This protocol allows for the non-destructive imaging of intact or soft lab micron resolution. It can be used to understand mechanisms of animal physiology, anatomy, and development. Because the flies are imaged in an intact state without the need for tissue dissection, the spatial architecture of tissue and organs are maintained in their native state and are easily observed.
Micro CT is an ideal tool for studies aimed at modeling human diseases in flies, or for understanding developmental defects that affect quality of life. It has also proven to be useful in other disciplines, such as engineering, physics, materials science, geology, and anthropology. After selecting the developmental time point of interest for imaging, transfer 5 to 20 animals into one milliliter of PBST in a 1.5 milliliter micro-centrifuge tube, and incubate the tube for five minutes at room temperature with periodic tapping to assist the removal of hydrophobic coating and allow the animals to become fully submerged.
At the end of the incubation, replace the PBST with one milliliter of Bouin solution and tap the tube to fully submerge the flies. For larval and pupal samples, after a two hour incubation at room temperature, wash the animals three times for five minutes and one milliliter of fresh PBS per wash. After the last wash, transfer the samples to a multi-well dissecting dish containing fresh PBS per well under a dissecting microscope, and use a small minutien pin attached to a holder to poke a hole in the anterior and posterior cuticle of each sample, taking care not to disrupt the underlying soft tissue.
After penetrating the cuticles, incubate the samples in one milliliter of fresh Bouin solution in a new 1.5-milliliter tube for 24 hours at room temperature before washing the sample three times for 30 minutes and one milliliter of one micro CT wash buffer per wash. After the last wash, add one milliliter of the appropriate staining solution to the tube and incubate the samples at room temperature for two to seven days. If longer image preservation is necessary, dehydrate the samples within ascending ethanol series in one milliliter of ethanol for one hour per concentration as indicated, to prepare the samples for critical point drying.
To mount critical point dried samples, place the samples in plastic or glass capillary tubes before mounting the tubes onto the rotating stage holder. For hydrated sample mounting, fill a P10 pipette tip with water and secure the narrow end with paraffin film. Use forceps to transfer a single specimen to the pipette tip and use a long, slender object to carefully push the specimen down into the tip until it just contacts the wall of the tip to hold the sample in place.
After covering the open end of the pipette, mount the P10 pipette onto a holder designed to fit within the chock of the rotating stage. For scanning, click the Open Door icon in the scanner software to gain access to the rotating stage chock and tighten the collar around the base of the sample holder to attach the sample. Set the scanning parameters in the software for optimal resolution and contrast and click Options and X-Ray Source to open the x-ray source power control.
Use the slider bars to set the x-ray voltage to 30 to 40 kilovolts and the current to 100 to 110 micro amps to produce an x-ray beam with three to four Watts of power and a small spot size. To set the number of projection images to be acquired during the scans, select Options and Acquisition Modes to set the camera exposure time to 500 to 800 milliseconds and use the slider bar to set the desired image pixel size according to the camera settings and position. Click and drag the slider bar to move the sample along its 360-degree rotation path, keeping the sample within the field of view.
Click the begin acquisition icon. A dialog box will appear allowing additional scanning parameters to be set. Name the file and the output folder to which the scan will be saved.
Set the random movement to 10 and the average to four to six frames. The rotation step will be automatically calculated depending on the camera settings used, then click Okay to begin the acquisition. A progress bar dialog box showing the scan time will appear and the scanner will begin acquiring a series of projection images of the specimen along the rotation path.
To generate the tomograms, open the projection images in the reconstruction software and select the shift correction algorithm to perform an initial image alignment. Select the best value that properly aligns the projection images and click Fine tuning to fine tune each reconstruction parameter. A series of previews will be generated that can be used to select the correct values.
Confirm that the optimal parameter values are displayed in the Settings tab, and in the Output tab, adjust the file parameters such as bit depth, file name, and folder where the data will be saved. A region of interest can be used to reconstruct only the structures of interest. If multiple reconstructions are required, click Start and Add to batch to add the current image to the batch manager and repeat the reconstruction for the remaining images.
For morphometric analysis of the reconstructed images, under the Segment tab, in an appropriate morphometric analysis software program, click Basic and New, give the image a new name, and select an appropriate color. Check the Define range box and adjust the slider to adjust the threshold range that encompasses the structure of interest. To paint an area defined by the threshold, press and hold the Control key while holding the left mouse key.
To remove an area, press and hold the Shift key while holding the left mouse key. Ensure that the measurements of the mesh region of interest are displayed in the information panel for basic morphometric analysis. Render the mesh object and the entire tomogram image, and visualize the resulting image in 3D, then right click to select Show Movie Maker to generate a video of the object using individual frames from the viewer.
In these images, an embryo, third instar larva, pupa at the ferrate adult stage, and adult female fly stained with iodine and imaged in water using a commercial bench top scanner can be observed. Excellent preservation and even staining of the delicate tissue are apparent, allowing all of the major organs to be readily identified. Here, a comparison of the same adult fly headcase, acquired in both slow and fast projection scan mode, is shown.
Importantly, although the resolution in the slow images is higher, the morphometric analysis does not differ between the slow and fast scans. Using the rendering software, any tissue or organ system of interest can be segmented and used for morphometric analysis and 3D visualization. In these images, a fly abdomen stained with phosphotungstic acid and imaged while water-hydrated or following critical point drying on an x-ray microscope, can be observed.
Using phosphotungstic acid, individual epithelial cells of the midgut and sperm bundles within the testes are easily resolved. While the critical point drying images show marginally increased resolution compared to the hydrated sample, better preservation of the ultrastructure of delicate tissues is achieved with hydrated samples, such as the fat cells near the cuticle. For the best resolution, limit sample movement and excess vibrations.
Securing the sample to the stage is critical for this, although creativity and resourcefulness may be required in order to achieve this. Additional imaging methods, such as light and electron microscopy, can be used as part of an imaging workflow in order to understand a given gene mutation from the nanoscale to the whole organism.
