging in Cuttlefish
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Histological and immunostaining images of the optic lobe slices were acquired on an upright fluorescent microscope (Axioskop 2 mot plus, Zeiss) using either a 5X (A-Plan, 0.12 NA, Zeiss) or a 10X (Plan-Neofluor, 0.3 NA; Zeiss) objective lens depending on the sample size, or on a fluorescent dissecting microscope (Stemi SV11, Zeiss). The high resolution fluorescent images of showing nuclei and neuropil were acquired on a confocal microscope (LSM 510, Zeiss) using a 40X objective lens (Plan-NEOFLUAR, NA 0.75, Zeiss). In addition, the left optic lobe of a sub-adult cuttlefish S. pharaonis (ML = 16 cm) was subjected to the MRI scanning at the Kaohsiung Chang Gung Memorial Hospital (9.4T, Bruker BioSpec 94/20 USR) to obtain its 3D structure. Before scanning, the sample was embedded in agar containing ferric ions to reduce background noise. The MRI scanning system is made up of a self-shielded magnet with a 20 cm clear bore and a BGA-12S gradient insert (12 cm inner diameter) that offered a maximal gradient strength of 675 mT m−1 and a minimum slew rate of 4,673 Tm−1s−1. The optic lobe was imaged at high resolution with TurboRARE-3D-torun sequence (TR/TE = 3,000/48 ms, NEX = 2). The stack of MRI data was then processed to make a movie of the stereo image of the optic lobe.
3
Quantitative Mitochondrial Morphology Imaging
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Cells were seeded in 35 mm Fluorodishes (World Precision Instruments GmbH) and after exposure loaded with loaded with tetramethylrhodamine methyl ester (100nM) (TMRM, Thermo Fisher Scientific) for 25 minutes at 37 °C, 5% (v/v) CO2. Next, cells were washed twice using Krebs-Henseleit buffer supplemented with HEPES (10 mM, KHH) (pH 7.4), and images were captured using a temperature-controlled chamber connected to an inverted microscope (Axiovert 200M, Carl Zeiss) using a x63, 1.25 NA Plan NeoFluor oil immersion objective. As a positive control, the known uncoupling agent carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) was used at the end of each measurement. Images were corrected for background and uneven illumination followed by analysis where images were masked with a binarised image for mitochondrial morphology using Image Pro Plus software (version 6.3, Media Cybernetics) as previously described48 (link).
4
Measurement of Ca2+ Sparks in Cardiomyocytes
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Ca2+ sparks were measured as previously described7 (link) using a laser scanning confocal microscope (LSM-510, Carl Zeiss) equipped with an argon-ion laser coupled to an inverted microscope (Axiovert 100, Carl Zeiss) with a Zeiss 40X oil-immersion Plan-Neofluor objective (numerical aperture, 1.3; excitation at 488 nm; emission at > 505 nm). Briefly, intact cardiomyocytes were loaded with fluo-4 AM (20 μmol/L; Molecular Probes) for 30 min at 24 °C. Line-scan mode was used, where single cardiomyocytes were scanned repeatedly along a line parallel to the longitudinal axis, avoiding the nuclei. To monitor Ca2+ sparks, cardiomyocytes were stimulated until the Ca2+ transient reached a steady state. Stimulation was then stopped, and Ca2+ sparks were recorded during the subsequent ~10 s rest. Data were analyzed with SparkMaster, an automated analysis program that allows for rapid and reliable spark analysis31 (link). The variables analyzed included general image parameters (like a number of detected sparks and spark frequency) as well as individual spark parameters (amplitude, full width at half maximum [FWHM], and full duration at half maximum [FDHM]). To assess SR Ca2+ content, caffeine (10 mmol/L) was rapidly perfused to discharge SR-loaded Ca2+.
5
Autophagy Visualization in Plants and Cells
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WT, atg2, and atg5 RGS1-RFP/GFP-ATG8a seedlings (7 days old) were treated with 0% or 6% D-glucose (w/v) for 30 min. Root epidermal cells located 2–4 mm below the cotyledon were imaged (Z stacks obtained) using a Zeiss LSM710 confocal laser scanning microscope equipped with a 20 × Plan-NeoFluor (N.A. = 0.5) objective and a 40 × C-Apochromat (N.A. = 1.20) water immersion objective. GFP fluorescence was excited by a 488 nm argon laser and detected at 505–550 nm by a photomultiplier detector, whereas RFP fluorescence was excited by a 543 nm HeNe laser and detected at 565–621 nm.
CA (1 µM) and LTR (1 µM) Red (Invitrogen) were added into BY-2 media for 12 and 3 h, respectively, before confocal imaging. GFP fluorescence was excited by a 488 nm argon laser and detected at 505–550 nm by a photomultiplier detector, and LTR fluorescence was excited by a 543 nm HeNe laser and detected at 565–621 nm. At least 10 sets of images were obtained thrice BY-2 cells for quantification analysis. GFP or LTR punctae were counted per cell according to 10 sets of images field of vision.
CA (1 µM) and LTR (1 µM) Red (Invitrogen) were added into BY-2 media for 12 and 3 h, respectively, before confocal imaging. GFP fluorescence was excited by a 488 nm argon laser and detected at 505–550 nm by a photomultiplier detector, and LTR fluorescence was excited by a 543 nm HeNe laser and detected at 565–621 nm. At least 10 sets of images were obtained thrice BY-2 cells for quantification analysis. GFP or LTR punctae were counted per cell according to 10 sets of images field of vision.
6
Dissection and Imaging of Drosophila Fat Body
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Third instar larvae were dissected in phosphate-buffered saline (PBS) and fixed in 4% formaldehyde (Sigma) for 2 h (fat body) at room temperature, washed three times in PBS-0.1% Triton X-100, and mounted in 40% glycerol for direct visualization of tissues. When needed, 300 nM 4′,6-diamidine-2-phenylindole (DAPI) was added to the first washing step.
For LysoTracker staining, the reagent was added to unfixed tissues and directly visualized as previously described (Scott et al., 2004 (link)). Tissues were imaged using a Zeiss confocal microscope LSM 710, using a 20× or 63× Zeiss Plan-Apochromat objective (NA 1.0 and 1.4, respectively) or a 40× Zeiss Plan-Neofluor objective (NA 1.3). When needed images were deconvoluted using Huygens Professional deconvolution software from Scientific Volume Imaging. Omegasome three-dimensional reconstructions were performed with Imaris software from Bitplane (Oxford Instruments), using confocal Z-stacks comprising up to 32 optical slices. For counting GFP-2xFYVE foci inFigure 3 , a threshold was set using ImageJ to eliminate background signal. The cells were divided in two regions: a perinuclear region of 5 μm around the nuclei and a peripheral region in between the border of the cell and the perinuclear region (red and blue lines, respectively).
For LysoTracker staining, the reagent was added to unfixed tissues and directly visualized as previously described (Scott et al., 2004 (link)). Tissues were imaged using a Zeiss confocal microscope LSM 710, using a 20× or 63× Zeiss Plan-Apochromat objective (NA 1.0 and 1.4, respectively) or a 40× Zeiss Plan-Neofluor objective (NA 1.3). When needed images were deconvoluted using Huygens Professional deconvolution software from Scientific Volume Imaging. Omegasome three-dimensional reconstructions were performed with Imaris software from Bitplane (Oxford Instruments), using confocal Z-stacks comprising up to 32 optical slices. For counting GFP-2xFYVE foci in
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