Orca r2 ccd camera

For measurements of free intracellular calcium ([Ca²⁺]_i) the cells were loaded with 3 µM (HEK) or 1 µM (DRG) of the fluorescent calcium indicator FURA-2AM (1 mM in DMSO, Biotrend, Cologne; Germany) and 1 µl Pluronic F-127 (10% in DMSO, Calbiochem, Darmstadt, Germany) for 45–60 min (HEK) and 30 min (DRG) in Tyrode's solution containing 137.6 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl₂, 1.8 mM CaCl₂, 10 mM HEPES and 5 mM glucose (adjusted to pH 7.3 with NaOH; Roth). After washing them trice with Tyrode’s solution and at least 20 min resting, cover slips were mounted in an open bath chamber (Series 40 Quick Change Imaging Chamber; Warner Instruments, Hamden, USA) on an inverted microscope (Olympus IX81; Olympus, Tokyo, Japan) equipped with an image acquisition and analysis system (xcellenceRT; Olympus) and superfused with Tyrode’s solution (~ 2 ml min⁻¹). Cells were illuminated alternatingly with light of 340 and 380 nm wavelength (≥ 0.5 Hz) and the respective fluorescent signals at 510 nm were detected by an ORCA-R2 CCD camera (Hamamatsu Photonics, Hamamatsu, Japan; scheme see Fig. 1c). The ratio of fluorescence emission at 510 nm for excitation at 340 vs 380 nm excitation was used as relative change in [Ca²⁺]_i [22 (link)]. All experiments were carried out at room temperature.

CHO-K1 hIR/GLUT4-myc-GFP cells were grown in 96-well imaging plates (35,000 cells/well; Mobitec, Göttingen, Germany) overnight as previously reported [16 (link),17 (link)]. Cell culture medium was aspirated off and, after washing the cells with HBSS (VWR, Vienna, Austria), replaced by HBSS (Thermo Fisher, Waltham, MA, USA) for 3 h. The cells were incubated with insulin or Bellis perennis extracts dissolved in KRPH buffer and imaged on an Olympus IX-81 inverted microscope in objective-type total internal reflection (TIR) configuration via an Olympus 60× NA = 1.49 Plan-Apochromat objective as described earlier [27 (link),28 (link)]. The 96-well plates were placed on an x–y stage (CMR-STG-MHIX2-motorized table; Märzhäuser, Wetzlar, Germany). Scanning of larger areas was supported by a laser-guided automated focus-hold system (ZDC2). The 488 nm emission of the diode laser (Toptica Photonics, Munich, Germany) was used to image green fluorescent protein (GFP) fluorescence. After appropriate filtering, the fluorescence signal was recorded using an Orca-R2 CCD camera (Hamamatsu Photonics, Herrsching, Germany).

Adult hermaphrodites were anesthetized with tricaine and tetramisole and immobilized between a coverslip and agarose pad on a slide. The time-lapse images shown in Figure 2A–E and Figure 4A–B were captured on an Olympus (Center Valley, PA) IX71 microscope equipped with a 60× PlanApo NA 1.42 oil objective and an Orca R2 CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). Hg arc excitation light was shuttered by a Sutter Lambda 10-3 shutter controller (Sutter Instruments, Novato, CA). Images shown in Figure 5A were captured with an Intelligent Imaging Innovations (Denver, CO) Marianas Spinning Disk Confocal equipped with a Photometrics (Tucson, AZ) Cascade QuantEM 512SC EMCCD, and Zeiss 63× 1.4 objective. Image sequences in Figure 6 were captured with a Perkin Elmer-Cetus (Waltham, MA) Ultraview Spinning Disk Confocal equipped with an Orca R2 CCD and an Olympus 60× 1.4 objective.

Images of HE staining were obtained using an Olympus BX51 Microscope (Tokyo, Japan) equipped with a DFK 23U digital camera and Olympus Plan Apo 10× and 20× objectives. Fluorescence images were acquired using a fluorescence microscope (Olympus BX60) equipped with the appropriate filters (U-MNIB, U-MNG) and a Hamamatsu Orca R2 CCD camera.

To study the effect of HTT_ex1 sequence on assembly formation, [rnq-] yeast cells transformed with 25Q-GFP, 25QP-GFP, 43Q-GFP, 43QP-GFP, 97Q-GFP or 97QP-GFP plasmids were grown in parallel to exponential phase, then induced with galactose (see yeast methods). Thereafter, cells were examined by wet-mount on poly-lysine slides (VWR International LLC) at 4, 8, 12 and 24 hr post-induction, using an Axioscope A1 epifluorescence microscope (Carl Zeiss; Oberkochen, Germany), equipped with an X-Cite Series 120Q lamp and an Orca R2 CCD camera (Hamamatsu Photonics; Hamamatsu City, Japan), with a 63x Plan-Apochromat oil-immersion objective (NA 1.4). Fluorescence images were taken using the 470/40 nm and 525/50 nm excitation and emission filters. DIC images were also taken. Cells containing a) LAs b) SAs or c) diffuse fluorescence were manually counted for each time point and construct, and the data were plotted using R.