The NLS-RFP construct was subcloned from plasmid pML85 (gift of Michael Lisby) into C. glabrata plasmid pMJ22 (88 (link)) (obtained from Addgene) using XhoI and NotI restriction sites. Slides for time-lapse microscopy were prepared by pipetting warm YPD containing 1% low-melting-point agarose (with or without 0.03% MMS) onto glass slides and letting it solidify, forming YPD-agarose pads. Exponentially growing C. glabrata cells carrying the NLS-RFP plasmid were pipetted onto the YPD-agarose pads, covered with coverslips, and sealed using Biotium coverslip sealant (Fisher Scientific). The cells were imaged at room temperature for 6 h at 10-min intervals using a Nikon Eclipse Ti2 inverted microscope and Hamamatsu ORCA-Flash4.0 camera and analyzed using NIS-Elements software.
Eclipse ti2 inverted microscope
Manufactured by Hamamatsu Photonics
The Eclipse Ti2 inverted microscope is a versatile research-grade instrument designed for advanced microscopy applications. It features a high-resolution optical system, sophisticated illumination control, and a modular design to accommodate a wide range of sample types and experimental requirements. The core function of the Eclipse Ti2 is to provide researchers with a reliable and configurable platform for high-quality imaging and analysis of biological specimens.
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3 protocols using eclipse ti2 inverted microscope
1
Time-lapse Microscopy of C. glabrata
2
High-resolution Imaging and Colocalization Analysis
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Imaging was performed at the Nikon Imaging Centre at King’s College London. Z stacks were acquired at 0.12 µm step size on an Eclipse Ti-2 Inverted microscope with Vt-iSIM scan head and Hamamatsu Flash4.0 sCMOS camera using a ×100 oil immersion objective. Laser settings, image capture and Richardson-Lucy deconvolution were managed in NIS-Elements. Images were further processed and Pearson’s correlation coefficient and Mander’s colocalisation coefficient were calculated using the Colocalization Studio plugin73 (link) in Icy software. Maximum intensity projections are shown for better visualisation. A one-way ANOVA with Tukey multiple comparison correction was performed to test for statistical significance in GraphPad Prism v7.04.
3
TIRF Imaging of Fluorescent Proteins
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TIRF imaging was carried out on a Nikon Eclipse Ti2 inverted microscope with a CFI60 60× Apo TIRF objective and a Hamamatsu Orca-Flash 4.0 V2 sCMOS camera. eGFP and Tag.RFP-T fluorescence was excited using 488 and 561 nm lasers and detected using a Chroma HC TIRF Quad Dichroic (C-FL TIRF Ultra Hi S/N 405/488/561/638) and Chroma HC Quad emission filters BP 525/50 and BP600/50, respectively (Bellows Falls, VT). Unless mentioned specifically, channels were acquired sequentially at a 1.2 s interval and 400 ms exposure time over 4.8 to 6 min. Real-time acquisition was achieved by a National Instruments (PXI 1033; Austin, TX) controller. The system was controlled with NIS-Elements software and maintained at 37° C by an OkoLab environmental chamber (Burlingame, CA).
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