HeLa cells were cultured and plated in phenol red-free growth medium (Gibco). Before imaging, the cells were washed with PBS and compound PFF-1 (500 nM) was added 10 min prior to imaging. Read-out was achieved with a laser at 561 nm (0.25 kW cm−2, 20 ms) for a total of 100,000 frames. Initial pump acquisition was achieved with a single 405 nm laser pulse (2.6 W cm−2, 20 ms). Sequential pump acquisition was achieved with a 405 nm pulse irradiation (2.6 W cm−2, 20 ms) repeated every 10 min. An incubator with controllable temperature (37 °C) and CO2 (5%) was used to maintain the cells in an appropriate environment during these long measurements.
Phenol red free growth medium
Manufactured by Thermo Fisher Scientific
Phenol red-free growth medium is a type of cell culture medium used to support the growth and maintenance of cells in vitro. It does not contain the pH indicator phenol red, which can interfere with certain experimental applications.
Automatically generated - may contain errors
4 protocols using phenol red free growth medium
1
Single-Molecule Imaging of Compound PFF-1
2
Live-Cell Imaging with Optogenetic Stimulation
3
3D Single-Molecule Localization Microscopy
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HeLa cells were cultured and plated in phenol red-free growth medium (Gibco). Before imaging, the cells were washed with PBS and compound PFF-1 (500 nM) was added to the medium 10 min prior to imaging. An adaptive optics plug and play accessory MicAO 3DSR for SMLM (Imagine Optic, France) was used. This module was inserted between the microscope side port and the sCMOS, Hamamatsu Orca Flash 4 v3 camera. As a second step, a controllable and aberration-free astigmatism was introduced to the point spread function (PSF) of single molecules, which allowed us to perform 3D SMLM based on astigmatism by determining the z positions of the fluorescent molecules based on the ellipticity of their PSF41 (link). The laser excitation was at 561 nm with a laser excitation intensity of 0.25 kW cm−2.
4
Single-Molecule Localization Microscopy of HeLa Cells
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HeLa cells were plated in phenol red-free growth medium (Gibco). Cells were washed with PBS (0.5 mL) before imaging and incubated with compound PFF-1 at the indicated concentration for 10 min. Cells were imaged using a Nikon N-STORM, microscope (Nikon, UK Ltd.) equipped with an SR Apochromat TIRF 100 × 1.49 N.A. oil immersion objective lens. An excitation laser at a wavelength λ = 561 nm illuminated the sample in HILO mode40 (link). Fluorescence was detected with an iXon DU897 (Andor) EM-CCD camera (16 × 16 μm2 pixel size) for 2D SMLM and a Hamamatsu Orca Flash 4 v3 (6.5 × 6.5 μm2 pixel size) for 3D SMLM with an exposure time of 10 ms. An in-built focus-lock system (PFS) was used to prevent axial drift of the sample during data acquisition. The emission was collected and passed through a laser QUAD filter set for TIRF applications (Nikon C-NSTORM QUAD 405/488/561/647) comprising laser clean-up, dichroic and emission filters. The laser excitation at 561 nm had a power density of 0.25 kW cm−2. 2D and 3D SMLM camera frames were recorded at 46 or 100 frames s−1, respectively and for the later an adaptive optics plug and play accessory MicAO 3DSR for SMLM (Imagine Optic, France) was used. From each image stack, a reconstructed super-resolved image was generated using Thunderstorm (FIJI).
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