This article describes a method for live imaging of postnatal mouse retinal explants to study the morphology of starburst amacrine interneurons. The technique involves transfecting neurons with a fluorescent protein and capturing 3D images to analyze dendritic changes over time.
Begin with a live postnatal mouse retinal explant mounted on a membrane filter and placed in an incubation chamber under a confocal microscope.
The retina is transfected to express a fluorescent protein in the membranes of starburst amacrine interneurons.
Place a pre-wetted sample weight onto the retina to stabilize it.
Fill the chamber with buffer.
Maintain optimal buffer flow and temperature in the chamber to preserve neuronal viability.
Position a water immersion objective into the imaging chamber.
Start epifluorescence imaging.
The laser light, directed through the objective, excites the fluorescent protein in the amacrine cells.
The emitted fluorescence is captured through the same objective, allowing visualization of the labeled amacrine cells.
Identify a brightly fluorescent cell for imaging.
Set the imaging parameters.
Capture images at multiple Z-planes to achieve a 3D visualization of the complete dendritic morphology.
Perform time-lapse imaging to track changes in dendritic morphology over time.
Assemble the live imaging incubation chamber and fill it with oxygenated aCSF. Turn on the pump and temperature controller, making sure that the temperature does not rise above 34 degrees Celsius. It can take up to an hour for the chamber temperature to stabilize. It is recommended to set up the incubation chamber before beginning retinal dissection. Stable chamber temperature helps reduce sample drift.
To transfer the retinal flat mound into the perfusion chamber, stop the pump, and remove the aCSF that is in the chamber. Then, place the MCE disk with the retinal flat mount into the incubation chamber. Pre-wet a sample weight to break the surface tension, then place it onto the flat mound, ensuring that the sample weight is placed on the MCE paper to reduce sample drift. Refill the chamber with the warmed aCSF and circulate aCSF at approximately 1 milliliter per minute.
Position the nose piece with the 25 times water dipping objective into the imaging chamber. Screen for labeled cells of interest using epifluorescent light, and adjust the imaging volume to capture dendritic features of interest.
Using a lookup table that identifies both oversaturated and undersaturated pixels, adjust the laser power such that no pixels are oversaturated. Acquire the 3D imaging volume and repeat acquisition at the desired frame rate. Imaging volume can be adjusted between each time point to compensate for sample drift.
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