Shortly before the irradiation, cells were washed with PBS and the medium was replaced with warm low absorption medium (31053; Thermo Fisher Scientific). Cells were placed in an incubator chamber at 37 °C with 5% CO2 supplementation mounted on a Leica TCS SP8 X microscope system (Leica Microsystems). A Leica HC PL APO 40 ×/1.30 Oil CS2 objective and a zoom of 0.75 made it possible to irradiate up to 200 cells in one frame based on the cell size and the seeding. We placed 40 horizontal stripe masks (5 px wide) in a view field of 512 × 512 px and irradiated the cells 35 times with a 405 nm diode laser at 95% with FRAP booster with a pixel dwell time of 3.75 µs. One full frame irradiation lasted 1.985 s. Thus, a total irradiation with 35 iterations took 69.3 s (130 Hz bidirectional, frame rate 0.503/sec). We measured a laser power of 1.14 J/sec exiting the objective. Consequently, each pixel of the irradiation masks was exposed to 4.275 µJ per iteration resulting in a total energy of 149.625 µJ. After irradiation, cells were fixed and permeabilized with 3% formaldehyde, 0.2% Triton X-100 and 8% sucrose for 15 min at RT.
Tcs sp8 x microscope system
Manufactured by Leica
The Leica TCS SP8 X microscope system is a high-performance confocal microscope that enables advanced imaging of biological samples. It features a tunable supercontinuum white light laser, providing a broad range of excitation wavelengths for flexible fluorescence imaging. The system is equipped with high-sensitivity detectors and an advanced optical design to deliver high-resolution, high-contrast images.
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Lab products found in correlation
2 protocols using tcs sp8 x microscope system
1
Laser-Assisted Cell Irradiation Imaging
2
Laser-Induced DNA Damage Assay
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Laser micro-irradiation experiments were done using 8-well chamber slides (Ibidi # NC0704855). These slides were coated with collage O/N at 4C (10 ng/mL diluted in PBS). Collagen was aspirated and B16F10 were seeded at 100K–200K cells/well and allowed to adhere overnight. Cells were mounted on a Leica TCS SP8 X microscope system (Leica Microsystems) with an incubator chamber at 37°C with 5% CO2. For laser micro-irradiation cells were exposed to a 405 nm diode laser (95% with FRAP booster, 35 iterations, 150 μJ/pixel total power) and irradiation field of multiple 5-pixel wide stripes. At different time points after irradiation (1 minute, 5 minutes, 10 minutes, 15 minutes, and 20 minutes), cells were fixed with 4% PFA in PBS for 10 min at RT. γH2AX staining was done as described above. For analysis of micro-irradiation stripe intensity ImageJ was used.
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