Reducing soft-tissue shrinkage artefacts caused by staining with Lugol’s solution

Diffusible iodine-based contrast-enhanced computed tomography (diceCT) is progressively used in clinical and morphological research to study developmental anatomy. Lugol’s solution (Lugol) has gained interest as an effective contrast agent; however, usage is limited due to extensive soft-tissue shrinkage. The mechanism of Lugol-induced shrinkage and how to prevent it is largely unknown, hampering applications of Lugol in clinical or forensic cases where tissue shrinkage can lead to erroneous diagnostic conclusions. Shrinkage was suggested to be due to an osmotic imbalance between tissue and solution. Pilot experiments pointed to acidification of Lugol, but the relation of acidification and tissue shrinkage was not evaluated. In this study, we analyzed the relation between tissue shrinkage, osmolarity and acidification of the solution during staining. Changes in tissue volume were measured on 2D-segmented magnetic resonance and diceCT images using AMIRA software. Partial correlation and stepwise regression analysis showed that acidification of Lugol is the main cause of tissue shrinkage. To prevent acidification, we developed a buffered Lugol’s solution (B-Lugol) and showed that stabilizing its pH almost completely prevented shrinkage without affecting staining. Changing from Lugol to B-Lugol is a major improvement for clinical and morphological research and only requires a minor adaptation of the staining protocol.


Supplemental Figure 2. Effect of addition of paraformaldehyde (PFA) to Lugol's solution (Lugol) and buffered Lugol's solution (B-Lugol).
Eppendorf tubes (2 ml) were filled with 1.5 ml of 3.75% Lugol or 3.75% B-Lugol and supplemented without or with different amounts of PFA in 0.5 ml PBS as indicated in the legend of the graph. Without addition of PFA the Lugol becomes gradually acidic (pH = 4.4 after 147 hours), while the pH of B-Lugol remains stable (pH = 7.1 after 147 hours). With addition of PFA, pH decreases in both Lugol and B-Lugol, though to a much higher extent in Lugol (pH = 3.2 -3.7) compared to B-Lugol (pH = 5.7 -6.6). This effect is larger with the addition of more PFA.
Supplemental PDF: formulas and protocols

Lugol's solution
The success of staining is mainly dependent on three factors: 1) specimen size, 2) staining solution concentration and 3) staining time. In larger specimens the staining fluid has to penetrate deeper to reach the core of the sample. Staining concentration and time are interdependent factors. A higher concentration results in faster diffusion of the staining solution, enabling shorter staining exposure times. However, extended exposure time (to ensure complete and even staining) with higher concentration can result in overstaining and loss of tissue differentiation and/or tissue shrinkage. See Grind the solid I2 using a mortar and pestle for quicker dissolvent. Put the I2 and KI (and PBS) in a glass Erlenmeyer and bring to an end volume of 100 ml with bi-distilled water. For convenience use magnetic stirrer. The powder should be dissolved in a couple of minutes, depending on the concentration and quantity of the solution. Always work under a fume hood and store the Lugol's solution in the dark.

Buffered Lugol's solution (B-Lugol)
Before preparing the buffered Lugol's solution, prepare a 266mM Sorensen's buffer (pH 7.2). See below for the appropriate amounts and ratio.

Preparation of B-Lugol
The easiest way is to prepare a 15% Lugol's solution (see table 1) and combine and dilute appropriately (table 2).

Staining with B-Lugol
Rinse the samples prior to staining with B-Lugol using PBS. After washing, take the samples out and immerse in B-Lugol. See table 2 for preparation of B-Lugol. We use a 3.75% B-Lugol; however depending on the size of the sample, lower concentrations can also be used. NOTE: Put the sample in an amount of volume at least equal to 20 times the weight of the sample. The higher the amount of volume of B-Lugol, the more stable the pH will remain.
Staining time depends on the size of the sample and the concentration of B-Lugol used. If you have ready access to a medical or micro-CT scanner, running shorter, low-quality scans iteratively is useful for checking the progress of staining before investing more time and money into longer, high-quality scans. During staining measure the pH level, osmolarity and optical density (OD). We would advise to replace the solution with new B-Lugol if the pH drops below 6.8

Supplemental PDF: SPSS output
Pearson correlation shows that most variables are correlated to each other. However, this is because they are all, except osmolarity , correlated with time. Partial correlation analysis , controlling for the effect of time, shows that only pH is correlated significantly with tissue volume. This regression analysis showed that the intercept and slope parameters (values on the line (constant) and pH, respectively , in the above table) have overlapping 95% confidence intervals . This shows that the relation between pH and tissue volume follows similar 'rules' in each of the staining solutions .

Correlations
To further dissect the role of osmolarity a regression analysis of the residual volume on Osmolarity was performed . This regression analysis showed that in most solutions the tissue volume could not be further explained by a role for osmolarity . The intercept and slope parameters were not, or hardly, different from 0. This regression analysis shows that the 95% confidence interval of the slope of the relation b etween pH and tissue volume is overlapping between Lugol and B-Lugol indicating that the effec t of pH on tissue volume is similar. And similar to the relation found in Experiment 1. The di fference in intercept reflects the strong difference in shrinkage occurring during staining in the B-Lugol solution.

Regression
To complete the analysis as done in Experiment 1 a linear regression analysis of residual tiss ue volume on osmolarity was performed . The linear regression analysis of residual tissue volume on osmolarity showed a small, but significant , positive effect of osmolarity on tissue volume. The slightly increasing osmolarity , despite the already very hypertonic nature of B-Lugol seems to counteract some of the shrinkage caused be the small decrease in pH.