Second Harmonic Generation Imaging in a Rat Model to Study Tubulin Defects

Begin with a rat brain tissue slice mounted on a glass slide and immersed in a buffer.

The rat suffers from tubulinopathy, a condition caused by defects in tubulin, the building blocks of microtubules, leading to their abnormal accumulation and disruption.

This also affects myelin production in the central nervous system.

Place the slice under a covered two-photon excitation microscope for detailed imaging.

Regularly hydrate the slice to maintain its integrity.

Set the microscope to a non-descanned imaging mode, a label-free technique for detecting weak signals.

Expose the tissue to a pulsed infrared laser.

Microtubules emit light as detectable signals known as second harmonic generation or SHG signals.

Areas with abnormal microtubule accumulation amplify these signals, increasing their detectability.

Apply optical filters to refine these signals.

The captured image reveals microtubule density and abnormalities, wherein high SHG intensity and clustering indicate tubulin defects and reduced myelin production.

Place the sample under the microscope and position it appropriately under the objective by direct observation through the oculars with transmitted light. Remove the excess HBSS so that a thin liquid film covers the entire sample. Visually check the liquid film every few minutes to avoid excessive evaporation and drying of the sample.

Prepare the microscope stage for non-descanned imaging, which includes closing all the doors of the dark incubation chamber, or covering the incubation chamber with a black nylon polyurethane coated fabric. Select the non-descanned imaging mode along the transmission path. This way, the capture of the weak SH signal of tubulin will be optimized.

Then select the objective. Next, set a laser power with a pixel dwell time of 12.6 microseconds. Take images no bigger than 512 by 512 pixels, with speed 5 and averaging 2 for an average acquisition time of 15 seconds. Capture images first using a 485-nanometer short pass filter. And in a second step, add a sharp 405-nanometer bandpass filter.