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