Apo tirf 1.49na

For imaging of PI(4,5)P₂ on glass, cg-PI(4,5)P₂ was added to imaging buffer (HBSS with 5% FCS) to a final concentration of 20 µM. The high-affinity PI(4,5)P₂-sensor PLCδ₁-PH-GFP was stored in a 1.8 mg/ml PBS/20% Glycerol stock. This was added 1:20 to the cg-PI(4,5)P₂ solution (e.g. 5 µl in 100 µl). The solution was pipetted onto a glass coverslip and imaged using a TIRF microscope (Nikon Ti Eclipse), equipped with an incubation chamber (37°C), a x60 TIRF objective (Apo TIRF 1.49NA, Nikon), a sCMOS camera (Neo, Andor), four excitation laser lines: (405,488 nm, 568 nm, 647 nm) an appropriate dicroic mirror (Di01-R405/488/561/635),filter (FF01-446/523/600/677). The TIRF microscope was operated by open-source ImageJ-based micromanager software (https://micro-manager.org/). Images were captured at 1 s intervals using a 488 nm laser (200 ms exposure) at 50% power (30 MW). Image analysis was performed with Fiji (ImageJ). Each 488 nm excitation frame was immediately followed by an uncaging frame, performed using a 405 nm laser (200 ms exposure) at 100% power (60 MW). ROIs of cg-PI(4,5)P₂ on glass were selected in the 405 nm channel and the fluorescence intensities of the PLCδ₁-PH-EGFP sensor in the same ROIs in response to uncaging over time measured in the 488 nm channel

A ReAsH-attached SERCA1a was visualized under an inverted microscope (TE2000E; Nikon Instruments) equipped with a 100 × objective lens (Apo TIRF 1.49 N.A.; Nikon Instruments), a 532-nm laser (JUNO532-800; Showa Optronics) with custom-made dichroic mirror to keep the laser polarization after reflection (Chroma), two emission filters (NF03-532E & NF01-532U; Semrock), an EMCCD camera (iXon⁺ DU897; Andor), a highly stable customized stage (Chukosha), and an optical table (Newport). The detailed optical setup was described previously^{21 (link),25 (link)}. All systems were set into a compartment (Nihon Freezer) under which the temperature was stabilized at 23.0 ± 0.1 °C or 5.0 ± 0.1 °C by PID regulation with a heater and a cooler.
The isotropic TIRFM was constructed using a diffractive diffuser (D0740A, Thorlabs) based on the method reported previously^{21 (link),25 (link)}. To observe defocused images, the distance between the objective lens and the specimen was kept constant by the perfect focus system (Nikon Instruments)^{21 (link)}. The custom-made piezoelectric stage (P-620.ZCL; Physik Instrumente GmbH & Co) was used for calibration of the exact distance⁵⁰ .
In the observation performed at 5 °C, to decrease the aberration, the objective lens was typically placed ~ 640 nm closer beyond the best focal plane, and the correction collar was set at the position fully turned counterclockwise.