This technique can image myelin in brain tissue sections. myelination plays an important role in the conduction of action potentials and understanding myelin will help us understand brain function. The main advantage of the CARS technique over, for example, electron microscopy is that CARS is a light microscopy technique, and can easily be combined with other fluorescence imaging, such as used in immunohistochemistry.
Several medical conditions are due to alterations in myelination, for example, multiple sclerosis, aging, and autism. Understanding these alterations will help us understand the underlying medical conditions. In this case, the CARS laser is tuned to image lipids or, specifically, CH2 bonds.
In the brain, the largest source of lipids is myelin. Thus, this is a technique to image myelin in brain tissue. It should be possible to use the same method for other types of tissue when imaging lipids is of interest.
Tuning and aligning the CARS laser requires significant expertise, and is best performed by a laser or light microscopy expert. After preparing the tissues as described in the manuscript, stain free-floating sections for nisel and antibody media to visualize cell bodies, then incubate the sections on a standard laboratory shaker for 30 minutes at room temperature. Before bringing the samples to the microscope, turn on and warm up the CARS laser for at least one hour, then align the CARS laser by spatially overlapping the pump and stokes laser beams, and adjust the delay between the two laser beams.
Color the condenser optics and the diaphragm of the microscope for forward CARS imaging, then adjust the external periscope to center the spatially overlapped two lasers onto the scanning head mirrors of the microscope. For best forward CARS non-descanned detection. make sure the condenser is color lured.
For immunofluorescence, confocal imaging, and cars imaging, fit the CARS laser with both forward and epi CARS non-descanned detectors by incorporating a confocal microscope equipped with visible lasers for fluorescence imaging. Now, place the sections in a culture dish with a cover slip bottom, and PBS to avoid drying the tissue. Also, use a glass weight to keep the tissue near the cover slip.
Using the graphic user interface, set the image acquisition parameters for cars and missile fluorescence confocal imaging. Place the sample on the microscope stage, focus the sample, and capture the images. CARS'spectra range from 640 to 660 nanometers and there appears to be no overlap in spectra with nisel-tagged immunofluorescent marker, indicating that CARS signals can be used with immunofluorescent signals.
Nisel was used to visualize the cell bodies of Mongolian gerbil and mouse brains, and CARS'imaging was used to visualize the myelin sheath, indicating that this technique could be used across species. Both sets of images show a section of the medial nucleus of the trapezoid body in the brain stem. The resolution of CARS is lower than with electron microscope and is on the order of several hundred nanometers.
The images can be analyzed with standard image analysis software, such as ImageJ to quantify the parameters of interest, for example, myelin thickness or length.
