A protocol for 3D visualization of microscopic tissue structures by using an X-ray specific staining method designed for X-ray computed tomography is presented.
We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described protocol is easy to apply, fast and suitable for larger soft-tissue samples. The presented methodology enables the characterization of crucial tissue structures in three dimensions and is demonstrated on a whole mouse kidney. The multiscale approach allows to image the entire mouse kidney and supports the selection of further volumes of interest, which are acquired with higher resolutions ranging into the nanometer range. Thereby, soft-tissue morphology with a similar detail level as the corresponding histological light microscopy images is reproduced. Deeper insights into the 3D configuration of tissue structures are achieved without impeding further investigations through histological methods.
Full characterization of soft-tissue specimens requires information about the 3D tissue microstructure. The current gold standard for soft-tissue sample analyses is histopathology. The tissue and cellular morphology of the specimen are explored in 2D within selected regions of interest (ROIs) using optical microscopy1. This method, however, has several drawbacks. The preparation of the sample is time-consuming, complicated, destructive and prone to artifacts. The produced microscopic slides provide only 2D information parallel to the sectioning plane. Often the number of histological sections, which are investigated, is restricted due to time constraints2,3.
In recent years, the field of 3D histology has evolved. Here, virtual tissue slices from any desired spatial plane are accessible. This allows for the tracking of structures throughout the sample, which leads to a deeper understanding of the 3D tissue architecture and structural changes associated with different pathologies. Various methods have been developed to achieve the generation of 3D volume data. They range from serial-section based approaches, which use either light or electron microscopy4,5,6,7,8, to block-face imaging methods, such as episcopic 3D imaging or block-face scanning electron microscopy7,8,9. All the mentioned methods, however, involve either sectioning or destruct the sample completely, which does not allow for further investigations. The obtained resolution is highly dependent on the sectioning process being prone to artifacts as described in conventional histology. These methods suffer also from alignment artifacts.
3D X-ray imaging techniques such as microscopic and nanoscopic computed tomography (microCT and nanoCT) aspire to generate 3D high-resolution data without destruction of the tissue sample. So far, the weak X-ray attenuation contrast of soft tissue and the limited access to high resolutions in a laboratory environment has impaired their use for 3D visualization of microscopic tissue structures. Recent advances towards laboratory-based, high-resolution X-ray CT allow for resolutions well below 1 µm10,11,12,13.
The lack of contrast in soft tissue in conventional attenuation-based X-ray imaging is compensated by staining agents, which enhance the X-ray attenuation contrast. Staining agents known from other imaging techniques such as osmium tetroxide (OsO4), iodine potassium iodide (IKI) or phosphotungstic acid (PTA) are often used14,15,16,17,18,19,20,21,22,23,24,25. Staining agents that allow for (i) specific biological targeting, (ii) homogenous and complete staining, (iii) easy handling, (iv) fast penetration of the tissue without creating artifacts such as diffusion rings, (v) large and dense tissue staining, and (vi) full compatibility with histopathology are required to establish X-ray CT as tool for 3D visualization of microscopic tissue structures. In this work, we show how soft-tissue samples are prepared for X-ray CT imaging with a cytoplasm-specific X-ray stain based on eosin that fulfills the requirements stated above26.
The multiscale imaging approach ensures the assessment of staining quality through an overview microCT measurement and the selection of volumes of interest (VOIs) for further high-resolution investigations. Staining quality is analyzed focusing on staining parameters such as (i) completeness, (ii) appearance of diffusion rings, (iii) contrast enhancement, (iv) appearance of CT artifacts such as streaks and (v) homogeneity. The laboratory-based nanoCT setup, which uses geometric magnification to reach resolutions down to 100 nm, visualizes soft-tissue morphology on (sub)-cellular level10,27. A comparative analysis of the nanoCT slices with corresponding histological light microscopy images confirms the reproduction of tissue architecture with similar detail on a microscopic level in 2D, enabling histopathological characterization of the tissue sample. This detailed video protocol is intended to raise the awareness and to highlight the potential of this methodology as non-destructive 3D soft-tissue imaging tool being of interest to a wide scientific community such as zoologists, biologists and health professionals.
Caution: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in the protocol are acutely toxic and carcinogenic. Please use all appropriate safety practices when performing the staining protocol including the use of engineering controls (fume hood, glovebox) and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).
Animals Used:
Animal housing was carried out at the Klinikum rechts der Isar, Technical University of Munich in accordance with the European Union guidelines 2010/63. Organ removal was approved from an internal animal protection committee of Klinikum rechts der Isar, Munich, Germany (internal reference number 4-005-09). All procedures were in accordance with relevant guidelines and regulations. All laboratories are inspected for accordance with the OECD principles of good laboratory practice.
1. Eosin staining protocol
2. X-ray microCT imaging
NOTE: The X-ray microCT measurements were performed with a microCT scanner, which offers overview CT measurements (the ability to image the entire sample within the field of view (FOV)) and the performance of high-resolution CT measurements (the ability to focus in on one desired volume of interest (VOI) of the very same sample) down to 1 µm.
3. X-ray nanoCT imaging
NOTE: The X-ray nanoCT scanner has been developed inhouse. The lens free instrument is equipped with a nanofocus X-ray source and a single-photon counting detector. 3D data with resolutions down to 100 nm can be generated10. Generally, nanoCT systems including those with X-ray optics are commercially available and not limited to the described nanoCT scanner.
Figure 1 shows CT slices and volume rendering of low-resolution microCT data highlighting contrast enhancement after staining. Figure 2 shows CT slices and volume rendering of high-resolution microCT data derived from a local tomography of the whole mouse kidney. Figure 3 shows CT slices of nanoCT data in comparison to the corresponding histological sections. Figure 4 shows CT slice and volume rendering of nanoCT data highlighting structural details at cellular level. The low-resolution microCT measurement allows for an overview of the whole organ and helps to identify volumes of interest (VOIs) for the high-resolution microCT measurement. Through this multiscale approach, the VOI for the nanoCT is determined. The nanoCT enables a very detailed view of the soft-tissue sample on cellular level. The comparative study with the corresponding histological section highlights full compatibility with histopathology. Here, the multimodal imaging approach is confirming the results obtained with both modalities.
Figure 1. CT slices and volume rendering of the low-resolution microCT data. (a,b) Overview images of the same mouse kidney before and after staining, respectively, highlighting the contrast enhancement obtained after application of the eosin-based staining protocol. Both microCT data sets were acquired using identical acquisition parameters. The voxel size in both data sets is 12 µm. The contrast enhancement achieved in (b) enables the identification of the following anatomic structural regions: Cortex (I), outer medulla (II) with further distinction in outer stripes of outer medulla (IIa) and inner stripes of outer medulla (IIb), inner medulla (III), papilla (IV) and renal pelvis (V). (c) Volume rendering of microCT data showing a virtual sagittal section through the whole mouse kidney. This figure has been modified from Busse and Müller et al.26 Please click here to view a larger version of this figure.
Figure 2. CT slice and volume rendering of high-resolution microCT data derived from the same mouse kidney after application of the developed eosin-based staining protocol. (a) The left corner shows the overview microCT image highlighting the ROI (blue box) for the displayed high-resolution image. The following anatomic structural regions are identifiable: Cortex (I), outer medulla (II) with further distinction in outer stripes of outer medulla (IIa) and inner stripes of outer medulla (IIb), inner medulla (III), minor calyx (IV) and vessels (V and VI). (b) Volume of interest rendering of the high-resolution microCT data acquired with a voxel size of 3.3 µm. The medulla region and a virtual section through a vessel derived from a local tomography of the whole kidney is shown. This figure has been modified from Busse and Müller et al.26. Please click here to view a larger version of this figure.
Figure 3. CT slices of nanoCT data (a,b) in comparison to the histological sections (c,d) derived from the same mouse kidney after application of the developed eosin-based staining protocol. (a) The nanoCT image of the same mouse kidney sample after staining, dissecting and CPD shows detailed structures of region (IIb) seen in Figure 1 and Figure 2. These are known as thick ascending limbs of the loop of Henle. (b) Minimum intensity projection slice derived from the same nanoCT data set shown in (a) with a virtual slice thickness of approximately 7 µm, which allows for clear visualization of the cell nuclei. (c) Representative histological section displaying thick ascending limbs of the loop of Henle with clear visualization of cell nuclei and brush border. The histological section has an approximate thickness of 7 µm and was obtained from the same mouse kidney sample after the applied eosin-based staining and embedding in a paraffin block. (d) Representative histological section with application of counter stain hematoxylin highlighting the cell nuclei in purple. Preparation of the histological section close to the section shown in (c) with approximate thickness of 7 µm. This figure has been modified from Busse and Müller et al.26 Please click here to view a larger version of this figure.
Figure 4. CT slice and volume rendering of nanoCT data. (a) The nanoCT image of the same mouse kidney sample showing the structures known as thick ascending limbs of the loop of Henle. This is a detailed view of region (IIb) seen in Figure 1 and Figure 2 acquired from a small piece of the kidney with a voxel size of approximately 400 nm. The preparation of the sample involved staining, dissecting and CPD. (b) Volume rendering of nanoCT data visualizing the 3D structure of thick ascending limbs of the loops of Henle. This figure has been modified from Busse and Müller et al.26 Please click here to view a larger version of this figure.
Currently, eosin is used as the standard histological protocol to label the cell cytoplasm. The staining agent is applied as a 0.1% (w/v) aqueous solution to microscopic slices of soft tissue (generally cut with a thickness of 2-10 µm)33. The application of this standardized histological protocol to 3D tissue samples such as a whole mouse kidney does not result in an attenuation contrast enhanced CT image. On the one hand, this can be attributed to the low intrinsic attenuation properties of soft tissue for typically used X-ray energies of laboratory-based microCT systems. Usually, soft tissue is composed of mainly carbon, hydrogen, oxygen and nitrogen34, and therefore, does not result in contrast enhancement. On the other hand, the low concentration of eosin used for staining was the limiting factor. Even though one eosin molecule holds four bromide atoms (high atomic number element bromine with Z = 3534), the sensitivity levels required for X-ray CT imaging were not met.
To overcome this challenge of low attenuation contrast, several concentrations of eosin were investigated. A limitation is here the maximum solubility of eosin in water, which is 30% (w/v) in an aqueous solution. Best attenuation contrast enhancement within the soft tissue was observed with the highest eosin concentration, which was expected according to the Lambert-Beer Law. Therefore, the final staining protocol was carried out with the highest concentration.
The question how to prepare the soft tissue optimally on a molecular level for the staining procedure to further improve contrast enhancement was answered by pH adjustment. Here, the acidification of the soft-tissue sample during fixation or before staining was found to be crucial. This was also shown by Hong et al.35. The higher accumulation of staining agent within the cell cytoplasm by the acid was achieved through improved ionic interactions, which were a result of the protonation of amino acid side chains of proteins and peptides present within the cell cytoplasm. A representative result highlighting the contrast enhancement in comparison to an unstained soft-tissue sample is shown in Figure 1a,b. Here, a structural overview of a whole mouse kidney visualizing crucial anatomical regions such as cortex, medulla, papilla and renal pelvis was achieved.
The presented staining protocol is simple to apply and contains only three steps. The required reagents are easily accessible. The overall staining time of 24 hours is fast for a whole-organ staining, which enables the 3D visualization of soft-tissue samples (Figure 1c, Figure 2b and Figure 4b) in a laboratory environment at multiple scales down to cellular level. It should be noted that the overall staining time and volume of the staining solution needed might request some adaptations depending on the nature of the sample. Nevertheless, the eosin-based staining protocol is suitable for whole-organ staining, which then enables high-resolution microCT imaging of whole organs. Shrinkage artifacts due to the solvent ethanol, which was used to keep the sample moist during the microCT measurements, were not observed. Additional preparation steps are required for nanoCT imaging, which allows for the investigation of smaller tissue pieces retrieved from the original sample. With respect to future histopathological applications, the overview scans will provide valuable insights into altered anatomical regions and structures, which allow for the determination of ROIs as demonstrated in Figure 2a. Those can be studied in 3D by microCT (Figure 1c and Figure 2b) or nanoCT (Figure 4b) and evaluated in 2D with histology (Figure 3).
Another strength of the protocol is seen in the full compatibility with histopathology with respect to the H&E staining procedure. The application of the eosin-based staining procedure to bulk samples does not impede further histological investigations (Figure 3), even though the applied eosin concentration is much higher compared to the histological staining solution. The nanoCT slice with a virtual thickness of approximately 400 nm (Figure 3a) compares already very well with the histological section (Figure 3c), which was derived from the corresponding soft-tissue sample. Considering the approximate thickness of a histological section with 7-10 µm, the generation of minimum intensity projection slices of the nanoCT data (Figure 3b), which correspond to a virtual thickness of approximately 7 µm, allows for a better comparison with the histological section (Figure 3c). Here, the cell nuclei are clearly revealed as non-attenuation area as eosin specifically stains proteins and peptides in the cell cytoplasm33.
The application of further counter staining with standard histological methods is possible, even though the order of the staining compared to the standard histological staining procedure was reversed. Starting first with the developed eosin-based staining protocol for CT, followed by counter staining of those eosin-based histological sections with hematoxylin, allows for full compatibility and results in a high-quality staining displaying the expected form of appearance. The cell nuclei-specific staining with Mayer’s sour hematoxylin was applied to the histological section highlighting the cell nuclei in purple (Figure 3d). The application of histological counter staining is currently limited to the H-stain. Other standard histological counter stainings such as periodic acid Schiff’s base, Elastica van Gieson or Gomori silver have to be evaluated as well as the compatibility with immunohistological techniques needs to be tested.
The eosin-based staining protocol allows for (i) cell cytoplasm-specific targeting, (ii) homogenous and complete staining, (iii) easy implementation, (iv) fast penetration of the tissue without creating artifacts such as diffusion rings, (v) the staining of large and dense soft-tissue samples, and (vi) full compatibility with histopathology in respect of the H&E stain. These requirements are important to allow high-resolution X-ray CT visualization of soft tissue down to cellular level. In combination with the recently developed nanoCT devices12,36,37, nondestructive generation of virtual histological slices that are comparable in contrast and resolution to conventional histological data is rendered possible. This combined approach will enable the establishment of X-ray CT as a valuable tool for the 3D visualization of microscopic tissue structures.
The authors have nothing to disclose.
We thank Dr. Enken Drecoll for histological discussions and the extremely helpful team at Excillum AB, Sweden. We acknowledge financial support through the DFG Cluster of Excellence Munich Center for Advanced Photonics (MAP) and the DFG Gottfried Wilhelm Leibniz Program. Furthermore, this research project has received funding from the European’s Union Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. H2020-MSCA-IF-2015-703745-CONSALT.
50-ml centrifuge tube by Falcon | VWR | 734-0453 | |
Formaldehyde solution, 37% | Carl Roth | CP10.2 | acid-free, stabilized with ~10% MeOH |
Glacial acetic acid | Alfa Aesar | 36289.AP | |
Eosin Y disodium salt | Sigma-Aldrich | E4382 | certified by Biological Stain Commission |
Phosphate Buffered Saline (PBS) | Merck | L1825 | Dulbecco's formualtion, w/o calcium and magnesium |
Sample Tubes by Nalgene | Carl Roth | ATK5.1 | |
Rocking Shaker ST5 | CAT | 60281-0000 | |
Cellulose tissue paper | VWR | 115-0600 | |
Forceps, by USBECK Laborgeräte | VWR | 232-0096 | |
Microcentrifuge tubes by Eppendorf | VWR | 211-2120 | safe-lock, 2.0 ml |
Ethanol absolute by Baker Analyzed | VWR | 80252500 | |
Disposable safety scalpel by Aesculap | VWR | AESCBA210 | |
Petri dish by Sterilin | VWR | 391-2019 | |
Plastic pasteur pipette | Carl Roth | EA68.1 | graduated, 1 ml |
Desiccator by Duran | VWR | SCOT247826954 | |
Silicone grease by Bayer | Sigma-Aldrich | 85404 | high-vacuum |
Carbon dioxide cylinder with standpipe | Linde | 3700113 | 10 kg, short |
micro-porous treatment capsule | PLANO GmbH | 4614 | pore size 78 µm (B) |
Bal-Tec CPD 030 | Bal-Tec AG | CO2 as drying agent | |
Stemi 2000-C stereomicroscope with KL 1500 LCD | Zeiss | this stereomicroscope has been updated(1) | |
Zeiss Xradia Versa 500 | Zeiss | this microCT scanner has been updated(2) | |
Avizo Fire 8.1 | Thermo Fisher Scientific | ||
PILATUS detector as part of the nanoCT scanner | Dectris | single-photon counting detector(4,5); there are commercially availble nanoCT systems available (6,7) | |
nanofocus X-ray source as part of the nanoCT scanner | Excillum | high-flux nanofocus X-ray transmission tube(3); there are commercially availble nanoCT systems available(6,7) | |
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(3) Nachtrab, F. et al. Development of a Timepix based detector for the NanoXCT project. Journal of Instrumentation 10 (11), C11009, (2015). | |||
(4) Kraft, P. et al. Performance of single-photon-counting PILATUS detector modules. Journal of Synchrotron Radiation 16 (3), 368-375, (2009). | |||
(5) Kraft, P. et al. Characterization and calibration of PILATUS detectors. IEEE Transactions on Nuclear Science 56 (3), 758-764, (2009). | |||
(6) Germany, Z. ZEISS product information: ZEISS Xradia 810 Ultra https://www.zeiss.com/microscopy/int/products/x-ray-microscopy/xradia-810-ultra.html> (April 9 2019). | |||
(7) Company, G. E. GE product information: Phoenix nanotom m, https://www.gemeasurement.com/sites/gemc.dev/files/geit-31344en_nanotom_m_0517.pdf> (April 10, 2019). |