Cells were imaged in the confocal mode of a Nikon Ti-R/B inverted microscope equipped with Yokogawa CSU-X1 spinning disk unit (5000 rpm). Fluorescence proteins were excited, and the emission was monitored and imaged on an iXon ULTRA 897BV back-illuminated deep-cooled electron multiplying charge coupled devices camera. Live cell imaging was performed using a 60×, 1.4 numerical aperture oil objective using 445 (5 mW) and 50 mW, 488-, 515-, 595-nm solid-state lasers. Sensors were imaged using the following settings: GFP: 488 nm at 56 μW/515 nm, YFP and Venus: 515 nm at 22 μW/540 nm, and mCh: 594 nm at 20 μW/630 nm (excitation/emission). Imaging of fluorescence sensors (PIP2, mGq, β-arrestin2, DBD, PKCδ, and Gγ9) was performed at 2 Hz.
Ti r b inverted microscope
Manufactured by Yokogawa
The Ti-R/B inverted microscope is a high-performance optical instrument designed for advanced microscopy applications. It features a high-quality optical system that provides clear and detailed imaging of samples. The microscope's inverted design allows for easy sample manipulation and observation. The Ti-R/B is capable of various microscopy techniques, including brightfield, phase contrast, and fluorescence imaging, making it a versatile tool for a wide range of research and analysis tasks.
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2 protocols using ti r b inverted microscope
1
Live-Cell Imaging of Fluorescence Sensors
2
Live-cell TIRF Imaging of Gβγ/PIP3 Dynamics
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A spinning-disk XD confocal TIRF (total internal reflection) imaging system with a Nikon Ti-R/B inverted microscope, a Yokogawa CSU-X1 spinning disk unit (5000 rpm), an Andor FRAP-PA (fluorescence recovery after photobleaching and photoactivation) module, a laser combiner with 40−100 mW four solid-state lasers (with 445, 488, 515, and 594 nm wavelengths), and an iXon ULTRA 897BV back-illuminated deep-cooled EMCCD camera were used to capture time-lapse image series of live cells. In Gβγ translocation and PIP3 generation experiments, imaging was performed using a 60×, 1.4 NA (numerical aperture) oil objective. To examine the Gβγ translocation, GFP fluorescent tags on Gγ subunits (WTs and mutants) were imaged in every 1 s interval using 488 nm excitation−515 nm emission for 10 min. In PIP3 generation and PIP2 hydrolysis experiments, mCherry-tagged PIP3 and PIP2 sensors, Akt-PH and PH, were imaged using 594 nm excitation−630 nm emission red laser.
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