Second-Harmonic Generation imaging of biological samples is an optical technique that allows the visualization of non-centrosymmetric molecules and their assemblies without the need of a tag or a dye. The images generated with this method have low background since very few biological molecules could act as a harmonal force. This protocol describes the application of the technique for diagnostic imaging of Tubulin beta-4A in the taiep animal model.
Tubulinopathies are recently described diseases. For this reason, a limited amount of information is available about the basic mechanisms underlying the pathophysiology at the cellular level. To begin, turn on the pulse laser to guarantee it will be ready to lase at an optimal and steady power level.
To study tissular microtubules, 10 to 20%of the available laser power is used, which in the described system corresponds to 13 to 26 milliwatts measured at the back focal plane of the objective. Prior to imaging, turn the laser to 810 nanometers. Then ensure that the microscope is Koehler-aligned with the objective used for the Second-Harmonic Generation, or SHG imaging.
Remove unwanted filters from the optical detection path and ensure that the condenser diaphragm is fully opened and no light is unnecessarily stopped. Prepare a 25x by 0.8 numerical aperture oil immersion objective with a small drop of immersion oil in the lens. Following the steps reported in the table here, except for the laser power which is less than 5%for cornstarch.
Image dry cornstarch sandwiched between glass slides. with SHG parameters to generate SHG images, they reveal the laser polarization direction. Capture one image of sparse cornstarch grains and mark the orientation of the SHG lobules in the XY plane corresponding to the laser polarization orientation.
For control, take another image of the same sample inserting a half-wave plate into the optical path to alter the laser's oscillation direction. The resulting image displays rotated SHG signal lobules. If using a different bandpass filter for the SHG, ensure that it has optimal transmission properties by comparing the images and the signal intensities pixel-by-pixel along the scan line.
Prepare a vibratome buffer tray filled with a warm Hanks'Balanced Salt Solution or HBSS. Fix the brain to the specimen plate using cyanoacrylate glue via a piece of masking tape. The most caudal portion contacts the glue so that the usable coronal sections from the opposite rostral portion can be cut.
Transfer the specimen plate onto its magnetic support inside the buffer tray. Start cutting 300 to 500 micron sections until the slices encompass the entire brain surface. Then reduce the thickness of the section to 160 microns.
Once a 160 micron section is cut at the level of the corpus callosum, recover it with a modified glass Pasteur pipette with a large flanged orifice. Transfer the section to a clean Petri dish with new warm HBSS or directly onto the cover slip or glass bottom dish for microscopy. 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 five and averaging two for an average acquisition time of 15 seconds. Capture images first using a 485 nanometer shortpass filter, and in a second step, add a sharp 405 nanometer bandpass filter.
Cut the cerebellum with a scalpel into two hemispheres and fix them to the vibratome support by the middle portion. Section and image the cerebellum using the same vibratome, blade, and microscope settings as described for the brain. When the fibers of the corpus callosum are imaged, fiber-like short structures and rounded elements are observed in the taiep brain.
In contrast, the corpus callosum of the control brain shows a much more heterogeneous and isotropic signal throughout the brain region. The origin of the differential signal lies specifically in the Second-Harmonic Generation phenomenon since adding the narrow bandpass filter only decreases the non-specific signal intensity from the control images, while selectively removing this low diffuse signal from around the soma-like and the short elongated structures in the taiep images which always generate intense SH light. In cerebellar white matter control tissue, the complete absence of SH signal was observed.
The Purkinje cells are barely visible when using the short pass filter, and the elongated and rounded structures persist in the SH image from the taiep tissue. A critical step to obtain the best image is to cut tissue sections as thin as possible. Moreover, working with acute tissue sections requires fast protocols to prevent tissue deterioration.
Therefore, it is crucial to set up and check the optical system beforehand. Second-harmonic signal from microtubules is weaker than second-harmonic signal from starch. Therefore, more laser power has to be used for imaging microtubules.
The laser power should be increased carefully because, as a high numerical aperture objective is used for imaging, the intensity at the sample would rapidly increase With Second-Harmonic Generation imaging, the pathological changes in the central myelinomatic of the tubulinopathic model are quickly and easily identified. Potential applications of this technique include its use for the study of the basic mechanisms of H-ABC tubulinopathy and for the assessment of pharmacological therapies to treat patients. On the long term, the technique has the potential to become a complimentary diagnostic tool to be used intracranially or on fresh biopsies.
