1 To begin, obtain ligand stain cells2 expressing halo-tagged protein of interest and halo H2B. 3 After turning on the microscope system, 4 set the live cell incubation parameters 5 and let the system equilibrate. 6 Put a drop of the oil onto 7 a 100x TIRF objective on the microscope 8 and load the cell sample onto the microscope stage.
9 To identify a morphologically healthy cell, 10 adjust the Z position 11 and image the cell sample under brightfield illumination. 12 Crop the field of view to capture the target cell nucleus. 13 Using laser illumination, adjust the TIRF angle 14 to achieve hilo illumination with an optimal 15 signal to noise ratio of a single molecule.
16 Then, set the exposure time to 500 milliseconds. 17 During the dead time between frames, 18 photo activate the molecules 19 with a 111 microwatt 405 nanometer beam, 20 and during each exposure, excite H2B halo molecules 21 with a 9.1 milliwatt 640 nanometer beam. 22 Initiate image capturing for 2000 frames continuously 23 and gradually increase the power of the 405 nanometer beam 24 upon reduction of photo activated molecules.
25 For a halo-tagged protein of interest, 26 split emission wavelengths between two cameras 27 using a long pass dichroic mirror. 28 To the same acquisition parameters demonstrated earlier, 29 add JFX549 channel to acquire one frame every 10 seconds 30 and capture 2000 frames continuously.
