Contrast limited adaptive histogram equalization (CLAHE) is a procedure for enhancing local contrast in an image or stack of images. In contrast to standard histogram equalization that applies single formula for enhancing contrast across the entire image, CLAHE applies multiple equalizations within partitions of an image, resulting in more localized and subtle contrast enhancements. This results in digital contrast enhancement that is not dominated by overly deep blacks or excessively bright whites.
For diceCT, CLAHE is very useful for improving edge recognition for digitally segmenting regions of interest (ROI) based on your CT data. It is particularly helpful when applied to sub-optimally stained specimens.
CLAHE is implemented in FIJI (ImageJ) and the script is available freely and openly. To perform it on a stack of CT images:
Drag and drop the folder that contains a stack of CT images into FIJI (download here: https://fiji.sc). Wait until FIJI reads in the entire image stack.
Copy the CLAHE script from the “Tips” section on the website.
In FIJI, go to “Plugins” > “New” > “Macro”. A new window will open. In the text field, paste the CLAHE script.
Select “Run”. One can specify the “block size,” “histogram bins,” and “max slope” parameters (the details of which are outlined on ImageJ.net), but I have found that the default parameters do a fine job.
Once CLAHE has gone through the entire stack, save the new image stack in a different folder to keep the original CT image stack intact (you never know when you’ll need them).
In CT data processing program of your choice (e.g., Avizo, VGStudio), read in the modified image stack.
Here is a before and after example to illustrate how CLAHE enhances diceCT images.
CLAHE processes image stacks fairly quickly, so I recommend trying it with all diceCT image stacks. Generally, it improves edge recognition for all 3-D rendering programs, thus, greatly reducing the time spent on segmenting ROIs. As mentioned above, CLAHE is particularly useful for sub-optimally stained specimens, which is helpful when one cannot devote time or reserve frequent CT scanning sessions for checking and optimizing the stain concentration and duration.
Potential issues with CLAHE include the increase in file size associated with CT data from having both original and modified image stacks. In addition, CLAHE may accentuate unwanted artifacts like beam hardening, so there is further motivation to minimize such scanning artifacts.
Those interested in learning more about the method can visit Wikipedia and read through the original article (Zuiderveld, 1994).
ice-cold 4% (wt) formaldehyde (PFA) (We used freshly made solution. Also, note that commercial formalin solutions often contain methanol, which may cause shrinkage.)
4% (wt/vol) acrylamide/bisacrylamide (hydrogel monomers) (We used a pre-made solution of 38% Acrylamide and 2% Bis-acrylamide by Amresco.)
0.25% (wt) VA-044 (thermally-triggered initiator) (This one can be hard to find; we source ours from Wako Australasia.)
0.05% (wt/vol) of saponin (This is useful if you want to increase specimen permeability, or if the specimen is large.)
Mix all ingredients together. Ingredients should be kept on ice during preparation of the solution to prevent premature polymerisation.
Immerse specimens in hydrogel solution for 1-2 weeks at 4° C. Larger specimens should be immersed for longer, and specimen type may be important too (e.g., lizards probably take longer than tadpoles). Users should be aware that the immersion time here is longer than for the original STABILITY protocol (Wong et al., 2013). This may be due to longer fixation in formalin, which seems to use up some biomolecule binding sites.
Any type of vegetable/nut/canola oil can be used for curing (approximately 3 ml). Carefully pour the oil on the surface of the hydrogel solution to form an airtight layer. Some researchers use a vacuum pump nitrogen replacement step, but we found the oil easier. The vials are placed in a water bath set at 37º C for three hours, triggering polymerisation of the gel. After polymerisation, remove excess gel (now solidified) with clean gloves and lint-free wipes, revealing the specimen as a gel/tissue hybrid. Specimens were then placed directly into iodine solution.
For staining we used 1.75% IKI solution, for about 30% longer than we would normally stain—but this depends on specimens and needs to be refined a bit more. Lower-concentration solutions may yield even less shrinkage. Aki Watanabe (pers. comm.) reported slower uptake of iodine at room temperature, which he found advantageous for a more controlled stain. We also found that staining in the fridge reduced the number of high-density “pockets” of iodine producing interference during scanning.
Please feel free to use our protocol. If you do so, we would appreciate a citation:
Carlisle, A., Weisbecker, V. (2016) A modified STABILITY protocol for accurate retrieval of soft-tissue data from micro-CT scans of IKI-stained specimens. Published online at https://dicect.com/2016/08/09/stability/, August 09, 2016.
Wong, M. D., Spring, S., & Henkelman, R. M. (2013). Structural stabilization of tissue for embryo phenotyping using micro-CT with iodine staining. PloS ONE, 8(12), e84321. [doi:10.1371/journal.pone.0084321]
“μCT scanning of low density porous polymer scaffolds leads to insufficient imaging ability due to poor contrast. In order to enhance their X-ray absorption, one might trial several approaches using contrast agents. This methods paper is aimed at guiding scientists in applying the most appropriate contrast enhancement method for CT imaging of such materials, by reporting both the positive and the negative results. Iodine staining coupled with chemical drying was the most efficient approach, allowing for contrast enhancement, morphology preservation, and high resolution all at once.”
Diffusible iodine-based contrast-enhanced computed tomography (diceCT) and related imaging techniques for research in evolutionary morphology
Organizers: P. M. Gignac1*, A. N. Herdina2, N. J. Kley3, A. Morhardt4, J. A. Clarke5, and M. Colbert5
The ability to visualize hard tissues (e.g., bone, dentine, enamel) rapidly in three dimensions using X-ray computed tomography (CT) has been one of the most important advancements in the field of vertebrate morphology in the last half-century. Until recently, however, comparably valuable advances in soft-tissue imaging have been difficult to realize fully due to the inherently low X-ray absorption of non-mineralized structures. Pioneering work in this area has demonstrated that Lugol’s iodine (I2KI) is a highly effective contrast agent for rapidly differentiating many types of soft tissues (e.g., epithelial, muscular, and neural structures) in micro computed tomography (μCT) images. Vertebrate morphologists have become a driving force in advancing this technique and utilizing the remarkable data generated from it to reconstruct phenotypes and functional anatomy in three and four dimensions.
Given the broad potential for iodine-enhanced CT imaging to become a major tool for soft-tissue reconstruction in vertebrate morphology, we will hold a symposium at the 2016 ICVM meeting to exhibit the wide range of taxa and questions that can be examined using these approaches. Through our symposium, Diffusible iodine-based contrast-enhanced computed tomography (diceCT) and related imaging techniques for research in evolutionary morphology, we will feature the newest and ongoing applications of contrast-enhanced three-dimensional (3-D) imaging already being undertaken by researchers within the International Society of Vertebrate Morphology. We further propose to hold a student-focused, combined poster session with the Hartstone-Rose and Marchi symposium on muscle functional morphology, to bridge the related techniques of our respective presenters. Our goal is to spur the further adoption of these methods by vertebrate morphologists. We will achieve this by (1) highlighting recent methodological advances in contrast-enhanced CT and μCT imaging, (2) demonstrating active research that integrates diceCT and related imaging techniques into toolkits addressing macroevolutionary questions, and (3) generating discussions of future directions and the long-term place for contrast-enhanced imaging in the study of extant vertebrates. We have assembled a diverse group of speakers who have enthusiastically agreed to participate in this symposium. They include well-established researchers, emerging early-career scientists, and graduate students in the fields of functional morphology, biomechanics, phenotypic integration, and vertebrate paleontology, whose academic contributions have already brought important insights to evolutionary biology. Demonstration of the high-level inferences that can be garnered with diceCT will spur collaborations among labs already exploring this powerful new tool with those who are considering how to apply it to their own research questions.
Green — Physiological examination of ratite orthopedic disorders and soft- tissue visualization via micro-CT
Herrel — Contrast-enhanced versus phase-contrast imaging: costs and benefits of different methods
Holliday — DiceCT and its applications for understanding the reptile musculoskeletal system
Introduction & Neurological Visualization
Gignac — DiceCTing the future: new horizons for 3-D visualization of vertebrate morphology (30 min)
Weisbecker — Using the STABILITY protocol prior to IKI-staining to provide the first accurate, in situ quantification of mammalian brain proportion scaling using marsupials (15 min)
Watanabe — Mind the Gap: ontogenetic shape differences between brains and endocasts in archosaurs (15 min)
Hughes — Incorporating diceCT into multi-scale structural studies of the brain for highly divergent lineages of acrodont Lizards: validation of preservation methods conducted in the field (15 min)
Gold — Applying diceCT to PET: new tools for correlating morphology to function in living animals (15 min)
Charles — Musculoskeletal modelling and simulations of the mouse hind limb during locomotion: the role of high-resolution scanning and contrast Imaging (15 min)
Lautenschlager — The evolution of the mammalian jaw adductor musculature – inferences from soft-tissue imaging of extant taxa (15 min)
Cox — Masticatory muscle anatomy of African mole-rats revealed by diceCT (15 min)
Orsbon— Integration of diceCT with XROMM and fluoromicrometry enhances functional morphology and biomechanics research: a case study of the macaque (Mammalia: Primates) feeding apparatus (15 min)
Stanley —Contrast-enhanced CT provides insight into amphibian lingual morphology (15 min)
Pardo —Studying metamorphosis of the cranial musculoskeletal system in the axolotl using contrast-mCT (15 min)
Porro —Contrast-enhanced micro-CT imaging of fish and frogs: digital dissections and biomechanical applications (15 min)
Yohe —The curious case of the vomeronasal organ in bats: genetics asks questions only anatomy can answer (15 min)
Vander Linden —Comparative morphology of bat cranial muscles using contrast enhanced micro-CT imaging (15 min)
Advancements and Infrastructure for Contrast-enhanced Imaging Techniques
Herdina — Advantages and difficulties of alcoholic iodine staining for correlative 2-D and 3-D micro-CT imaging and histomorphology in bat developmental studies (15 min)
Mahlow — DiceCT and the staining of old museum specimens, exemplified by the analysis of venom glands in viperid snakes (15 min)
Morhardt — Diffusible iodine-based contrast-enhancement of large, post-embryonic, intact vertebrates for CT scanning: staining, destaining, and long-term storage (15 min)
Li — An evaluation of the efficacy and mechanism of contrast-enhanced X-ray computed tomography for avian cranial material utilizing iodine through experimental and simulation approaches (15 min)
Starck —The publishing and archiving of microscopic anatomy (15 min)
1*Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Sciences, Tulsa, OK, USA (corresponding organizer: firstname.lastname@example.org)
2Department of Theoretical Biology, University of Vienna, Vienna, Austria
3Department of Anatomical Sciences, Stony Brook University, Stony Brook, NY, USA
4Department of Biomedical Sciences, Ohio University, Athens, OH, USA
5Jackson School of Geosciences, The University of Texas at Austin, Austin, TX, USA
“Use of iodine as a contrast agent for computed tomography visualizations has been a growing area of research for the last several years. In this open access study we provide a summary of the diceCT literature and synthesize the elements of successful approaches: we analyze factors that govern success at each step of the diceCT imaging pipeline; we recommend a standard for reporting details of specimen preparation, storage, and imaging that will be pivotal to the repeatability and broader adoption of diceCT going forward; and we discuss cutting-edge applications and future directions for diceCT techniques. We anticipate robust growth within the diceCT community and the realization of new horizons in contrast-enhanced imaging—and it is our explicit purpose to spur the adoption of this relatively nascent approach by the morphology community as a whole.”
“Our understanding of the diversity and function in the vertebrate brain has been limited in part by the reliance upon laboratory facilities to preserve brain tissue under optimal conditions. In this study, we field-tested two standard laboratory-based techniques for brain preservation in an African biodiversity hotspot. We validated these protocols across multiple scales of analysis through cytoarchitectonic and immunohistochemical comparisons between field and laboratory-fixed tissue sets and diceCT imaging. In particular, diceCT images revealed excellent contrast of brain tissue structures, including myelinated and unmyelinated portions of the brain. Our protocol should serve as a malleable framework for researchers intending to explore the brains of poorly known and often inaccessible vertebrate species.”
“The morphological adaptations that led to flight in dinosaurs are understood, but the accompanying neuroanatomical changes are still elusive. We used Positron Emission Tomography scanning and diceCT to identify the parts of the brain used during flight in starlings. We found that the anterior Wulst and entopallium are the most used, probably creating a short-term conflict alert system that connects visual and somatosensory input to adjust flight paths. This gives us our first insight into how birds use their brains to fly and what that could mean for the evolution of volancy in theropod dinosaurs.”
“There has been very limited data available to inform detailed protocols for staining large specimens using diceCT. In our study, we systematically assessed the efficacy and mechanism of diffusion-based contrast-enhanced X-ray Computed Tomography (CT) with serial experiments and the validation of numerical modeling. We have applied a Diffusion-Sorption model to explain the CT contrast increasing pattern within the cranial tissues of a goose specimen over a long staining duration. We identified attributes of different tissues that affect the effective diffusion rate and staining efficacy, including partition coefficient, bulk density and tissue porosity. Based on our results, specific protocols—customized by tissue size and type—can be designed for diceCT to maximize visualization of these soft tissues contrasts.”
Comparisons of MRI to diceCT mouse brains (left), showing displacement heat maps (right). Gray areas in the heat map indicate regions of large differences (>0.5 mm) either due to extreme shrinkage or difference in segmentation.
“Phenotypic screens for brain defects traditionally used either histology or high-resolution magnetic resonance imaging (MRI) to look for structural abnormalities. The former is tedious and slow, while the latter is expensive. By coupling the excellent contrast offered by the iodine with the tissue preserving properties of hydrogel, we demonstrate that whole mouse brains can be effectively and rapidly imaged by μCT at a fraction of a cost of high-resolution MRI. Our methodology is best suited for rapid phenotyping applications where large numbers of samples need to be screened for gross differences in the overall brain shape using computational morphometric approaches.”