Roots of two-week-old Arabidopsis lines expressing MVLG_06175ΔSP-mCherry, MVLG_06175-mCherry, and mCherry alone, leaves of four- or six-week-old Arabidopsis line expressing MVLG_06175ΔSP-mCherry, MVLG_06175-mCherry, MVLG_05122ΔSP-CFP, mCherry alone, and CFP alone, and their corresponding WT controls were observed by confocal microscopy. Images were acquired by an Olympus Fluoview FV-1000 confocal coupled to an Olympus 1 × 81 inverted microscope ((Olympus, East Syracuse, NY, USA), a PlanApoN 60× objective, and FV-10 ASW 2.1 software. A single channel scanning configuration was set up for the acquisition of mCherry (excitation 587 nm, emission 610 nm) and CFP (excitation 458 nm, emission 476 nm) using a 543 nm HeNe laser and a 458 line of argon laser, respectively. Scanning was set at a speed of 2 μs/pixel to acquire z-stacks of each visual field. Images are presented as either single plane images or stacked images.
Planapon 60 objective
Manufactured by Olympus
Sourced in United States
The PlanApoN 60× objective is a high-quality microscope objective lens designed for use in various laboratory applications. It provides a high numerical aperture and a long working distance, allowing for detailed observation and analysis of samples. The lens is optimized for both phase contrast and fluorescence imaging, making it a versatile tool for a wide range of microscopy techniques.
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4 protocols using planapon 60 objective
1
Confocal Imaging of Arabidopsis Root and Leaf
2
Quantitative Fluorescence Microscopy Analysis
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Image z-stacks (every 0.2 µm in the z direction, 20–25 sections in total) of the immunostained samples were obtained at room temperature by using the DeltaVision Elite microscopy with Olympus PlanApoN 60× objective (NA 1.42) and sCMOS camera. InsightSSI light (∼50 mW) and the four-color standard filter set were equipped. Acquisition software was Softworx in the DeltaVision. Because the signals were not distributed homogeneously across z-stacks, some images in the stacks were projected to cover whole nucleus (usually seven images) using Softworx and used as a source images. Nucleoplasm regions were extracted on the basis of the DNA (DAPI) staining regions. For active RNAPII staining, the mean intensities of the nuclear signals after background subtraction (the nuclear signals without primary antibody treatment or the signals outside nuclei) were calculated and plotted. For immunostaining with Ki67 and Topo II, the numbers of nuclei with intensity higher than a threshold value were counted and plotted.
3
Chromosome Counting in HeLa Cells
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To count the chromosome numbers of HeLa cells, mitotic chromosome spreads were made. Cells were treated with colcemid (0.2 μg/ml) (045-16963, Wako) for 1 hour at 37°C. The following steps were performed at room temperature except the final fixation step. After washing with PBS, cells were trypsinized and resuspended in hypotonic buffer (75 mM KCl) for 10 min. Cells were then gently fixed by repeating the following treatment three times: fixation buffer (MeOH: acetic acid, 3:1) for 5 min, centrifuged at 850g for 4 min, and then resuspended in new fixation buffer. Last, the cells were completely fixed by treatment with 200 μl of fixation buffer at −30°C for at least 30 min. For imaging, 5 μl of this fixed cell suspension was dropped onto coverslips after mixing and completely dried by incubating at 60°C for 30 min. Dried cells were stained with DAPI (0.5 μg/ml) for 5 min, followed by PPDI mounting. Images of mitotic chromosome spreads were obtained using DeltaVision Elite microscopy with an Olympus PlanApoN 60× objective (NA 1.42) and an sCMOS camera. The number of chromosomes in one cell was counted manually.
4
ATP and Magnesium Quantification in Cells
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For ATP reduction, cells were incubated with 10 mM sodium azide and 50 mM 2-deoxyglucose in DMEM for 40 min at 37°C. To measure ATP, aliquots of 0.5 × 105 cells were seeded into a 12-well culture plate (IWAKI) and Cell ATP Assay Reagent (300-15363, Toyo B-Net Co. Ltd.) was used according to the manufacturer’s instructions. Bioluminescence was measured using a Lumat LB 9507 tube luminometer (EG&G Berthold). A standard plot of ATP concentration versus bioluminescence intensity verified that our measured ATP concentrations fell within a linear range. Both the reaction and measurement were performed at 23°C in the dark. The incubation time was 5 min from the addition of assay reagent to measurement.
Magnesium Green AM (Kd ∼ 1.0 mM; M3735, Thermo Fisher Scientific) was applied to the DMEM culture medium at 10 μg/ml with 0.02% Pluronic F-127 (P3000MP, Thermo Fisher Scientific), and the cells were incubated at 37°C for 1 hour. Magnesium Green fluorescence was measured by DeltaVision equipped with an Olympus PlanApoN 60× objective (NA 1.42) and an sCMOS camera with a FITC filter by using Softworx software. Cell culture conditions (37°C, 5% CO2, and humidity) were maintained in a live-cell chamber under the microscope. The nucleoplasm intensity was measured using Softworx software and plotted after subtracting background signals outside the cells.
Magnesium Green AM (Kd ∼ 1.0 mM; M3735, Thermo Fisher Scientific) was applied to the DMEM culture medium at 10 μg/ml with 0.02% Pluronic F-127 (P3000MP, Thermo Fisher Scientific), and the cells were incubated at 37°C for 1 hour. Magnesium Green fluorescence was measured by DeltaVision equipped with an Olympus PlanApoN 60× objective (NA 1.42) and an sCMOS camera with a FITC filter by using Softworx software. Cell culture conditions (37°C, 5% CO2, and humidity) were maintained in a live-cell chamber under the microscope. The nucleoplasm intensity was measured using Softworx software and plotted after subtracting background signals outside the cells.
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