SLBs were imaged using a commercial LSM 710 (Carl Zeiss, Jena, Germany) at 20 °C with a Zeiss C-Apochromat 40X, NA = 1.2 water immersion objective. Photobleaching experiments were conducted at 20 °C using the same microscope set-up described above. Control images were acquired before bleaching, then a 10 × 10 µm area was bleached at nominal 100% laser transmission, and a series of images (every 4 s) were captured immediately after bleaching. Domain size analysis was carried out by processing images with the “analyze particles” tool of ImageJ (http://imagej.nih.gov/ij/ ). Circularity was calculated as 4π (area/perimeter2) for every measured domain using ImageJ software.
C apochromat 40x
Manufactured by Zeiss
Sourced in Germany
The C-Apochromat 40x is a high-performance objective lens designed for microscopy applications. It features a numerical aperture of 1.2 and is optimized for fluorescence imaging. The lens is intended to provide superior chromatic correction and high-resolution imaging capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Lsm 880 airyscan confocal microscope, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Phenol red free dmem, by Merck Group (1 mentions)
Eight well chambers, by Ibidi (1 mentions)
Lsm 710 confocor3 microscope, by Zeiss (1 mentions)
Plan apochromat 20x, by Zeiss (1 mentions)
Juli br live cell movie analyzer, by NanoEnTek (1 mentions)
Plan apochromat 10x, by Zeiss (1 mentions)
Ds ri2, by Nikon (1 mentions)
8 protocols using c apochromat 40x
1
Imaging and Analysis of Lipid Domains
2
Real-time Ferroptosis Monitoring in Cells
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Cells were seeded in DMEM in IBIDI eight-well chambers (Ibidi, Germany) 24 h before the experiment. The day after, cells were washed with PBS to replace the media by phenol red-free DMEM (Sigma-Aldrich, Germany) supplemented with FBS and antibiotics. Cells were loaded with 1-µM Fluo-4 AM, 2-µg/mL PI, or 1 µM of C11 BODIPY 581/591 for 30 min at 37 °C. All images were acquired with a Zeiss LSM 710 ConfoCor3 microscope (Carl Zeiss, Jena, Germany) or a gSTED Leica confocal microscopy equipped with incubator at 37 °C and 5% CO2. Time-lapse imaging with z-stack acquisition was carried out before and after ferroptosis induction. Transmitted light and fluorescence images were acquired through a Zeiss C-Apochromat 40X, NA = 1.2 water immersion objective or a 63X, NA = 1.2 oil immersion objective onto the sample. Excitation light came from Argon ion (488 nm) or HeNe (561 nm) lasers. All images were processed in Fiji. The percentage of Fluo-4 AM-positive cells was calculated at each time point. For this, individual cells were automatically detected based on the fluorescence of the Fluo-4 AM. To define cells as positive, we arbitrarily set a fluorescence threshold in which the percentage of Fluo-4 AM-positive cells did not exceed 5% in the negative control. The total number of cells in each condition was determined in the transmitted light images.
3
Fluorescence Microscopy for Salivary Gland Analysis
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SMG morphological evaluation was performed using a digital inverted fluorescence microscope (Nikon, Tokyo, Japan; Ti) equipped with a digital camera (Nikon, DS-Ri2) and a CFI Plan Fluor 4x objective (Nikon) or JuLI Br live cell movie analyzer (NanoEnTek, Seoul, Republic of Korea). Immunofluorescence images were taken by confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany;LSM700) equipped with Plan-Apochromat 10x, Plan-Apochromat 20x, and C-Apochromat 40x objectives (Carl Zeiss) and with 405, 488, and 555 nm wavelength excitation lasers. Live imaging of epithelial rudiments of SMG and SMG-C6 cells were conducted through a confocal microscope (Carl Zeiss) with a customized live cell chamber (Live Cell Instruments, Seoul, Republic of Korea) that maintained 5% CO2 and 37 °C conditions. To visualize peripheral cell movement (Fig. 4I,J ), the epithelial rudiments of SMGs were briefly stained with 1 μg/ml Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA; H3570) –culture media solution for 1 h. After staining, cells were washed with culture medium two times.
4
Confocal Microscopy Imaging and FRAP Analysis
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These experiments were carried out using a Zeiss LSM510 Meta confocal microscope equipped with C-Apochromat 40X(NA = 1.20, water immersion) objective and confocal images were acquired with 512 × 512 pixel (pinhole aperture ~1 airy units). Flash-EDT2 dye was excited using an argon laser at 488 nm. Hoechst 33342, for nuclear counterstaining was excited with a 405 nm diode laser. A 563 nm laser was used to excite Lysotracker red. All live cell imaging was performed in 20 mM HEPES-based medium.
For the FRAP measurements, the scanning laser power was set to 5%, whereas the bleaching at the region of interest (ROI, ~2 µm in diameter) was carried out using 100% of the laser power. Pre-bleaching was carried out by taking 10 images, which was followed by photo-bleaching with 20 iterations at 100% laser power. Eighty (80) post-bleaching images were acquired.
For the FRAP measurements, the scanning laser power was set to 5%, whereas the bleaching at the region of interest (ROI, ~2 µm in diameter) was carried out using 100% of the laser power. Pre-bleaching was carried out by taking 10 images, which was followed by photo-bleaching with 20 iterations at 100% laser power. Eighty (80) post-bleaching images were acquired.
5
Fluorescence Fluctuation Microscopy Protocol
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FCCS was performed on a Zeiss LSM 880 Airyscan confocal microscope equipped with a Zeiss C-Apochromat 40x, numerical aperture 1.2, water immersion objective. Image acquisition and measurement point selection were controlled by Zen Black software. Excitation was provided by the 488 nm laser line of an Argon ion laser and 561 nm laser line of HeNe laser. The laser power, measured before the objective, was 3 μW for 488 nm and 9 μW for 561 nm. This unequal power was selected to reduce the relative magnitude of green fluorescence bleed-through into the red channel (Jülich et al., 2015 (link)). The emitted light passed through a 34 μm pinhole and was separated by MBS 488/561/633 into two different detection ranges of 508–535 nm for the green channel and 606–668 nm for red channel set for internal 32-Channel GaAsP array. The correlator was set as 0.2 ms binning with 8 tau channels. The acquisition time for a measurement was 10 s.
6
Imaging Aggregated Alpha-Synuclein Proteins
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These experiments were carried out using a Zeiss LSM 510 Meta confocal microscope equipped with a C-Apochromat 40 X (NA = 1.20, water immersion) objective and confocal images were acquired with 512 × 512 (pinhole aperture ~1 airy units). The α-Syn-EGFP protein was excited using an argon laser at 488 nm.
7
Fluorescence Fluctuation Microscopy Protocol
8
Live Cell Fluorescence Correlation Spectroscopy
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FCCS was performed with a Zeiss LSM 880 confocal microscope. Image acquisition and analysis were controlled by Zen black software. GaASP and PMTr detectors for single fluorescence molecule detection and dynamic characterization were used. Measurements were performed with a Zeiss C-Apochromat 40x, numerical aperture 1.2 water immersion objective. For FCCS in live cells, two simultaneous fluorescence channel detection coupled with transmission T-PMT was used. Fluorescence emission was detected in the range of 500-560 nm and 650-710 nm. Nunc TM Lab-Tek Q5 Chambered Coverglass (Thermo Fisher Scientific, USA) were used. QuickFit 3.0 [35] free software was employed for FCCS data analysis. To fit data, the global fitting with 1-2 components 3D normal diffusion model was applied. If a satisfactory fit was not obtained, a 1-component 3D normal diffusion model was applied only to the FCCS curve, and is specified in the caption of the table reporting the data. The confocal volume was determined using 25 nM Rhodamine 123 solution.
Hydrodynamic radii were calculated applying the Stoke-Einstein equation:
where D c is the diffusion coefficient derived from the FCCS fitting, k B the Boltzmann constant, T the absolute temperature, 𝜂 the viscosity and 𝑟 𝐻 the hydrodynamic radius.
Further details on FCCS theory and experimental data are reported in the Supplementary Information.
Hydrodynamic radii were calculated applying the Stoke-Einstein equation:
where D c is the diffusion coefficient derived from the FCCS fitting, k B the Boltzmann constant, T the absolute temperature, 𝜂 the viscosity and 𝑟 𝐻 the hydrodynamic radius.
Further details on FCCS theory and experimental data are reported in the Supplementary Information.
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