Ix83 confocal microscope

To determine the subcellular localization of TaClpS1 in N. benthamiana leaves, A. tumefaciens carrying pCAMBIA1302: TaClpS1–GFP, pCAMBIA1302: TaClpS1Δ–GFP or pCAMBIA1302: GFP vector at a final OD₆₀₀ of 0.5 was infiltrated into N. benthamiana leaves. Vectors pCAMBIA1302: TaClpS1Δ–GFP and pCAMBIA1302: GFP were used as negative controls. The infiltrated N. benthamiana were maintained in growth rooms at 21–25 °C with a 16-h/8-h light/dark cycle. At 48 h after agroinfiltration, confocal images were obtained with an Olympus IX83 confocal microscope (Japan) using excitation wavelength of 488 nm and emission wavelength of 520 nm for GFP, and excitation wavelength of 561 nm and emission wavelength of 640 nm for chloroplast autofluorescence, respectively.
For testing the localization of TaClpS1 in wheat cells, Triticum aestivum Suwon11 seedlings were grown in the glasshouse at 25 °C for 2–3 weeks. The fusion constructs pCaMV35S: TaClpS1-GFP, pCaMV35S: TaClpS1Δ-GFP and pCaMV35S: GFP were independently transformed into wheat protoplasts by polyethyleneglycol (PEG)-calcium method as described previously [38 (link), 39 (link)]. The mixtures containing pCaMV35S: TaClpS1-GFP or pCaMV35S: GFP and wheat protoplasts were incubated at 22 °C. Images were obtained with an Olympus IX83 confocal microscope (Japan) at 24 h after incubation.

HepG2 cells were transfected essentially as described^{47 (link)} with a vector for expression of GFP (pEGFP-N1, clonetech, #6085-1) or the above described vectors for expression of GFP fused to the C-terminus of Tspan15, Tspan15 Iso2, CD53 or CD53 Iso2. Cell-free membrane sheets were produced by short ultra-sound pulses^{47 (link)}. If not stated otherwise, epi-fluorescence microscopy was employed for imaging membrane sheets and whole cells that in this case additionally were visualized with the membrane dye TMA-DPH (Invitrogen, #T204). TMA-DPH and GFP-fluorescence were imaged by epi-fluorescence microscopy essentially as described^{47 (link)}. For confocal microscopy, cells additionally expressed KDEL-RFP and were stained as described below. They were imaged in the confocal mode of a 4-channel easy3D superresolution STED optics module (Abberior Instruments) coupled to an Olympus IX83 confocal microscope (Olympus, Tokyo, Japan), equipped with an UPlanSApo 100x (1.4 NA) objective (Olympus, Tokyo, Japan). For imaging details see below. Additionally, GFP was excited with a 485 nm laser and recorded with a 525/50 nm filter.

For confocal and STED microscopy, coverslips mounted on microscopy slides were imaged with a four-channel easy3D super-resolution STED optics module (Abberior Instruments) combined with an Olympus IX83 confocal microscope (Olympus) using an UPlanSApo 100× (1.4 numerical aperture) objective (Olympus). GFP and Vybrant DiO were excited with a 485 nm laser and recorded with combined 500 to 520 nm and 532 to 558 nm filters. Alexa594 and Rhodamine Phalloidin were excited with a 561 nm laser and recorded with a 580 to 630 nm filter. Atto647N and STAR RED were excited with a 640 nm laser and detected with a 650 to 720 nm filter. For STED microscopy, a pulsed 775 nm STED laser was used for depletion of Alexa594, STAR RED, and Atto647N.
A pinhole size of 60 μm was used, and the pixel size was 25 nm for all images. Confocal images were recorded with time-gated detection with 0.78 ns delay and 8 ns gate width. STED micrographs were recorded with six line accumulations and time-gated detection with 0.75 ns delay and 8 ns gate width.