The overall goal of this protocol is to rapidly acquire high-resolution 3D digital images of biological specimens using standard laboratory equipment. This method can answer key questions in the field of developmental biology through the visualization and dynamics of complex morphological features. The main advantages of this technique are that it uses standard lab equipment and requires minimum prior experience.

Demonstrating the procedure will be Jungang Huang, a graduate student from my lab. After dissection and fixation of the embryo as described in the text protocol, replace the fixative with clearing solution, and gently rotate the specimen on a rocker at room temperature for one to two weeks. At the end of the incubation, transfer the specimen to 10%formalin and leave on the rocker for another two days.

On the third day, wash the sample in PBS for three five-minute washes. Then dehydrate the samples in an ascending ethanol series at room temperature, on a gentle rocker for two hours for each concentration. After the 100%ethanol dehydration, transfer the specimen to staining solution for one hour, protected from light.

Then, replace the solution with fresh staining solution for overnight labeling of the specimen. The next morning, immerse the sample in a one to one volume to volume mixture of freshly prepared infiltration and staining solutions for at least three hours on a rocker at four degrees Celsius. At the end of the incubation, transfer the embryo into infiltration solution for another three-hour rocking incubation.

Then replace the supernatant with fresh infiltration solution for three more days of rocking at four degrees Celsius. On the fourth day, orient the embryo in an embedding mold and place the mold on ice. Mix solution B with the infiltration solution and then gently submerge the mold in cold embedding solution at four degrees Celsius.

After about three hours, use a tip to confirm that the solution has solidified, and push the specimen out of the mold. Trim any excess embedding material, then reorient the specimen block in a new mold in a cross orientation. Next, place the entire setup on ice in a fume hood.

Gently add embedding solution to the new mold until the mold is submerged. Then, place a block holder on top of the mold. After three to four hours, peel away the embedding mold and place the block under a stereo microscope with oblique illumination to determine the position of the specimen within the block.

On the block face, mark grid lines around the specimen and use the lines as guides for trimming any excess embedding material. To image the specimen, first clamp the block to a microtome, and obtain a fresh surface cut of the specimen. Then mount a stereo zoom microscope horizontally facing the block face of the specimen, and turn on the microscope and the accompanying computer.

Next, start the image acquisition software and use the live view mode in imaging software to focus the optics to the block face. Align the microscope and microtome as needed, so that the block face is at the center of viewfinder. One of the most critical steps of this procedure is to acquire images at the fixed home position of the microtome.

Adjust the zoom to make sure the entire block face is within the viewfinder. Then select the green fluorescent protein filter. Proceed to pre-cut the block until the specimen tissue appears, and re-focus the image as needed.

After measuring and setting the exposure time manually, acquire and save images of the block face after each fresh cut, and record the important image acquisition parameters. At the end of the imaging session, export and convert all of the image files to JPEG format, and use the appropriate image processing software to invert all of the original images, adjusting the brightness and contrast of the images as necessary. Upload all the processed images to a 3D visualization software, following the instructions.

Align all images using the automatic mode, and show the 3D and 2D results. In this representative image of a whole-mount surface view of an embryonic day 15.5 mouse embryo prepared and imaged as just demonstrated. The detailed morphological features of the skin, including the whisker pads and developing hair follicles can be observed.

Importantly, the resolution of the virtual section view of an embryonic day 15.5 specimen imaged by this method is similar to a standard histological section. Once master the entire procedure after the pretreatment with the clearing solution can be completed in one week, if it is performed properly. While attempting this procedure, it is important to remember to keep the staining solution protect from light at four degree during infiltration and embedding step.

Following this procedure, other methods, like scale staining, can be performed to answer additional question about cell types'specific distribution patterns. After watching this video, you should have a good understanding of how to use laboratory equipment to acquire high-resolution 3D images of biological specimens for their morphometric analysis. Don't forget that working with fixation, staining, and infiltration solution can be extremely hazardous, and that precaution such as operating in a fume hood and wearing gloves should always be taken while performing this procedure.