Plan apochromat 63 1.4 oil dic m27

CLSM fluorescence imaging spectrum was performed with a confocal laser-scanning microscope ZEISS LSM 880 (Carl Zeiss AG, Oberkochen, Germany) equipped with an Airyscan detection unit and a high sensitivity GAsP detector for visible detection. To maximize the resolution enhancement, we used a high numerical aperture (NA) oil immersion objective (Plan-Apochromat 63×/1.4 Oil DIC M27; Zeiss). All imaging was performed using Immersol 518 F immersion media (Carl Zeiss). Laser gain, detector gain, and pixel dwell times were adjusted to maintain the lowest laser power and highest signal to noise ratio in order to avoid saturation and bleaching effects. Three spectral channels were used to record the fluorescence of Laurdan (laser excitation: 405 nm; channel 1: 412–463 nm; channel 2: 472–535 nm) and A5-Alexa 647 (laser excitation: 633 nm; emission: 640–750 nm). CLSM experiments were performed in replicates for more than three times on different samples and days.

For live-cell imaging, cells were grown in MatTek dishes and imaged in an Elyra 7 with Lattice SIM2 microscope (Zeiss) equipped with an environmental chamber (temperature controlled at 37°C, humidified 5% CO₂ atmosphere), two PCO.edge sCMOS version 4.2 (CL HS) cameras (PCO), solid-state diode continuous-wave lasers, and a Zeiss Plan- Apochromat 63×/1.4 Oil DIC M27, all under the control of ZEN black software (Zeiss). Biotin (400 μM), Janelia Fluor 646 (200 nM), and/or Alexa Fluor 647-conjugated human transferrin (25 μg/ml; Invitrogen) were added to the cells for some experiments.

Four sequential brain sections per animal were selected for immunohistochemical analysis. Brain sections were permeabilized with PBS-1% Triton-X100 for 12.5 min and blocked for 2 h with 10% fetal bovine serum (Biochrom, London, UK). Then, sections were incubated for 72 h, at 4 °C, with a rabbit anti-glial fibrillary acidic protein (GFAP) antibody (DAKO Z0334, 1:200), or a rabbit anti-ionized calcium binding adaptor molecule 1 (Iba-1) antibody (WAKO 019-19741, 1:2000). Then, the sections were incubated for 2 h, at room temperature, with the secondary antibodies (anti-rabbit A488 Molecular Probes A11008, 1:1000 or anti-rabbit A546 Invitrogen A11010, 1:1000). Nuclei were stained with Hoechst 33342 (Invitrogen H1399, 1:1000). Images of the peri-infarct area were acquired using the LSM 710 AxioObserver Microscope (Zeiss, Jena, Germany) with a 40× objective (EC Plan-Neofluar 40×/1.3 Oil DIC M27) for the quantification of mean fluorescence intensity and integrated density, and a 63× objective (Plan-Apochromat 63×/1.4 Oil DIC M27) for the evaluation of glial cell morphology. Mean fluorescence intensity and integrated density were quantified with ImageJ software in four non-overlapping fields of view of the peri-infarct area (or equivalent region in the control group) per section.

Images of cells were acquired using a confocal microscope (Zeiss LSM 980 with Airyscan 2) with a Zeiss Plan Apochromat ×63/1.4 oil DIC M27 objective controlled by ZEN software (version 3.3 blue). Cells were seeded on 35 mm plates (FD-35-100, World Precision Instruments [WPI]) coated with polylysine. Confocal images were taken of T-REx-293 cells transfected with pCDNA4-hTRPV1^WT-GFP or pCDNA4-hTRPV1^N331K-GFP. Laser irradiation at 488 nm was used to excite the GFP and imaged using a 490–550 nm band-pass emission filter. Detector gain and laser intensity were constant for all experimental groups. Superresolution images were taken from T-REx-293 cells cotransfected with either pCDNA4-hTRPV1^WT-GFP or pCDNA4-hTRPV1^N331K-GFP together with pCDNA4-hTRPC3^WT-mCherry using the Airyscan mode to achieve a resolution of up to 140 nm. Laser irradiation at 488 nm and 561 nm was used to excite the GFP and mCherry, respectively. GFP was imaged using 420–480 nm and 495–550 nm band-pass emission filters, and mCherry was imaged using a 574–720 nm band-pass emission filter.

The fluorescence and DIC imaging were performed using a Zeiss LSM 710 laser scanning confocal microscope, equipped with a 63× oil immersion objective (Plan-Apochromat 63×/1.4 oil DIC M27) and a Zeiss Primovert inverted microscope. Samples were prepared and imaged using tween-coated (20% v/v) Nunc Lab-Tek Chambered Coverglass (ThermoFisher Scientific Inc.) at room temperature (22 ± 1 °C) unless otherwise noted, with ~ 1% labeled protein samples within the mixture of unlabeled proteins. All the samples were allowed to equilibrate in the chambered coverglass for ~30–45 min before imaging. For Alexa488-labeled samples, the excitation and emission wavelengths were 488 nm/503–549 nm; for Alexa594-labeled samples, the excitation and emission wavelengths were 595 nm/602–632 nm. Fluorescence recovery after photobleaching (FRAP) experiments were performed using the same confocal set up. The images and data were analyzed using Fiji software [70 (link)] and the FRAP curves were plotted and analyzed using origin software (OriginPro 2018).

Second harmonic generation signal from three‐dimensional spheroids in 3D collagen matrix or from tumour tissue was detected upon simultaneous excitation with a 920‐nm laser (MaiTai). The signal was collected using a BP 460/50 filter, MBS 690. The images were acquired using the LSM 7 MP microscope (Carl Zeiss, Jena, Germany) using the Zeiss W Plan‐Apochromat 20×/0.8 M27 or Plan‐Apochromat 63×/1.4 Oil Dic M27 objective lens.

Overnight cultures of MR10 wild type and ∆icaA were diluted 1000-fold in BHINC and 100 µL of the suspension was incubated with 19R-Alexa 488 (25 µg/mL) at 37 °C for 24 h under static conditions on a 35 mm polystyrene dish (Thermo Fisher Scientific). After incubation, the supernatant was removed by pipetting. Fifty microliters of 10% paraformaldehyde were mounted on the biofilm to fix the biofilm for 10 min at 25 °C. Then, 50 mM NH₄Cl was added to the sample to quench the paraformaldehyde and immediately removed. The samples were washed with 50 µL of PBS. For polysaccharide staining, the fixed biofilm was incubated with WGA-Alexa 594 (5 µg/mL) for 20 min at 37 °C. After incubation, excess WGA-Alexa 594 was removed, and the biofilm was washed with 50 µL of PBS. DNA was then stained with 50 µL of DAPI solution (1 µg/mL) for 5 min at 25 °C. A coverslip was placed on the sample. The biofilm and extracellular structures were observed with LSM880 in the Airyscan super-resolution mode (Carl Zeiss, Oberkochen, Germany). All the images were digitalized at a resolution of 16 bits into an array of 512 × 512 pixels with four averages. The optimal conditions of fluorescent samples were obtained using an argon laser (wavelength 488 nm, 3%), diode 405–30 laser (405 nm, 1%), and DPSS 561–10 laser (561 nm, 1%) with a Plan-Apochromat 63×/1.4 Oil DIC M27 (Carl Zeiss).