An inverted microscope (IX81; Olympus) equipped with an UPLANSAPO 100× objective lens (numerical aperture 1.40; Olympus) and a charge-coupled device camera (DP30BW; Olympus) was used for fluorescence microscopy. The filter sets used to acquire fluorescence signals were GFP-4050B (Semrock) for GFP and a U-MRFPHQ unit (Olympus) for FM 4–64 signals. Acquired images were processed using MetaMorph version 7.0 (Molecular Devices).
Uplansapo 100 objective lens
Manufactured by Olympus
The UPLANSAPO 100× objective lens is a high-magnification, oil-immersion objective lens designed for use in microscopy. It provides a 100× magnification, with a numerical aperture of 1.4, which allows for high-resolution imaging. The lens is made from superior optical materials and is engineered to deliver excellent image quality.
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2 protocols using uplansapo 100 objective lens
1
Fluorescence Microscopy of GFP and FM 4-64
2
Time-lapse Fluorescence Microscopy Protocol
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The procedures for time-lapse fluorescence microscopy were as described previously (Tanaka et al., 2010 (link)). Time-lapse images were collected at 25°C. Images were acquired using a DeltaVision Core or Elite microscope (Applied Precision), an UPlanSApo 100× objective lens (Olympus; NA 1.40), SoftWoRx software (Applied Precision) and a CoolSnap HQ camera (Photometrics). We acquired 7–9 (0.7 µm apart) z-sections, which were subsequently processed through deconvolution, and analysed with Volocity (Improvision) software. CFP, GFP, and mCherry signals were discriminated using the 89006 multi-band filter set (Chroma). For the image panels in figures, z-sections were projected to two-dimensional images. Statistical analyses were carried out using Prism (Graph Pad) software.
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