Skip to main content
NCBI home page
Journal of Anatomy logoLink to Journal of Anatomy
. 2013 May 30;223(2):185–193. doi: 10.1111/joa.12068

Concentration-dependent specimen shrinkage in iodine-enhanced microCT

Paula Vickerton 1, Jonathan Jarvis 2, Nathan Jeffery 1
PMCID: PMC3724211  PMID: 23721431

Abstract

Iodine potassium iodide (I2KI) solution can be employed as a contrast agent for the visualisation of soft tissue structures in micro-computed tomography studies. This technique provides high resolution images of soft tissue non-destructively but initial studies suggest that the stain can cause substantial specimen shrinkage. The degree of specimen shrinkage, and potential deformation, is an important consideration when using the data for morphological studies. Here we quantify the macroscopic volume changes in mouse skeletal muscle, cardiac muscle and cerebellum as a result of immersion in the common fixatives 10% phosphate-buffered formal saline, 70% ethanol and 3% glutaraldehyde, compared with I2KI staining solution at concentrations of 2, 6, 10 and 20%. Immersion in the I2KI solution resulted in dramatic changes of tissue volume, which were far larger than the shrinkage from formalin fixation alone. The degree of macroscopic change was most dependent upon the I2KI concentration, with severe shrinkage of 70% seen in solutions of 20% I2KI after 14 days' incubation. When using this technique care needs to be taken to use the lowest concentration that will give adequate contrast to minimise artefacts due to shrinkage.

Keywords: iodine, iodine potassium iodide, microCT, shrinkage, stain

Introduction

Micro-computed tomography (microCT) is an established modality for the non-destructive analysis of mineralised tissue in a co-registered space. First introduced in the 1980s by Elliott & Dover (1982), it uses X-ray attenuation data acquired from multiple projection angles to create high resolution, 3D data on bone density, structure and micro architecture. Over the subsequent 20 years it has become a widely used technique, replacing many laborious methods for assessing bony structures in 3D. Standard microCT does not lend itself well to soft tissue investigation. The low coefficient of X-ray attenuation of tissues such as muscle, liver or brain, results in amorphous silhouettes containing little internal detail.

Recently, however, iodine potassium iodide (I2KI) solution has been used as a contrast agent to improve the imaging of soft tissues via microCT (Fig. 1). This technique was originally used alongside a number of other staining solutions for the study of embryonic tissues (Metscher, 2009) and has since been employed by Jeffery et al. (2011) to image, in exquisite detail, whole mouse heads, as well as more detailed studies on fascicular arrangement in porcine samples. In follow-up papers the authors have used I2KI to reveal the three-dimensional muscle architecture of the masticatory apparatus in several species of rodent (Cox & Jeffery, 2011) and the complex arrangement of cardiac muscle in rodents and lagomorphs (Stephenson et al. 2012). The technique has also been employed to study sesamoid structures in the alligator (Tsai & Holliday, 2011) and the soft-tissues within the orbit of spiny rats (Hautier et al. 2012). Most recently, there has been a detailed anatomical investigation of the murine masticatory apparatus on the basis of enhanced microCT (Baverstock et al. 2013). The data generated by I2KI enhanced microCT has proven particularly useful in the creation of computational models that simulate muscle forces and activity patterns (Aslanidi et al. 2012; Cox et al. 2012).

Fig. 1.

Fig. 1

Open in a new tab

MicroCT scans of (a) a mouse knee joint, (b) I2KI-enhanced mouse head, (c) I2KI-enhanced ream (Abramis brama) heart, (d) I2KI-enhanced mouse hindlimb. Scale bars: 2 mm. In all scans the greyscale on the images reflects the X-ray attenuation of the material; the brighter the tissue, the higher the X-ray absorption. The iodine contrast stain increases the X-ray attenuation of the muscle; the more stain taken up, the higher the absorbance and so the brighter the image.

Remarkably, despite the long history of I2KI staining for starch in histology (Wick, 2012), the exact mechanism of I2KI staining remains unknown. The use of iodine as a colour-changing starch indicator test (Ameen & Abedin, 1975) implies that the iodine is trapped in the structure of complex carbohydrate molecules such as those found in glycogen (see Jeffery et al. 2011). Recent work to investigate I2KI distribution using backscattered electron scanning microscopy is likely to provide more definitive answers in the future (Boyde, 2012).

A potentially major limitation of I2KI-enhanced microCT is soft-tissue shrinkage. Initial studies have suggested that this staining process is associated with shrinkage beyond that normally found with, for example, tissue fixation. Specimen deformation during specimen preparation is a problem common to many tissue studies (Boyde & Maconnachie, 1980, 1981, 1983; Boyde & Franc, 1981). Understanding the degree of shrinkage and factors that may introduce variability in the shrinkage are key to the continued application of the technique to imaging soft tissues. The aim of this study is to quantify the degree of shrinkage in dissected mouse skeletal muscle, cardiac muscle and cerebellum, over a range of I2KI concentrations and incubation periods. Alternative formulations of iodine and fixatives used for imaging, for example, embryos (Metscher, 2009) have yet to be proven effective for imaging the comparatively larger samples described in the aforementioned papers. Hence, the current paper will focus only on solutions of I2KI and formalin and compare findings against generally accepted levels of shrinkage associated with conventional forms of preservation (i.e. freezing, formalin, ethanol and glutaraldehyde).

Materials and methods

Specimens

Tissue samples were harvested from post-mortem BL6 mice, selected because they represent the most commonly used strain of the most commonly used mammalian model species. The soft tissues investigated were skeletal muscle, cardiac muscle and brain. These represent a broad range of tissues that have been the focus of recent I2KI studies. The skeletal muscle samples consisted of whole tibialis anterior, triceps surae, gluteus medius and rectus femoris muscles. These were dissected from five previously frozen (−10 °C, for in excess of 6 months), and seven non-frozen mice. Cardiac muscle samples consisted of seven dissected and then bisected hearts. These were washed to remove any residual blood. Brain tissue was represented by seven dissected cerebelli. A total of 76 samples from 12 mice were investigated for tissue shrinkage.

Volume measurements

Volumes were measured using a microvolumeter (Fig. 2) based on the apparatus described by Douglass & Wcislo (2010) but with the addition of a laser level to improve the precision of our measurements of fluid displacement. The samples were first immersed in water, then blotted dry. They were then placed in a water-filled specimen cylinder, which was attached via a water-filled polypropylene tube to a 1-mL measurement syringe. The level of the water in the specimen cylinder was measured with a laser beam, which was reflected off the surface of the water onto a wall. As specimens were placed in the receptacle cylinder, the reflected laser point was deflected. The measurement syringe was then withdrawn until the laser point was back to its original position, giving the volume of the sample. Each sample was immersed in water for less than a minute while it was measured to reduce the effect of osmotic gradients upon accuracy. To ensure that movement of water molecules was not significantly altering the measurements, unstained and stained tissues (20% I2KI for 14 days) were measured repeatedly using the microvolumeter filled firstly with water (hypotonic relative to the samples), then with phosphate-buffered saline (PBS) (hypertonic) and finally with 20% I2KI (to represent the most extreme hypertonic solution).

Fig. 2.

Fig. 2

Open in a new tab

A diagram of the equipment used to measure the volume of the muscle specimens. Las, laser source; Lasp, reflected laser point; T, tubing; Ms, measurement syringe; Rc, receptor cylinder; S, specimen. Apparatus and figure adapted from Douglass & Wcislo (2010).

The apparatus was validated with reference to standard samples (steel bearings) of known diameter from which the volumes were calculated [v = (4/3) π (d/2)³]. Five repeated volume measurements were taken for each bearing. To validate the microvolumeter apparatus at lower volumes, two muscles which had been previously stained using I2KI and scanned via microCT were measured using computational stereological methods, and were then dissected out and measured using the microvolumeter. Findings were compared on the basis of least-squared regression analysis.

Baseline volumes for all samples were measured immediately post dissection. Each measurement was taken as the average of five repeats with the specimen being blotted between each repeat to remove excess fluid. Samples representing each tissue type were then immersed in 10% formaldehyde in PBS, 3% glutaraldehyde in PBS, 70% ethanol or solutions of 2, 6, 10 and 20% iodine potassium iodide (I2KI) dissolved in 10% PBFS. Samples were then measured at intervals of 1, 2, 7, 14 and 28 days following the procedure outlined above. Full experimental conditions are outlined in table 1. Statistical analyses were conducted in excel office 2007 and past v2.15 (Hammer et al. 2001).

Table 1.

The experimental conditions

Tissue type Previous treatment Solution Sample Measurement
Skeletal muscle Frozen, −10 °C for an excess of 6 months PBFS, 2, 6, 10 and 20% Muscles, tibialis anterior, triceps surae, gluteus medius and rectus femoris for each conc. Days 0, 1, 2, 7, 14, 21, 28 post dissection
Skeletal muscle None PBFS, glutaraldehyde, 70% ethanol, 2, 6, 10 and 20% Muscles, tibialis anterior, triceps surae, gluteus medius and rectus femoris for each conc. Days 0, 1, 2, 7, 14, 21, 28 post dissection
Cardiac muscle None PBFS, glutaraldehyde, 70% ethanol, 2, 6, 10 and 20% A bisected heart, for each conc. Days 0, 1, 2, 7, 14, 21, 28 post dissection
Cerebellum None PBFS, glutaraldehyde, 70% ethanol, 2, 6, 10 and 20% A cerebellum sample at each conc. Days 0, 1, 2, 7, 14, 21, 28 post dissection
Open in a new tab

Results

Repeated measurements in different volumeter reservoir solutions showed no significant difference (P > 0.05, t-test of means) for either stained or unstained samples. This demonstrates that the hypotonic water reservoir has no significant effect on the measurements.

Findings for the standard volumes showed that the mean repeated microvolumeter readings were not significantly different from the microCT estimates of muscle volume or the calculated steel-bearing volumes. Regression analysis for the combined data gave a slope through the origin of 0.998 (R² = 0.9997). This means that the microvolumeter has an average error of only ± 0.00262 mL (ranging from +0.005 to −0.007 mL). This level of error (± 1.36% average, ± 0.21–2.8% range) is low relative to the changes of tissue volume under investigation (see below).

Tissues immersed in the three different fixatives showed markedly different changes in volume. After immersion in PBFS, all of the samples underwent an initial rapid increase in volume., and a subsequent, far more gradual decrease in volume (Figs 3 and 4a–c). Immersion in 70% ethanol resulted in a gradual decrease in tissue volume. The tissue samples immersed in glutaraldehyde initially swelled and then began to shrink, but on average remained a little larger than their initial volume (Fig. 3).

Fig. 3.

Fig. 3

Open in a new tab

A graph showing the average percentage of the initial volume of skeletal muscle, cardiac muscle and cerebellum samples immersed in 10% PBFS, 70% ethanol or 3% glutaraldehyde over for a period of 28 days (n = 6 for each fixative).

Fig. 4.

Fig. 4

Open in a new tab

Graphs showing the percentage of the initial tissue volume after immersion in different concentrations of I2KI solution or PBFS over a 28-day incubation period for: (a) skeletal muscle, (b) cardiac muscle, (c) cerebellum.

Immersing specimens in I2KI solution (dissolved in PBFS) results in a rapid decrease in volume in both freshly dissected and previously frozen specimens of skeletal muscle. There was no significant (P > 0.05) difference between the degree of volume reduction at any incubation time or solution concentration between previously frozen and non-frozen samples (Fig. 5). Subsequent analysis therefore used both frozen and unfrozen skeletal muscle data in order to maximise the number of datum points.

Fig. 5.

Fig. 5

Open in a new tab

A graph showing the proportional decrease in skeletal muscle volume over 14 days in 20% I2KI in both previously frozen (solid grey line, n = 4) and unfrozen (dashed grey line, n = 4) specimens. The average of the frozen and unfrozen specimens is displayed in bold.

All three tissue types showed a concentration-dependent shrinkage after immersion in I2KI solution (Fig. 4a–c) which was more extensive than that brought about by immersion in PBFS alone. The higher the I2KI concentration, the larger the volume decrease, as illustrated by the bisected hearts in Fig. 6. The cerebellar samples showed the least shrinkage, whereas the skeletal and cardiac samples showed similar levels of shrinkage (Fig. 4a–c). The rate (% per day) at which all three types of tissue shrank was also concentration-dependent and, again, showed similar patterns for both skeletal and cardiac muscle (Fig. 7a,b). The muscle samples undergo an initial rapid phase of specimen shrinkage, with the rate of shrinkage slowing after the first 2 days and beginning to reach a plateau at around 7 days. The higher concentrations of I2KI brought about a more rapid shrinkage than the lower concentrations, but the rates for different concentrations converge after 7 days. The cerebellar samples appear to undergo a more rapid period of shrinkage which then plateaus earlier at around 2 days (Tables 1 and 2.

Fig. 6.

Fig. 6

Open in a new tab

Bisected mouse hearts, in a similar orientation on day 4 of immersion in: (a) 10% PBFS, (b) 2% I2KI, (c) 6% I2KI, (d) 10% I2KI, (e) 20% I2KI. On day 0, all five samples were measured as being between 0.07 and 0.09 mL. Image obtained by photography, and to scale. Scale bar: 5 mm.

Fig. 7.

Fig. 7

Open in a new tab

Graphs showing the average rate of tissue shrinkage after immersion in different concentrations of I2KI solution over a 14-day incubation period in: (a) skeletal muscle (n = 8), (b) cardiac muscle (n = 2), (c) cerebellum (n = 2). The skeletal muscle samples included both previously frozen and previously unfrozen samples in order to maximise the dataset.

Table 2.

Means (SD) displaying the percentage of skeletal muscle volume remaining after 14 days' immersion in PBFS or I2KI

Fixative Average/percentage of original volume after 14 days (SD) Upper limit/percentage of original muscle volume after 14 days Lower limit/percentage of original muscle volume after 14 days
PBFS 81 (15.35) 99 61
70% Ethanol 78 (7.24) 88 71
Glutaraldehyde 105 (8.18) 117 94
2% I2KI 74 (7.08) 82 59
6% I2KI 58 (11.40) 72 43
10% I2KI 53 (9.52) 65 41
20% I2KI 33 (9.34) 47 23
Open in a new tab

There is a linear relationship between the original volume of the tissue sample and the volume after 14 days of immersion (Fig. 8a) with the gradients dependent upon the concentration of I2KI used. Figure 8b illustrates that there was no correlation between the original tissue volume and the percentage remaining of the original volume after 14 days of immersion in each solution. Some concentrations in Fig. 8b appear to show weak correlations between original muscle size and the degree of shrinkage experienced (see 2% I2KI) but none was found to be statistically significant. There were insufficient data to make individual analyses for each tissue type (n = 3 for cardiac and cerebellar samples), so cardiac and cerebellar samples were analysed alongside skeletal muscle samples to maximise the number of datum points.

Fig. 8.

Fig. 8

Open in a new tab

(a) Plot of skeletal muscle volume after 14 days' incubation in PBFS (n = 10), I2KI of concentration 2 (n = 10), 6 (n = 10), 10 (n = 10), and 20% (n = 10), against the initial tissue volume. Least-squared regression slopes and equations are shown. (b) Comparison of initial skeletal muscle sample volume vs. the percentage of sample volume remaining after 14 days' incubation in I2KI of concentration 2 (n = 10), 6 (n = 10), 10 (n = 10), 20% (n = 10) and PBFS (n = 10). Both previously frozen and unfrozen material was used to maximise the data set. Spearman rank correlation coefficients were 0.48 ns for PBFS, 0.55 ns at 2%, 0.35 ns at 6%, 0.41 ns at 10%, 0.19 at 20%.

Least-squared multiple regressions were calculated to capture the interdependency of shrinkage on the three principal variables of, initial sample size, I2KI concentration and incubation time. The R2 values (coefficient of determination) are given in Table 3, and demonstrate that in all three tissues the percentage reduction in volume is most closely related to I2KI concentration. Only in cardiac samples was there a secondary relationship, with initial sample size; however, the range of sample sizes had very little influence beyond 14 days as the shrinkage plateaus between 3 and 7 days (refer to Fig. 7a–c). The multiple regressions can be used to create a formula for correction of tissue shrinkage as follows: percentage of original skeletal muscle volume = 90.66 (o.vol) −2.18 (conc.) −0.66(t) + 75.39; percentage of original cardiac muscle volume = 463.77 (o.vol) −2.06 (conc.) −0.97 (t) + 49.32; percentage of original cerebellar volume = −102.51 (o.vol) −1.70 (conc.) −0.79 (t) + 92.28 where o.vol is original volume in mL, conc. isI2KI concentration in %, and t is incubation time in days.

Table 3.

Multiple regression statistics

Tissue type n Multiple R2 Original volume (mL) [R2] I2KI concentration (%) [R2] Incubation (days) [R2] Intercept
Skeletal 128 0.77 90.66 [0.06] −2.18 [0.70] −0.66 [0.04] 75.39
Cardiac 16 0.91 463.77 [0.73] −2.06 [0.83] −0.97 [0.07] 49.32
Cerebellar 16 0.83 −102.51 [0.02] −1.70 [0.71] −0.79 [0.10] 92.28
Open in a new tab

R2 values indicate the strength of the relationship between each variable and the overall percentage shrinkage of the tissue. Individual R2 values for each determinant given in square brackets. n reflects the number of samples, each of which is recorded for each variable.

The linear regression and Spearman's rank correlation coefficient values of each variable (original volume, I2KI concentration and time) against the percentage shrinkage at 14 days was also calculated for each tissue type (Table 4. This also shows that the variable which showed the strongest correlation with the degree of shrinkage was the concentration of I2KI, with highly significant P-values.

Table 4.

Linear regression statistics, Spearman's rank correlation coefficient and P-value statistics indicating the relationship between the overall percentage shrinkage of the tissue and each of the variables independent of the other variables investigated

Regression series R rank P-value RMA slope (a) 95% confidence interval b
% of original skeletal muscle vol. vs. original vol. 0.21 * 444.55 520.2 to 387.4 19.634
% of original skeletal muscle vol. vs. I2KI conc. −0.82 *** −2.6412 −2.434 to −2.864 83.763
% of original skeletal muscle vol. vs. time −0.23 ** −3.4322 −2.974 to −3.892 79.265
% of original cardiac muscle vol. vs. original vol. 0.74 *** 1980.5 2377 to 1443 −98.57
% of original cardiac muscle vol. vs. I2KI conc. −0.90 *** −2.9187 −2.32 to −3.568 89.087
% of original cardiac muscle vol. vs. time −0.36 ns −3.7929 4.306 to −5.738 84.116
% of original cerebellum vol. vs. original vol. 0.28 ns 713.94 1413 to −936.4 33.034
% of original cerebellum vol. vs. I2KI conc. −0.84 *** −1.9077 −1.32 to −2.624 84.712
% of original cerebellum vol. vs. time −0.38 ns −2.479 2.175 to −4.033 81.463
Open in a new tab

R-rank gives the strength of correlation between the variables (± 1 reflects a strong correlation, 0 reflects a weak correlation). P-values indicate the significance of this correlation (P < 0.05 is significant). a and b values represent coefficients for constructing a corrective formula in the format y=ax+b.

Discussion

The technique of visualising complex 3D shapes by microCT with an iodine-based contrast agent was originally employed by Metscher (2009) to follow the embryonic development of soft tissue structures. One of these iodine-based contrast agents (I2KI) has now been extensively applied to examine skeletal muscle (Cox & Jeffery, 2011; Jeffery et al. 2011; Hautier et al. 2012). It allows for high-contrast, high-resolution, 3D visualisation on commercially available microCT systems. This technique has since found many other applications, visualising, for example, the conduction system of the heart (Stephenson et al. 2012) and the development of the cardiovascular system (Degenhardt et al. 2010), and the technique has proved particularly useful in the development of biomechanical computational models (Cox et al. 2011) as well as the anatomical description of complex morphotypes (e.g. Cox & Jeffery, 2011; Baverstock et al. 2013). With increasing interest in this contrast technique it is likely to find many more applications, particularly in anatomical and biomechanical studies. It is therefore important to determine the alteration from the normal morphology resulting from its use.

Fixation of tissues aims to prevent cellular breakdown while preserving the tissue's shape, volume and components as close to their living state as possible. No fixative is capable of fulfilling all of these criteria and their use is commonly associated with volume changes in the tissues. The mechanisms behind this are not fully understood (Hopwood, 1982). Figure 3 characterises the volume changes measured when tissues were immersed in three commonly used fixatives – PBFS (formalin), glutaraldehyde and ethanol. Immersion in PBFS or glutaraldehyde resulted in an initial swelling of the tissue, followed by a shrinkage phase, whereas immersion in ethanol resulted in continual tissue shrinkage. Preserving whole organs is presently predominated by formalin, which has long been known to cause tissue deformation (Fox et al. 1985).

Of the three fixatives, glutaraldehyde produced a fixed sample closest to the specimen's original volume. It has been reported that 3% formaldehyde solution has a far higher osmolarity (approximately 1000 mOsm) than that of a 3% solution of glutaraldehyde (approximately 300 mOsm) (Bacallao et al. 2006) and so a far larger degree of shrinkage would be expected from tissue immersed in PBFS than glutaraldehyde. However, it is important to note that earlier studies have suggested that aldehydes exert no effective osmotic pressure (Young, 1935). In a number of studies, cells immersed in glutaraldehyde have been noted to swell (Arborgh et al. 1976), as Fig. 3 shows. However most studies show that glutaraldehyde fixation causes tissue shrinkage (Gusnard & Kirschner, 1977). The difference between results shown here and those in the literature may be due to a difference in the buffer (PBS instead of sodium cacodylate) or a difference in the timescale over which the volume changes were recorded. Ethanol immersion resulted in a prolonged shrinkage of the tissue. This is expected, as ethanol fixes tissues by rapidly changing their hydration state. Figure 3 shows a similar (although not quite as extensive) degree of shrinkage as that recorded by Boyde & Maconnachie (1980), who also measured the relationship between the concentration of ethanol and degree of tissue shrinkage.

The next challenge was to determine whether there was any shrinkage over and above that observed by formalin fixation with the addition of I2KI to the PBFS solution and, if necessary, to document the influence of I2KI concentration and incubation period on the shrinkage observed. With the addition of I2KI, the swell phase was replaced by a more pronounced period of shrinkage. The rate and amount of shrinkage varied with tissue type, probably due to differences of cellular organisation and composition, altering the diffusion into and out of the tissue. For example, Weisbecker (2012) found that the cerebellum underwent the least shrinkage as a result of standard formalin fixation and suggested this was due to smaller cell size and higher neuron density. In the present study, shrinkage also varied in relation to incubation time, with the greatest change occurring in the first few days. By far the greatest influence on shrinkage, however, was I2KI concentration. Samples in solutions of PBFS containing 20% I2KI shrank by as much as 70% (cardiac muscle) compared with a 20% reduction at 2% I2KI. The effects of concentration are clearly shown in Fig. 6. In comparison, the influence of initial sample size on percentage shrinkage experienced was negligible. Although the I2KI solution probably diffuses more rapidly into the smaller samples, due to greater surface area to volume ratios, the resulting shrinkage as a proportion of original sample size is not statistically different from that experienced by larger specimens after 14 days (Fig. 8b).

As it is not always practical to fix all specimens as soon as they are obtained, storage of specimens by freezing and then defrosting is a common and convenient alternative. We therefore wanted to establish whether freezing specimens further exacerbated tissue shrinkage. Our findings suggest that freezing and subsequent thawing of specimens before treatment with fixative and I2KI does not alter the overall magnitude or rate at which the skeletal muscle specimens shrank after immersion with I2KI (Fig. 5).

The microCT data seen in Fig. 1(b,d) and in Jeffery et al. (2011) show large dark bands within the skeletal muscle, which cannot be seen in cardiac muscle (Fig. 1c). These dark bands may indicate connective tissue in this region or may signify air-filled gaps as a result of tissue cleavage (as specimens were scanned in air, not a fluid). The possibility of tissue cleavage was commented on by Degenhardt et al. (2010), in which stained embryos showed a large black region between the diaphragm and the heart. This region was larger in embryos stained at higher concentrations of I2KI. This would imply that these bands represent air or retained rinsing buffer (samples are routinely rinsed and blotted before scanning to remove any surface coating of I2KI solution). Cleavage of the tissues would not be unexpected given the degree of shrinkage reported here.

Metscher's paper (2009) demonstrated that a range of solutions were capable of increasing soft-tissue contrast in small specimens a few millimetres in size: 1% iodine in ethanol, Phosphotungstic acid, and osmium tetroxide. No doubt these other compounds together with appropriate fixatives also result in tissue deformation (see for example Schmidt et al. 2010, embryo work). However, shrinkage associated with the above contrast has not been investigated here or elsewhere, as these stains have not been used successfully for contrast-enhanced imaging of larger specimens.

The tissue samples measured in the present study were isolated tissues; it is not clear whether the same degree of shrinkage would occur in, for example, a whole head or indeed a whole animal. It is possible that there would be the same volume reduction but that the bony restraints would create tissue cleavage in the soft tissues saturated with I2KI. It also seems likely that shrinkage would be less in situ than reported here, as the tissues, particularly muscle, are attached, enclosed or otherwise supported by surrounding structures such as bone.

The large volume changes characterised here show a dependency upon the concentration of the iodine solution. As the exact chemistry of fixation and staining are not fully understood it is not possible to give a definitive biochemical explanation for the tissue shrinkage documented in this paper. A possible explanation for the concentration-dependent pattern is that increasing the iodine content in the solution creates an increasingly hypertonic solution so that water moves along the osmotic gradient, out of the tissue, causing shrinkage. This is supported by Degenhardt et al. (2010), who found that tissue distortion was reduced but not prevented by using isotonic solutions of I2KI.

In conclusion, the evidence presented here suggests that when quantifying soft-tissue phenotypes using I2KI enhanced microCT, particular care must be taken to compare only similar tissue types as well as to standardise the concentration and incubation times for both experimental and control specimens. Researchers should ensure that the standardised concentration and incubation time are sufficient to fully saturate the largest specimens under investigation and allow percentage changes to converge and stabilise across the sample. If these conditions cannot be met, then some adjustment of the results may be necessary with reference to the values reported in this paper.

Acknowledgments

The authors would like to thank H. Sutherland for her help in specimen preparation, and two anonymous reviewers for their constructive and helpful comments. P.V. was supported by a scholarship from the Institute of Ageing and Chronic Disease, University of Liverpool.

References

  1. Ameen M-U, Abedin N. Anatomy, histology, and secretions of the salivary glands of Sphaerodema rusticum (F.) (Hemiptera: Belostomatidae) Int J Insect Morphol Embryol. 1975;4:35–47. [Google Scholar]
  2. Arborgh B, Bell P, Brunk U, et al. The osmotic effect of glutaraldehyde during fixation. A transmission electron microscopy, scanning electron microscopy and cytochemical study. J Ultrastruct Res. 1976;56:339–350. doi: 10.1016/s0022-5320(76)90009-5. [DOI] [PubMed] [Google Scholar]
  3. Aslanidi O, Nikolaidou T, Zhao J, et al. Application of micro-computed tomography with iodine staining to cardiac imaging, segmentation and computational model development. IEEE Trans Med Imaging. 2012;32:8–17. doi: 10.1109/TMI.2012.2209183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bacallao R, Sohrab S, Phillips C. Handbook of Biological Confocal Microscopy. New York, USA: Springer; 2006. Guiding principles of specimen preservation for confocal fluorescence microscopy; pp. 368–380. [Google Scholar]
  5. Baverstock H, Jeffery N, Cobb S. The morphology of the mouse masticatory musculature. J Anat. 2013;223:46–60. doi: 10.1111/joa.12059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boyde A. Staining plastic blocks with triiodide to image cells and soft tissues in backscattered electron SEM of skeletal and dental tissues. Eur Cells Mater. 2012;24:154–161. doi: 10.22203/ecm.v024a11. [DOI] [PubMed] [Google Scholar]
  7. Boyde A, Franc F. Freeze-drying shrinkage of glutaraldehyde fixed liver. J Microsc. 1981;122:75–86. doi: 10.1111/j.1365-2818.1981.tb01244.x. [DOI] [PubMed] [Google Scholar]
  8. Boyde A, Maconnachie E. Treatment with lithium salts reduces ethanol dehydration shrinkage of glutaraldehyde fixed tissue. Histochemistry. 1980;66:181–187. doi: 10.1007/BF00494644. [DOI] [PubMed] [Google Scholar]
  9. Boyde A, Maconnachie E. Morphological correlations with dimensional change during SEM specimen preparation. Scan Electron Microsc. 1981;4:27. doi: 10.1002/sca.4950040104. [DOI] [PubMed] [Google Scholar]
  10. Boyde A, Maconnachie E. The Science of Biological Specimen Preparation. Chicago: Scanning electron microscopy Inc; 1983. Not quite critical point drying; pp. 71–75. [Google Scholar]
  11. Cox PG, Jeffery N. Reviewing the morphology of the jaw-closing musculature in squirrels, rats, and guinea pigs with contrast-enhanced microCt. Anat Rec. 2011;294:915–928. doi: 10.1002/ar.21381. [DOI] [PubMed] [Google Scholar]
  12. Cox PG, Fagan MJ, Rayfield EJ, et al. Finite element modelling of squirrel, guinea pig and rat skulls: using geometric morphometrics to assess sensitivity. J Anat. 2011;219:696–709. doi: 10.1111/j.1469-7580.2011.01436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cox PG, Rayfield EJ, Fagan MJ, et al. Functional evolution of the feeding system in Rodents. PLoS ONE. 2012;7:e36299. doi: 10.1371/journal.pone.0036299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Degenhardt K, Wright AC, Horng D, et al. Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining: clinical perspective. Circ Cardiovasc Imaging. 2010;3:314–322. doi: 10.1161/CIRCIMAGING.109.918482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Douglass JK, Wcislo WT. An inexpensive and portable microvolumeter for rapid evaluation of biological samples. Biotechniques. 2010;49:566. doi: 10.2144/000113464. [DOI] [PubMed] [Google Scholar]
  16. Elliott JC, Dover SD. X-ray microtomography. J Microsc. 1982;126:211–213. doi: 10.1111/j.1365-2818.1982.tb00376.x. [DOI] [PubMed] [Google Scholar]
  17. Fox CH, Johnson FB, Whiting J, et al. Formaldehyde fixation. J Histochem Cytochem. 1985;33:845–853. doi: 10.1177/33.8.3894502. [DOI] [PubMed] [Google Scholar]
  18. Gusnard D, Kirschner RH. Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying. J Microsc. 1977;110:51–57. doi: 10.1111/j.1365-2818.1977.tb00012.x. [DOI] [PubMed] [Google Scholar]
  19. Hammer Ø, Harper DA, Ryan PD. PAST: paleontological statistics software package for education and data analysis. Palaeontologia electronica. 2001;4:9. [Google Scholar]
  20. Hautier L, Lebrun R, Cox PG. Patterns of covariation in the masticatory apparatus of hystricognathous rodents: Implications for evolution and diversification. J Morphol. 2012;273:1319–1337. doi: 10.1002/jmor.20061. [DOI] [PubMed] [Google Scholar]
  21. Hopwood D. Fixation and fixatives. In: Stevens A, Bancroft JD, editors. Theory and Practice of Histological Techniques. New York: Churchill Livingstone; 1982. pp. 20–40. [Google Scholar]
  22. Jeffery NS, Stephenson RS, Gallagher JA, et al. Micro-computed tomography with iodine staining resolves the arrangement of muscle fibres. J Biomech. 2011;44:189–192. doi: 10.1016/j.jbiomech.2010.08.027. [DOI] [PubMed] [Google Scholar]
  23. Metscher BD. MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions. Dev Dyn. 2009;238:632–640. doi: 10.1002/dvdy.21857. [DOI] [PubMed] [Google Scholar]
  24. Schmidt E, Parsons T, Jamniczky H, et al. Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development. BMC Dev Biol. 2010;10:18. doi: 10.1186/1471-213X-10-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stephenson RS, Boyett MR, Hart G, et al. Contrast enhanced micro-computed tomography resolves the 3-dimensional morphology of the cardiac conduction system in mammalian hearts. PLoS ONE. 2012;7:e35299. doi: 10.1371/journal.pone.0035299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tsai HP, Holliday CM. Ontogeny of the alligator cartilago transiliens and its significance for sauropsid jaw muscle evolution. PLoS ONE. 2011;6:e24935. doi: 10.1371/journal.pone.0024935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weisbecker V. Distortion in formalin-fixed brains: using geometric morphometrics to quantify the worst-case scenario in mice. Brain Struct Funct. 2012;217:677–685. doi: 10.1007/s00429-011-0366-1. [DOI] [PubMed] [Google Scholar]
  28. Wick MR. Histochemistry as a tool in morphological analysis: a historical review. Ann Diagn Pathol. 2012;16:71–78. doi: 10.1016/j.anndiagpath.2011.10.010. [DOI] [PubMed] [Google Scholar]
  29. Young J. Osmotic pressure of fixing solutions. Nature. 1935;135:823–824. [Google Scholar]

Articles from Journal of Anatomy are provided here courtesy of Anatomical Society of Great Britain and Ireland

RESOURCES