Jan 13, 2026
PTA contrast enhancement and specimen preparation for micro-CT scanning of spiders and other small arthropods
- Francisco Andres Rivera-Quiroz1
- 1Naturalis Biodiversity Center
- Francisco Andres Rivera-Quiroz: https://orcid.org/0000-0001-8748-4930
Protocol Citation: Francisco Andres Rivera-Quiroz 2026. PTA contrast enhancement and specimen preparation for micro-CT scanning of spiders and other small arthropods. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8xjw9v2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 08, 2026
Last Modified: January 13, 2026
Protocol Integer ID: 238204
Keywords: ct scanning of spider, studying arthropod morphology, arthropod morphology, other small arthropods over the past decade, other small arthropod, pta contrast enhancement, collected specimen, staining method, specimen preparation, little impact on specimen, specimen, foremost imaging method, ct scanning, imaging, spider, pta, anatomy
Funders Acknowledgements:
Francisco Andres Rivera-Quiroz
Grant ID: VI.Veni.222.154
Disclaimer
This protocol is part of the project "Into the eyes of an eight-eyed hunter", funded by the NWO (The Netherlands) through the Talent Programme Veni Science Domain 2022 project VI.Veni.222.154.
Abstract
Over the past decade, Micro-CT scanning has gained traction as one of the foremost imaging methods for studying arthropod morphology and internal anatomy. This method combines versatility, high data output, and relatively little impact on specimens, making it ideal for studying freshly collected specimens as well as material from natural history collections. In addition to the capabilities of the scanners, the selection of a staining method has a major impact on the quality of the data produced. This protocol is a modification of a PTA staining method published in 2022 (see references). Here, I further simplify the method by removing unnecessary steps, such as dehydration through an ethanol series and the use of rotators and vacuum chambers.
Image Attribution
All the figures and plates are produced by the author and published under CC-BY 4.0
Guidelines
Read the document in full before starting. Note that this staining protocol takes significantly more time than Iodine staining. Plan your project, specimen preparation, and scanning times well in advance.
Materials
See thematerial lists in the steps.
Troubleshooting
Problem
Specimen staining times are longer than expected
Solution
I have noticed a wide range of variation in the staining times of specimens. Even specimens of the same species with roughly the same body size and prepared in exactly the same way can take drastically different times to stain (e.g. one week vs two weeks). This has been observed in other studies (see Penna-Gonçalves, et al. 2025). Although it does not occur often, it is something that should be considered.
Problem
PTA not penetrating in the specimen
Solution
Related to the previous point, sometimes scimens might not stain evenly after a one or two-week period. If this happens, try first refreshing the PTA solution and leaving it for a few more days. For some really large specimens like the wolf spider Hogna radiata, I have taken up to a month to stain the whole cephalothorax.
Safety warnings
This protocol uses ethanol and phosphotungstic acid. Please check the safety sheets and follow common rules for the use of these chemicals. Although I tested this protocol with good results on land snails, the acidic nature of PTA can negatively affect calcareous materials.
Before starting
5m
Prepare:
- 70 % volume EtOH
- 1 % volume PTA (Phosphotungstic Acid) diluted in 70 % volume EtOH
- One small Petri dish
- One 1.5ml Eppendorf tube
- Fine tweezers
- Small plastic pipettes
5m
Specimen Preparation
10m
To optimize the penetration of the staining, it is recommended to remove the legs and abdomen of the specimen. This will allow the PTA solution to penetrate and stain the tissues evenly.
10m
If the specimen cannot be dissected (e.g. rare species or type material), it can remain intact, although the staining process will take longer.
Specimen Staining
2w 0d 0h 1m
Once the specimen has been fixed and prepared appropriately in 70% EtOH, it can be stained in the PTA solution. To do so, transfer the specimen to a 1.5ml Eppendorf tube (Fig. 1) and cover it with the 1% PTA in 70%EtOH solution.
Figure 1. Small Linyphiid spider in PTA staining solution inside a 1.5ml Eppendorf tube.
1m
The specimen should stay in the solution for several days, which will depend on the size and preparation of the specimen. For example, small non-sclerotised specimens (around 1 or 2mm) with removed legs can take between two days and a week to be properly stained. Larger specimens (between 0.5 and 1cm) can take 1-2 weeks to stain if the legs are removed fully. Heavily sclerotized specimens that have not been dissected or larger body sizes can take more than two weeks to fully stain.
2w
Older protocols have used vacuum pumps (see Rivera-Quiroz & Miller 2022) or further removed the cuticle of the specimen (see Penna-Gonçalves, et al. 2025) to expedite the penetration of PTA in the tissue. This usually leads either to uneven staining or damage of the internal tissues. Therefore, it is advisable to be patient and give enough time to the PTA to diffuse passively in the tissues.
Specimen mounting
6m
To facilitate the scanning and observation of the specimens, it is always advisable to mount the specimen in a way that stabilizes it and keeps it from moving or shifting during the scanning. To do so, prepare:
- EtOH 70%
- Petri Dish
- Fine tweezers
- Small plastic pipettes
- Eppendorf tubes of various sizes
- Cotton
- Alcohol resistant paint markers (e.g. Edding 780)
Transfer the specimen from the PTA solution to a petri dish filled with 70%EtOH. This will help get rid of the excess PTA and function as a "washing step". No other washing steps are required.
1m
Place a tube of the appropriate size in the petri dish next to the specimen. The tube should be slightly wider than the specimen itself so it fits smoothly but does not create any distortion on the specimen or allow it to move laterally. The tube must be completely submerged in the ethanol to avoid the formation of air bubbles. The specimen can be gently placed inside the tube and then held in place with the help of the cotton (Fig. 2). This will prevent the specimen from shifting vertically during the scan.
If you intend to prepare and scan large batches of specimens, it is critical to correctly label each tube to accurately identify the specimens. I suggest using alcohol resistant markers since normal markers are easily removed with ethanol.
Figure 2. The selection of the tube will depend on the size of the actual specimen and the capabilities of the scanner. A larger working distance allows more freedom in the width of the tube. A shorter working distance requires a smaller tube to get as close as possible to the specimen.
5m
Staining Evaluation
15m
Since the PTA is completely transparent to the visual spectrum, and the specimen is not visibly altered by it (Fig. 3), it can be difficult to judge whether the specimen is properly stained or not. To check the status of the staining, I recommend using a desktop micro-CT scanner (e.g. Skyscan or Neoscan) to evaluate the tissue contrast under x-rays.
Figure 3. Staining evaluation under visible light. The specimen on the left is unstained,
the one on the right has been stained for seven days. Note that the change in coloration is minimal, with just some slight decoloration in the abdomen. Despite this, the specimen on the left is fully visible under X-Ray.
15m
To do so, the specimen can be placed inside the scanner in the same Eppendorf tube where it is being stained. This will allow observing if the straining of the specimen is even and complete, and if the contrast with the medium is sufficient for making meaningful observations of the internal anatomy (Fig. 4). This step usually takes less than five minutes per specimen and can save you several hours in preparation, mounting and scanning time.
Figure 4. Staining evaluation under X-rays. The specimen on the left is fully stained. Note the darker and even coloration on the legs, cephalothorax and abdomen. The specimen on the right is not correctly stained yet and needs more time submerged in the PTA solution. The semi-transparent legs and abdomen indicate that the staining has not penetrated those tissues yet.
If the specimens are not evenly or fully stained, they can be left in the same PTA solution for a few more days. In the case of larger specimens, you might consider renewing the PTA solution to make sure the PTA has not been depleted.
If the specimens are fully stained, you can move on to the next step.
Specimen scanning
1h
The tube with the specimen can be placed directly in the scanner. The selection of an appropriate tube size in the previous step might have an impact on the magnification you can achieve (Fig. 5). This is especially important for table-top scanners that have a shorter working distance. In the case of larger scanners (e.g. Zeiss X-radia), the selection of the tube is not crucial. Still, it is critical to hold the specimen in place and avoid it from moving or shifting, or the entry of air that might create bubbles.
Figure 5. A tube closer to the same width of the specimen will allow the source/detector to get closer, taking full advantage of the magnification.
1h
This protocol has been tested in a variety of samples from tiny spiders of the family Oonopidae and Tetrablemmidae to relatively large ones like Araneus, Nephila (Araneidae) or Hogna and Lycosa (Lycosidae). The scanning settings differ from scanner to scanner and depend on a variety of factors, including specimen size, magnification, and resolution, among others. Therefore, they are not covered here. Still, I include for reference some virtual slices and 3D renderings that have been obtained following this staining protocol (Fig. 6). Besides spiders, I have successfully used this same protocol with plant roots and land snails.
Figure 6. Virtual slices and 3D volume renderings of spider specimens for reference. A) Coronal slice of the cephalothorax focusing on the Central Nervous System. B) Axial view of the cephalothorax with emphasis on the venom glands. C) Axial view of the eyes, retinae and optic nerves. D) Volume rendering of the cephalothorax from a dorsal view. E) Volume rendering of the cephalothorax from an anterior view.
Final considerations
Although I have not personally tested thishere, Sumner-Rooney, et al. (2019) have shown that PTA staining and micro-CT scanning do not negatively affect DNA extraction, PCR amplification or sequencing, making this protocol compatible with the use of specimens for genetic examination.
Protocol references
Rivera-Quiroz, F. A., & Miller, J. A. (2022). Micro-CT visualization of the CNS: Performance of different contrast-enhancing techniques for documenting the spider brain. Journal of Comparative Neurology, 530, 2474–2485. https://doi.org/10.1002/cne.25343
Penna-Gonçalves, V., Willmott, N. J., Kelly, M. B. J., Black, J. R., Lowe, E. C., & Herberstein, M. E. (2025). Comparing microCT Staining and Scanning Methodology for Brain Studies in Various Sizes of Spiders. The Journal of comparative neurology, 533(1), e70017. https://doi.org/10.1002/cne.70017
Rivera-Quiroz, F. A., & Miller, J. A. (2021). Old Brains in Alcohol: The Usability of Legacy Collection Material to Study the Spider Neuroarchitecture. Diversity, 13(11), 601. https://doi.org/10.3390/d13110601
Sumner-Rooney, L., Kenny, N.J., Ahmed, F. & Williams, S.T. (2019). The utility of micro-computed tomography for the non-destructive study of eye microstructure in snails. Sci Rep 9, 15411. https://doi.org/10.1038/s41598-019-51909-z
Acknowledgements
Thanks to Jeremy Miller for his support, to Rob Langelaan for his help scanning the specimens and to Martin Rücklin for his help setting up and running the 3D workstations at Naturalis. The micro-CT scans were obtained with funding from the NWO (The Netherlands) through the Talent Programme Veni Science Domain 2022 project VI.Veni.222.154.