This article describes a method for imaging neurons using a thick brain section stained with Golgi-Cox solution. The technique involves using silicone oil to enhance imaging quality and capturing detailed three-dimensional representations of neuronal projections.
Start with a thick brain section that was stained with Golgi-Cox solution to selectively darken neurons and counterstained with cresyl violet to highlight the brain region.
Place them on a slide that has been pretreated with a clearing agent to enhance tissue transparency.
Place the slide on the microscope stage, apply silicone oil, and lower the objective lens until it contacts the oil.
Silicone oil matches the refractive index of the objective lens and slide, enabling detailed imaging.
Focus on the sample, then adjust the camera's exposure and white balance for optimal image quality.
Focus on the top layer to set the upper boundary, then on the bottom layer to set the lower boundary.
Adjust the spacing between images with sufficient overlap to capture details of neuronal projections.
Repeat imaging at different locations to capture areas of interest, then overlay these images to create a three-dimensional representation of the neurons.
To capture high resolution image stacks of the area containing the neuron of interest, first select a 30x 1.05 NA silicone oil immersion objective on the microscope. Then, apply 3 to 4 drops of silicone immersion oil to the slide. Place the objective over the slide, ensuring contact between the objective and oil. Focus the image and adjust the camera settings, including the exposure and white balance in the imaging software. Next, set the upper and lower boundaries for the image stack by focusing to the top of the section, and then selecting Set, next to top of stack, within the image acquisition window.
Then focus to the bottom of the section and select Set next to bottom of stack. Set the step distance to 1 micron by entering 1 micron next to distance between images within the image acquisition window. Lastly, capture the image stack by selecting acquire image stack within the image acquisition window. Repeat the previous steps to capture the entire area of interest, making sure that all image stacks overlap by at least 10% in the x and y-axes.
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