Lsm 780 clsm

Fluorescent-labeled strains of PA14 and K56-2 were co-inoculated under swarming conditions as described above. Whole plates were then scanned using the Typhoon FLA 9000 with LPR filter and 532 nm wavelength laser for mCherry detection and BGP1 filter and 473 nm laser for green fluorescence, although subject to interference by P. aeruginosa autofluorescence. Close-up of tendrils were visualized with an Olympus Stereoscope using darkfield for the swarming colony and RFP filter for the fluorescence. Tendrils tips were visualized with a Zeiss LSM 780 CLSM. Agar pads bearing a tendril tip were carefully cut from the agar gel with a scalpel and placed onto a 3.5-mm coverslip-bottom dish. A section of 4.25 mm × 8.50 mm was imaged using a 20X objective in tile acquisition mode with a resolution of 10,240 pixels by 20,480 pixels (0.415 μm per pixels). Images were processed using Zeiss Zen Black Edition.

The RenCa cells were seeded on glass coverslips at a density of 2.0 × 10⁵ cells per well in 2.0 mL of complete high glucose Dulbecco’s modified Eagle’s medium (HG-DMEM) in 6-well plates for 24 h. PDM/DOX, PLM/DOX, SCM/DOX, or free DOX · HCl with a DOX · HCl dosage of 10.0 μg mL⁻¹ was added to each well. After coincubation for 2 h, the medium was removed, and the cells on glass coverslips were washed with PBS five times and immobilized by 4% (W/V) PBS-buffered paraformaldehyde for 20 min at room temperature. And then, the cells were washed with PBS five times and reacted with 0.1% (V/V) Triton X-100 in PBS for 12 min at room temperature. The nuclei were then stained with DAPI for 3 min at 37°C, after which the cells were washed with PBS five times. At last, the filamentous actin was stained with Alexa 488 for 30 min at 37°C and washed with PBS five times. The fluorescence was observed with a LSM 780 CLSM (Carl Zeiss, Jena, Germany). Additionally, to further monitor the intracellular DOX release of these micelles with the time, PDM/DOX, PLM/DOX, SCM/DOX, or free DOX · HCl with a DOX · HCl dosage of 1.0 μg mL⁻¹ was coincubated with the cells. After coculture for 2, 6, 12, or 24 h, the cells were detected as mentioned above.

The cells were seeded on the coverslips in 6-well plates with a density of 2.0 × 10⁵ cells/well. A volume of 2.0 mL of DMEM was added into the cells per well and cultured for 12 h. Then 100.0 μL of PUM/C₆ was added to each well. After incubation for 1 or 6 h, the cells were fixed with 4% (W/V) formaldehyde for 15 min. Then, the fixed cells were incubated with DAPI for 3 min and washed with PBS. The images of cell localization were observed under LSM 780 CLSM (Carl Zeiss, Jena, Germany) with 10× eyepiece and 40× objective.

Living plants of L. gibba were analyzed non-invasively using the computer-assisted Zeiss LSM 780 CLSM (Carl Zeiss, Jena, Germany) and the commercial software package ZEN [101 ]. The helium–neon laser line 633 nm was applied for excitation. Auto-fluorescence of chlorophyll was detected with a 650 nm long-pass filter. The 3D in-depth imaging and 3D reconstruction of the chloroplast distribution in living cells was based on the optical serial sections, which were generated in Z-direction starting from the surface of the intact frond. In total, 133 sections (slice thickness 0, 36 µm) were integrated for the animated presentation.