Cells were imaged in an Olympus IX71 inverted microscope with a 1.40-NA 100× phase contrast oil immersion objective as previously described.[20 (link)], [26 (link)] Images were captured on a Photometrics Evolve EMCCD with a 40 ms integration time. Samples were excited with a 561-nm laser (Coherent Sapphire 561-50) with a power of 1.9 μW·μm−2. For Bs PY79 MutS-PAmCherry, PAmCherry was activated by cycles of exposure to a 406 nm laser (Coherent Cube 406) with a power of 0.15 μW·μm−2 for 200 ms. YFP-RecO and mCitrine-RecO fusions were imaged with a 488-nm laser (Coherent Sapphire 488-50) at 0.25 μW·μm−2. For 3D imaging, a cylindrical lens with 1000 mm focal distance (ThorLabs LJ1516RM-A) was placed in the optical path between the microscope and the camera. For each 3D experiment, a z-calibration curve was generated using a sample of immobilized 0.1-μm TetraSpeck™ microspheres (ThermoFisher Scientific) and collecting data at a series of known z-positions with a piezo stage (Physik Instrumente) controlled by PIMicroMove software as previously described.[38 (link)]
Piezo stage
Manufactured by Physik Instrumente
The Piezo stage is a high-precision positioning device that utilizes piezoelectric technology to achieve accurate and repeatable motion. It is designed for applications requiring precise positioning and control.
Automatically generated - may contain errors
Lab products found in correlation
Tetraspeck microsphere, by Thermo Fisher Scientific (1 mentions)
Ix71 inverted microscope, by Olympus (1 mentions)
Lj1516rm a, by Thorlabs (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Coolsnap hq2, by Teledyne (1 mentions)
Axioimager z2 epifluorescence microscope, by Zeiss (1 mentions)
Observer z1 microscope, by Hamamatsu Photonics (1 mentions)
Orcaflash 4lt camera, by Hamamatsu Photonics (1 mentions)
2 protocols using piezo stage
1
3D Single-Molecule Imaging of DNA Repair Proteins
2
Visualizing Cellular Redox State Changes
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RoGFP was expressed in primary fibroblasts using modified pEGFP-N1 (RRID:Addgene_38120) as the expression vector and JetPei as the transfection reagent. After the cells were incubated in culture medium treated with or without HU or APH for 72 h at 37 °C, the cells were washed twice with Hanks’ balanced salt solution. For pEGFP-N1/roGFP1, the cells were imaged on a Zeiss Observer Z1 microscope with a Hamamatsu ORCA Flash 4LT camera. Images were acquired using MetaMorph software (Molecular Devices). For dual excitation ratio imaging, excitation filters at wavelengths of 400 nm and 488 nm were used, and an emission filter at a wavelength of 535 nm was used. The fluorescence excitation ratio was obtained by dividing the intensities of the cells using excitation filters at 400 nm and 488 nm.
For pEGFP-N1/roGFP-NLS (nuclear localization), the cells were incubated with DAPI (1 μg/ml) and imaged using a microscope. The images were captured using the 63x oil immersion objective of a motorized Axio Imager Z2 epifluorescence microscope (Carl Zeiss) equipped with a high-sensitivity cooled interline CCD camera (Cool SNAP HQ2; Roper Scientific) and a PIEZO stage (Physik Instrumente). Images were acquired using MetaMorph software (Molecular Devices). In each case, 300–500 cells were analyzed per condition.
For pEGFP-N1/roGFP-NLS (nuclear localization), the cells were incubated with DAPI (1 μg/ml) and imaged using a microscope. The images were captured using the 63x oil immersion objective of a motorized Axio Imager Z2 epifluorescence microscope (Carl Zeiss) equipped with a high-sensitivity cooled interline CCD camera (Cool SNAP HQ2; Roper Scientific) and a PIEZO stage (Physik Instrumente). Images were acquired using MetaMorph software (Molecular Devices). In each case, 300–500 cells were analyzed per condition.
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