This study demonstrates a method for imaging motile cilia in ependymal cells of mouse brain ventricles using differential interference contrast (DIC) microscopy. The technique allows for the observation of ciliary activity and quantification of beating frequency, providing insights into cerebrospinal fluid circulation.
Take a sagittal section of a mouse brain to expose the ventricles, which are cavities filled with cerebrospinal fluid, or CSF.
The ependymal cells lining the ventricles possess motile cilia that propel CSF to facilitate its circulation.
Place the section in a glass-bottom dish containing a nutrient-rich medium to sustain ciliary activity.
Transfer the dish to the climate-controlled chamber of a differential interference contrast, or DIC, microscope to maintain cell viability during imaging.
DIC microscopy employs a prism to split polarized light, directing it through the specimen along different paths.
Variations in refractive index across the specimen introduce phase differences between the beams.
A second prism recombines the beams, creating interference that produces a high-contrast image with a three-dimensional effect, enhancing the visibility of fine structures.
Locate functional ependymal cells with motile cilia by observing bubble movement generated by their beating.
Using high-contrast DIC imaging, record the cilia to quantify their beating frequency.
To image the sections, place the brain slices in 30-millimeter glass bottom culture dishes containing 1 milliliter of high-glucose medium, and adjust the enclosed microscope chamber environment to 37 degrees Celsius and 5% carbon dioxide, using a 60x objective oil immersion lens.
Next, focus on the cells with regular DIC transmitted light. Then, following the direction of the DMEM bubble movement, as a guide to the location of the motile ependymal cilia, use the DIC filter to select an area containing healthy cells with motile cilia in the brain's lateral ventricle. Upon identification of the ependymal cilia, adjust the light, and focus to obtain a satisfactory image.
Next, set the live imaging parameters in the imaging software. For example, here 24-bit images are being acquired, with the camera binning set to 1 times 1, combined with the 60x objective, and a 5 to 10-millisecond exposure time.
Open the microscope aperture to the optimal level for a minimal exposure time, and collect the DIC images. Observe the live images stream to the camera to provide fast and immediate image acquisition without delay. The speed of cilia beating can then be calculated based on the requirement of the minimal exposure times for obtaining a sufficient image contrast.
繁體中文
