HeLa cells were transfected with pEGFP-N1 hMETTL9-GFP constructs using FuGENE transfection reagent (Promega) for 24 h and probed with ER-tracker and Mitotracker (ThermoFisher Scientific). Live cells were imaged with PlanApo ×100, numerical aperture 1.1 oil objective (Olympus).
Planapo 100
Manufactured by Olympus
Sourced in Japan
The PlanApo ×100 is a high-quality objective lens designed for microscopy applications. It features a plan-apochromatic optical correction, providing excellent image quality with minimal distortion and aberrations. The lens has a magnification of 100x and a numerical aperture of 1.40, enabling high-resolution imaging and detailed observation of specimens.
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Lab products found in correlation
Fugene transfection reagent, by Promega (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Er tracker, by Thermo Fisher Scientific (1 mentions)
Coolsnap hq camera, by Teledyne (1 mentions)
Softworx, by Cytiva (1 mentions)
Deltavision rt system, by Cytiva (1 mentions)
C9100 13, by Hamamatsu Photonics (1 mentions)
Aquacosmos, by Hamamatsu Photonics (1 mentions)
Ff01 525 50, by IDEX Corporation (1 mentions)
Ff01 593 40, by IDEX Corporation (1 mentions)
Em ccd c9100 13, by Hamamatsu Photonics (1 mentions)
Aquacosmos software, by Hamamatsu Photonics (1 mentions)
Celltirf, by Olympus (1 mentions)
5 protocols using planapo 100
1
Localization of hMETTL9 in HeLa Cells
2
Imaging Protein Localization and Cell Morphology
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The DeltaVision RT system (Applied Precision) consisting of an Olympus IX70 wide‐field inverted fluorescence microscope, Olympus PlanApo ×100 (numerical aperture 1.4) and oil‐immersion objectives, and a CoolSNAP HQ camera (Roper Scientific), was used for observing protein localization and cell morphology. The images were captured and processed by iterative constrained deconvolution using SoftWoRx (Applied Precision). To observe cell morphology, 1 μL of Calcofluor (5 mg/mL) was added in 500 μL of cell cultures (final concentration: 1 μg/mL) and briefly vortex, left at room temperature for 1 min. Cells were then collected by centrifuge (3000 rpm, 1 min) and mounted on 2% agar pad containing glutamate‐based minimal media (Moreno et al. 1991 ) with appropriate supplements. For live imaging of CRIB‐GFP, cells were mounted on 2% agar pad containing YE5S and left at 27 °C for 30 min, and then started imaging. The images were taken every 6 min in 10 Z‐sections of 0.4 μm thicknesses each otherwise stated. The max projection images were used for data analysis. Cell length was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Live Cell Imaging with HILO Microscopy
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Living cells were mounted in a glass-bottom culture dish coated with 2 mg/mL concanavalin A70 (link) and were imaged with HILO microscopy as previously described.46 (link) A solid-state laser (488 nm, 20 mW; Sapphire 488-20-OPS, Coherent) and an inverted microscope (IX-81, Olympus) equipped with an oil-immersion objective lens (PlanAPO 100×, NA 1.45, Olympus) were used. Images were captured with a back-thinned electron multiplying charge-coupled device camera (C9100-13; Hamamatsu Photonics) at a frame rate of 30 frames per second, and recorded on a digital hard-disk recorder (AQUACOSMOS, Hamamatsu Photonics). Fluorescence intensities were measured using Image J software.
4
Total Internal Reflection Fluorescence Microscopy
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A custom-built TIRF microscope was used, as described previously.19 (link) Briefly, the beam from a 100 mW, 488 nm laser or 150 mW, 561 nm laser (Light HUB-6, Omicron, Germany) was expanded using a Galilean beam expander and focused at the back focal-plane of a high numerical aperture, oil-immersion, objective lens (PlanApo, 100×, NA 1.45, Olympus, Japan) using a small, aluminium-coated mirror (3 mm diameter, Comar Optics, UK) placed at the edge of the back-aperture of the objective lens. The average laser power at the specimen plane was adjusted to ∼0.5 μW μm−2 and the incident laser beam angle was adjusted to ∼63° to create the evanescent field at the glass–aqueous medium interface. A second small mirror was placed at the opposite edge of the objective lens back-aperture to remove the returning (internally-reflected) laser beam from the microscope and a narrow band-pass emission filter FF01-525/50 or FF01-593/40 (Semrock, Rochester, NY) was used to block the scattered 488/561 nm laser light and other unwanted light. An EMCCD camera (iXon897BV, Andor, UK) captured video sequences at a rate of 20–50 fps, and the data were stored on a computer hard drive for analysis.
5
Fluorescence Microscopy Imaging Protocol
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Imaging by fluorescence microscopy32 (link) was performed on an inverted microscope (IX-83, Olympus) with a 100 × objective (PlanApo 100 × NA1.45 Oil, Olympus) using a HILO and TIRF illuminator (Cell TIRF, Olympus). Solid-state laser (488 nm, 20 mW, Sapphire 488-20-PS, Coherent) and CoolLED fluorescence light source (635 nm, Molecular Devices) were used for fluorescence excitation. Images were captured with two back-thinned electro multiplier charge coupled device cameras (EMCCD, C9100-13, Hamamatsu Photonics). Images were recorded with AQUACOSMOS software (Hamamatsu Photonics) and analyzed using ImageConverter software (Olympus Software Technology)33 (link).
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