This protocol is the first of its kind to apply photostatins on the living preimplantation mouse embryo, and regard uses to use light-switchable drugs on other 3D physiological systems. Light-switchable photostatins allow the manipulation of microtubule growth, in space and time, and to be converted back to an off state. Unintentionally exposing photostatins to blue light, prior to the experiment, can activate them prematurely.
So we advise working in complete darkness, or red light conditions, at all times when preparing your samples. In strict dark conditions, using only red light for illumination, begin making stock and intermediate working solution of PST-1P in ultrapure water. As described in the text manuscript.
Then, dilute PST-1P in fresh KSOM to achieve the final concentration of 40 M.To prepare the chamber slide for live imaging, add 10 L of PST-1P-treated KSOM into the center of one well, to form a hemispherical droplet. To prevent the droplet from evaporating, cover it with sufficient mineral oil. Then, loosely wrap the prepared culture dish in foil, or place the culture dish in a non-airtight opaque container in the incubator to ensure no light exposure.
After transferring the embryos into the PST-1P-treated KSOM drop, ensure that the embryos are clustered in the center of the droplet, and settled to the bottom of the dish. Once the immersion objective lens is prepared, mount the chamber slide on the microscope, inside an environmental chamber. Set at 37 C and 5%CO2, and in complete darkness, to ensure all PST-1Ps are in the inactive trans-configuration, and that embryos can sink to the bottom of the dish.
Use a red light torch to position the objective lens in contact with the immersion medium. Then, locate the edge of the droplet of the medium and position the objective directly over it. Using the live-scanning mode, and a 561 nm laser, locate the embryos within the droplet.
Use stage controllers and live-scanning mode to set the start and end points for acquiring a z-stack of the whole embryo. Adjust the laser power settings up to a maximum of 5%according to your signal. And set digital offset to a maximum of 0.900, according to your signal, to optimize the appearance of the EB3-dTomato comets.
Minimize background noise. Set the pinhole at 2 m. Pixel resolution at 512 by 512.
And the pixel dwell time at 3.15 s. And set the zoom to 2x. Acquire a z-stack of the whole embryo with 1 m section intervals to assess the areas of microtubule growth in the whole organism.
For EB3-dTomato comet tracking experiments, open the 3D z-stack image, increase the zoom to three times. And draw a rectangular ROI around the specific subcellular area of interest. Then, acquire the time lapse movie of a single z-plane, using the set parameters, with the time interval of 500 ms.
To activate PST-1P, switch to a 405 nm laser, and acquire another time-lapse, with the laser set to 10%power, pinhole opened maximally, pixel resolution of 512 by 512, the pixel dwell time of 3.15 s, zoom of three times, at a time interval of 500 ms, and set 20 frames to be acquired. Immediately after activation, switch back to 561 nm laser, and repeat acquisition, as demonstrated previously, to confirm the loss of EB3-dTomato comets after activation of PST-1P. Now, to reverse PST-1P back to its inactive trans-state, engage a 514 nm laser, and acquire another time-lapse with set parameters.
To visualize the recovery of EB3-dTomato comets, switch back to 561 nm laser, and repeat acquisition. In untreated control embryos, before illumination with a 405 nm laser, the EB3-dTomato signal was high within the entire cytoplasm of the cell, except the nuclear area. Post illumination, changes in the signal were not detected.
Indicating that the 405 nm laser did not cause photo damage to the embryo or microtubule dynamics. During PST-1P treatment of 16-cell stage embryos, under strict dark conditions, strong EB3-dTomato expression was detected in the 3D image, demonstrating that inactive PST-1P did not elicit inhibition of microtubule polymerization under dark conditions. Before activation of PST-1P, the time-lapse imaging of a single z-plane confirmed strong EB3-dTomato expression, and microtubule polymerization in a subcellular region.
After 405 nm light activation, within seconds, a reduction in the EB3-dTomato comet signal was detected. And recovery of the signal was achieved upon deactivation of PST-1P, using a 514 nm laser. The embryos micro injected with EB3-dTomato, and incubated with PST-1P, showed normal embryonic potential, developing to the blastocyst stage.
And normal microtubule dynamics during mitosis. Indicating non-toxicity of an active PST-1P to the embryo. In the acquired time-lapse movies, EB3-dTomato labeling appeared as comet-like structures, and enabled tracking of growing microtubule filaments.
In PST-1P treated embryos, kept in dark conditions, individual EB3-dTomato comets emanated from the interphase bridge, which acts as a non-centrosomal microtubule organizing center in the early mouse embryo. A t-stack shows EB3-dTomato comets in the region of the interphase bridge. Following activation of PST-1P, EB3-dTomato comets were reduced at the base of the interphase bridge and in the t-stack.
Remember that white light can activate PST-1P prematurely. So use only red light sources for visibility. Green light also deactivates PST-1P, so please avoid using green fluorescent label markers.
This technique has been used in drosophila, amoeba, mice, and multiple in vitro systems, to target microtubule growth in order to study cell migration and tissue formation.
