eclipse 90i dual laser scanning confocal microscope  (Nikon)

Journal: Scientific reports

Article Title: A high-precision wound healing assay based on photosensitized culture substrates.

doi: 10.1038/s41598-024-59564-9

Figure Lengend Snippet: Figure 3. Upper panel: Wound healing/migration assay conducted with NRK cells grown to confluence on functionalized polymer coatings after optical wounding with a laser of λ = 408 nm using an upright CLSM. The substrates were illuminated for 1 min. An ND4 filter adjusted the laser intensity. Phase contrast micrographs were recorded 1.5 h, 3.5 h, 5.5 h, 9.5 h and 24 h after wounding. The wound edges were marked with the help of the image analysis software ImageJ. Scale bar: 400 µm. Lower panel: Wound healing/migration assay as in the upper panel. However, cells were studied by fluorescence microscopy after a vital stain using calcein AM (living cells, green)/EthD-1 (dead cells, red). Samples were stained 0 h, 3 h, 6 h, 9 h or 24 h after wounding. Scale bar: 100 µm.

Article Snippet: After incubation, the staining solution was removed, the specimen was washed with PBS++ and then imaged using an upright Nikon Eclipse 90i Confocal Laser Scanning Microscope (CLSM) in combination with a 10 × objective (NA = 0.25). (ii) In a second assay the cells were stained with a solution containing 8 μg/mL fluorescein diacetate (FDA; λexc = 498 nm; λem = 517 nm) and 20 μg/mL propidium iodide (PI; λexc = 535 nm; λem = 617 nm) in serum-free medium after the optical wounding.

Techniques: Migration, Polymer, Software, Fluorescence, Microscopy, Staining

Journal: Scientific reports

Article Title: A high-precision wound healing assay based on photosensitized culture substrates.

doi: 10.1038/s41598-024-59564-9

Figure Lengend Snippet: Figure 5. Measured wound area for NRK cells (A) and RAT1 cells (B) on functionalized glass substrates over time. The substrates were either illuminated for 1 min using a CLSM (A) or 2 min using an epifluorescence microscope (B) with a wavelength of λ = 408 nm and a 10 × objective. In case of RAT1 cells the illuminated area was manually adjusted by a diaphragm. In case of the NRK cells an ND4 filter was inserted to reduce the intensity of the laser light. The red line shows the linear regression of the data between the first and the fore last time point. From the negative slope of the linear regression, migration rates can be determined. For RAT1 cells the migration rate is (21.1 ± 0.5)⋅103 µm2/h, for NRK cells it is (10.3 ± 0.4) ⋅103 µm2/h. (A) Data depicted for NRK cells: mean ± SD, n = 5.

Article Snippet: After incubation, the staining solution was removed, the specimen was washed with PBS++ and then imaged using an upright Nikon Eclipse 90i Confocal Laser Scanning Microscope (CLSM) in combination with a 10 × objective (NA = 0.25). (ii) In a second assay the cells were stained with a solution containing 8 μg/mL fluorescein diacetate (FDA; λexc = 498 nm; λem = 517 nm) and 20 μg/mL propidium iodide (PI; λexc = 535 nm; λem = 617 nm) in serum-free medium after the optical wounding.

Techniques: Microscopy, Migration

Journal: Scientific reports

Article Title: A high-precision wound healing assay based on photosensitized culture substrates.

doi: 10.1038/s41598-024-59564-9

Figure Lengend Snippet: Figure 7. CaAM (green)/EthD-1 (red) assay of NRK cells on functionalized glass substrates containing different vitamin E concentrations after optical wounding with a laser of λ = 408 nm at the upright CLSM. Vitamin E concentrations of 0%, 1%, 2%, 5% and 10% (w/w of PS) were used. The illumination time was set to 1 min. An ND4 filter was inserted. Scale bar: 100 µm.

Article Snippet: After incubation, the staining solution was removed, the specimen was washed with PBS++ and then imaged using an upright Nikon Eclipse 90i Confocal Laser Scanning Microscope (CLSM) in combination with a 10 × objective (NA = 0.25). (ii) In a second assay the cells were stained with a solution containing 8 μg/mL fluorescein diacetate (FDA; λexc = 498 nm; λem = 517 nm) and 20 μg/mL propidium iodide (PI; λexc = 535 nm; λem = 617 nm) in serum-free medium after the optical wounding.

Techniques:

Journal: Scientific reports

Article Title: A high-precision wound healing assay based on photosensitized culture substrates.

doi: 10.1038/s41598-024-59564-9

Figure Lengend Snippet: Figure 6. Fluorescence micrographs (CLSM) of confluent NRK cells grown on glass slides coated with PS/ PtTFPP (upper panel) or PS (lower panel) that were illuminated for 2 min with 408 nm, 488 nm or 543 nm laser light. The cells were stained by a calcein AM / ethidium homodimer vital stain as detailed in Materials & Methods. Significant wounding only occurs when the growth substrates are coated with PS/PtTFPP functional layer excited at the resonant wavelength of the Soret band at 408 nm. Illumination of control substrates (PS) does not introduce wounds into the cell layer at any of the exposure conditions. The scale bar corresponds to 100 µm.

Article Snippet: After incubation, the staining solution was removed, the specimen was washed with PBS++ and then imaged using an upright Nikon Eclipse 90i Confocal Laser Scanning Microscope (CLSM) in combination with a 10 × objective (NA = 0.25). (ii) In a second assay the cells were stained with a solution containing 8 μg/mL fluorescein diacetate (FDA; λexc = 498 nm; λem = 517 nm) and 20 μg/mL propidium iodide (PI; λexc = 535 nm; λem = 617 nm) in serum-free medium after the optical wounding.

Techniques: Fluorescence, Staining, Functional Assay, Control, Introduce

Journal: Scientific reports

Article Title: A high-precision wound healing assay based on photosensitized culture substrates.

doi: 10.1038/s41598-024-59564-9

Figure Lengend Snippet: Figure 9. (A) Effect of exposure time (texp) and light intensity on wound size when confluent layers of NRK cells were grown on standard photosensitizer-doped functional coatings upon irradiation by a 408 nm laser using an upright CLSM. The exposure time was gradually increased from 0.5 min, 1 min to 2 min. Light intensity was controlled by the use of neutral density filters (ND4 and ND8). Wound edges were delineated by white lines using the ImageJ software. (B) Changes in the size of the wounded areas in dependence on exposure time and light intensity. Mean ± SEM; n = 8.

Article Snippet: After incubation, the staining solution was removed, the specimen was washed with PBS++ and then imaged using an upright Nikon Eclipse 90i Confocal Laser Scanning Microscope (CLSM) in combination with a 10 × objective (NA = 0.25). (ii) In a second assay the cells were stained with a solution containing 8 μg/mL fluorescein diacetate (FDA; λexc = 498 nm; λem = 517 nm) and 20 μg/mL propidium iodide (PI; λexc = 535 nm; λem = 617 nm) in serum-free medium after the optical wounding.

Techniques: Functional Assay, Irradiation, Software