Our new protocol actually allows us to visualize cellular structures in 3D by using x-ray computed tomography. And we do this by staining tissue with eosin and in that way make the cytoplasm visible. Besides medical applications and research, we also think that our method can be applied in biology like, for example, zoology research or developmental biology.
The technique is relatively straightforward, very fast, and also suitable for larger tissue samples. We think that our approach combined with x-ray micro computed tomography can be integrated into the clinical workflow and then actually help pathologists make better decisions. Start by fixating the soft tissue samples.
Fill a 50 milliliter centrifuge tube with a fixative solution containing 9.5 milliliters of 4%formaldehyde and 0.5 milliliters of glacial acetic acid. Add the soft tissue sample to the centrifuge tube and refrigerate it for 24 to 72 hours. After refrigeration wash the soft tissue sample with DPBS solution for one hour.
Then stain the sample by placing it in two milliliters of Eosin Y staining solution and incubate it on a horizontal shaking plate for 24 hours. On the next day carefully take the soft tissue sample out of the sample container and remove excess staining agent with cellulose tissue paper. Place it in a conical container above an ethanol vapor phase for storage and further use.
Prepare an appropriate sample holder to mount the soft tissue sample according to manuscript directions and ensure a tight fit to prevent the sample from moving during the x-ray CT measurements. Once the adhesive has hardened and the sample holder is ready to use, transfer the mouse kidney into the intact centrifuge tube which holds a few drops of 70%ethanol at the bottom. Place the mounted sample into the x-ray CT scanner.
After carefully aligning the sample, choose acquisition parameters for the best image quality. For the presented micro CT data, acquire the scan at a peak voltage of 50 kilovolts and a current of 3.5 watts using 1601 projections equally distributed over 360 degrees. Then use the micro CT data from the overview scan to select the region of interest for the high resolution CT scan.
Prepare volumes of interest of the soft tissue sample by cutting it into very small pieces of approximately 0.5 millimeter edge length using a scalpel and a stereo microscope. If using a mouse kidney, cut it into two halves along the longest axis. Take one half and prepare different anatomical regions such as a renal cortex and renal medulla.
Transfer the small pieces to a new Petri dish for dehydration. Dehydrate the samples using a series of ethanol solutions according to manuscript directions, performing each dehydration step for one hour. Transfer the dehydrated samples into the microporous capsule and close it.
Keep the samples in contact with 100%ethanol at all times. Then critical point dry the small tissue pieces. Once the tissue samples are prepared, keep them in a new Petri dish stored in a desiccator prior to further use.
Mount the tissue pieces to an appropriate sample holder ensuring a tight fit. For a mouse kidney superglue the pieces to a sample holder using a stereo microscope. After careful alignment of the sample, choose acquisition parameters for the best imaging quality.
For the presented nano-CT data, acquire projections at a peak voltage of 60 kilovolts with 1599 projections equally distributed over 360 degrees and a voxel size of approximately 400 nanometers. This protocol was used for 3D visualization of microscopic tissue structures of a mouse kidney. Low resolution micro CT measurements allowed for an overview of the whole organ and helped identify volumes of interest for high resolution measurements.
The same mouse kidney was used to obtain a high resolution micro CT data. A more detailed view of the anatomical structures such as the cortex, outer medulla, and inner medulla among others is achieved. A volume of interest rendering shows the medulla region and a virtual section through the vessel.
The nano-CT was used to obtain a detailed view of the kidney sample on a cellular level. A small piece of tissue obtained from the whole mouse kidney was used to image the thick ascending limbs of the loop of Henle with a voxel size of about 400 nanometers. A comparative study was performed to ensure that the nano-CT is fully compatible with histopathology.
The multimodal imaging approach confirmed the results obtained with both methods. During the incubation step it is crucial that the sample is fully surrounded by the staining solution. Also when performing CT x-ray imaging, the most important fact is to ensure sample stability during data acquisition.
The soft tissue samples that have been analyzed with our protocol can be further analyzed using standard histological technique such as counterstaining hematoxylin. Our staining protocol will be substantially contributing to advancing 3D x-ray histology. Medical research will especially benefit from this nondestructive 3D imaging technique.
