Congratulations to Echols and Birch on winning the 2017 Wellcome Image Awards!


B0011001 Microvasculature of the African Grey Parr
African grey parrot with the intricate cervical and cranial blood supply reconstructed in 3D.

Avian vascular injection images by diceCT co-author Scott Echols and colleague Scott Birch were recently recognized as winners of the 2017 Wellcome Image Awards.

Congrats to both Scotts and kudos on the fabulous images—keep up the great work!

New Publication: Quantifying 3D Morphology & RNA from Individual Embryos

Iodine-enhanced CT scan projection of a mouse embryo (E11.5) in 3D (top) and an exemplar embryo RNA integrity analysis after staining and scanning, showing 18S and 26S bands as well as a lower marker (bottom).

“Here we present a method that generates high quality iodine contrasted CT images from embryonic soft tissue. This method has two primary advantages over previous methodologies. First, it allows extraction of high quality nucleic acids following the CT scan, generating a 1:1 correspondence between scan and gene expression or DNA profile. Second, it minimizes the shrinkage artifacts associated with fixation and contrasting. The ability to relate a single sample to its genetic profile will allow study of both partially penetrant models and also increase our understanding of how genotype and phenotype are directly related.”

– Lead author, Rebecca Green (Hallgrímsson Lab)

Go read more at Developmental Dynamics!

New Publication: Three-dimensional visualisation of the internal anatomy of the sparrowhawk (Accipiter nisus) forelimb using contrast-enhanced micro-computed tomography

“A three-dimensional model of the wing musculature of the sparrowhawk (Accipiter nisus) was built using μCT and a 3% iodine-buffered formalin solution to test the ability of the technique for visualising wing musculature and obtaining quantitative data of muscle geometry.  This model allows the identification of the individual muscles comprising the avian wing and can be useful for further biomechanical analysis of flight.”

– Lead Author, Fernanda Bribiesca-Contreras

Go read more about the project at PeerJ!

Transverse μCT images of a sparrowhawk (Accipiter nisus) wing (upper panel)—Columns: (A) Control . (B–F) ∼3% (w/v) iodine-buffered formalin solution after three (B), 10 (C), 15 (D), 18 (E), and 25 (F) days. Rows: (G) Corresponds to brachial area, (H) antebrachial area and (I) the manus (scale bar = 5 mm). Three-dimensional model of the wing muscles of a sparrowhawk (lower panel)—three-dimensional model of the wing muscles of a sparrowhawk reconstructed from CT images of the stained wing after 25 days in a ∼3% iodine-buffered formalin solution (superficial muscles are shown in dorsal (left) and ventral (right) view).



“Tour Through the Brain of Python” Named Winner of the FASEB BioArt Competition

“Tour Through the Brain of Python,” a diceCT visualized video fly-through of a Macklot’s python head by Paul Gignac and Nathan Kley, is a winner in the fifth annual FASEB BioArt Competition!

It’s so important to seize opportunities to share the wonder of discovery with the public. The BioArt images showcase the beauty of scientific research and are a great place to start the conversation —Hudson Freeze, PhD, FASEB President.

The Federation of American Societies for Experimental Biology holds an annual BioArt Competition to help engage Members of Congress and the general public about the immense value of biomedical and bioimaging research in the United States and the need for sustained support of federal funding agencies that facilitate life science and biomedical studies. To celebrate the competition, winning entries will be exhibited at the National Institutes of Health and online at

“Tour through the brain of a python”— Paul M. Gignac, Oklahoma State University Center for Health Sciences, and Nathan J. Kley, Stony Brook University School of Medicine

New Publication: Habitat-specific divergence of air conditioning structures in bird bills

“We used high precision computed tomography (CT) and traditional radiography to study the nasal conchae, complex structures within the nasal cavity that condition air via countercurrent heat exchange. Concha size differed between 2 subspecies of Song Sparrow (Melospiza melodia) that inhabit climatically distinct habitats, suggesting adaptation to local climates. The conchae and external bill are nested structures that were positively related in size and play functionally related roles in thermoregulation, therefore suggesting phenotypic integration. We hypothesize that the typically deeper and wider bill of the dune subspecies has evolved, at least in part, to accommodate larger conchae.”

– Lead author, Raymond Danner

Head over to The Auk to see the the published image sets!

DiceCT sparrow nasal cavities
Frontal cross sections along the bills in 2 Song Sparrows prepared using alcoholic iodine (I2E), illustrating the differences between (A) M. m. atlantica and (B) M. m. melodia. Sections are ordered sequentially, from caudal-most to rostral-most. Within each cross section, the top is dorsal and the bottom is ventral. (Scale bar is 2.0 mm.)

VA-044 Azo Initiator Supply for STABILITY

va-044For those diceCT users working STABILITY protocols into your specimen preparation regimes, we’ve been hearing reports that your thermally-triggered initiator, VA-044 (2,2′-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride), may be difficult to source. It tends to be sold in larger volumes than researchers practically require for STABILITY, which has made it difficult to purchase while leaving much of the chemical unused on researcher’s shelves.

Dr. Vera Weisbecker (University of Queensland, Queensland, Australia) has recently offered to share some of her supply with STABILITY users, who might not be able to get a hold of it from Wako Specialty Chemicals or elsewhere. Please email her directly if you are interested. 

We, @DiceCT, would like to offer an enormous thank you to Dr. Weisbecker for her generosity and openness, in particular, as well as for advancing STABILITY for our larger community!

DiceCT on Display at the Royal Society of London Online


A diceCT visualized ratsnake head and brain is a finalist in the 2016 Royal Society of London Scientific Photography Competition! This piece and many other contributions of remarkable scientific artwork are now part of a display at the Royal Society of London Online.

“The cranial anatomy of a diceCT imaged ratsnake in frontal view” by Nathan Kley and Paul Gignac. Ratsnake (genus Pantherophis) head in frontal view, prepared using diffusible iodine-based contrast enhanced computed tomography (diceCT) imaging, in which an iodine staining solution acts as a contrast agent that non-destructively renders soft tissues visible in X-ray micro-CT scans. The specimen was imaged using a GE Phoenix v | tome | x micro-CT scanner at 29.5 microns. Soft tissues including the brain and its internal structures, spinal cord, optic chiasm, roots and branches of cranial nerves, jaw and neck muscles, glands, nasal epithelia, cranial bones, and integument can all be clearly visualized simultaneously. (Image contrast and brightness were modified to render the background fully black and the neck was cropped to fit the image frame.)

The competition celebrates the power of photography to communicate science and the role images play in making science more accessible to a wide audience. To view featured photos at the Open House exhibition (9/17–9/18/2016), visit The Royal Society of London.

New Publication: Comparison and Evaluation of the Effectiveness of Two Approaches of Diffusible Iodine-Based Contrast-Enhanced Computed Tomography (diceCT) for Avian Cephalic Material


Screen Shot 2016-08-24 at 11.39.46 AM
Sagittal sections of the two tinamou heads processed using I2KI-formaldehyde (A) and I2E (B), respectively. Black arrows highlight the staining effect on the spinal cord; white arrows highlight the differential penetration of iodine in the two approaches (inset 3-D renderings for each specimen show the section positions).

I2KI-formalin (iodine-potassium iodine-formalin) treatment can obscure the natural X-ray contrast of the bone due to neighboring soft-tissues during staining of avian cephalic material. By contrast, we found the I2E (iodine-ethanol) treatment can increase the X-ray opacity of not only soft tissues (e.g., muscles), but also the bone. Mathematical simulations suggest that remarkably different results from I2E and I2KI-formalin staining are due to different partition coefficients and retardation factors of tissues, fixation effects, and distinct iodine diffusion and sorption patterns. We also found a clear positive relationship between glycogen concentration and grayscale values measured within muscle, epithelia, nervous tissues, and glands regardless of the staining solution used.

–Lead Author, Zhiheng Li

Head over to the Journal of Experimental Zoology to download the pub!

Get Your Brights Brighter & Darks Darker: CLAHE + diceCT

By Aki Watanabe (@akiopteryx)

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:

  1. Drag and drop the folder that contains a stack of CT images into FIJI (download here: Wait until FIJI reads in the entire image stack.
  2. Copy the CLAHE script from the “Tips” section on the website.
  3. In FIJI, go to “Plugins” > “New” > “Macro”. A new window will open. In the text field, paste the CLAHE script.
  4. Select “Run”. One can specify the “block size,” “histogram bins,” and “max slope” parameters (the details of which are outlined on, but I have found that the default parameters do a fine job.
  5. 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).
  6. 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.


Screen Shot 2016-08-01 at 6.22.53 PM
DiceCT, two-year old Alligator mississippiensis head in transverse views (left) unmodified, and (right) filtered using CLAHE.

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).

A modified STABILITY protocol for accurate retrieval of soft-tissue data from micro-CT scans of IKI-stained specimens.

Alison Carlisle1, Vera Weisbecker1*,

1School of Biological Sciences, University of Queensland.
*For correspondence, e-mail:


Modified from Wong, Spring, & Henkelman (2013)

**UPDATE: Also see the June 26, 2017 edition of Scientific Reports for the full study

Ingredients and Proportions:

  • 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.


(1) Incubation

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.

(2) Curing

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.

(3) Staining

Some potential issues: This brain was very large (~140g) and did not stain throughout; fridge staining, however, may have improved this. Also, note the high density “pockets” that occur much less frequently if staining is done in the fridge.

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.


Stain visualization and reconstruction of specimen in 3-D, using Mimics by Materialise.

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, 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]

Method presented at the 11th Meeting of the International Congress on Vertebrate Morphology