This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.
Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.
Most of the soft biological tissues appear to have viscoelastic properties that are important to understand their injury and development1,2. In addition, viscoelastic properties are important biomarkers in the diagnosis of a variety of diseases such as fibrosis and cancer3,4,5,6. Therefore, the characterization of viscoelastic properties of soft tissues is crucial. Among the many characterization techniques used, ex vivo mechanical testing of tissue samples and in vivo elastography using biomedical imaging are the two widely used methods.
Although various mechanical testing techniques have been used for soft tissue characterization, the requirements for sample size and testing conditions are not easy to be satisfied. For example, shear testing needs to have samples fixed firmly between the shear plates7. Biaxial testing is more suitable for membrane tissue and has specific clamping requirements8,9. A compression test is commonly used for tissue testing, but cannot characterize specific positions within one sample10. The indentation test does not have additional requirements to fix the tissue sample and can be used to measure many biological tissue samples such as the brain and liver. In addition, with a small indenter head, regional properties within a sample could be tested. Therefore, indentation tests have been adopted to test a variety of soft tissues1,3,11.
Characterizing the biomechanical properties of soft tissues in vivo is important for translational studies and clinical applications of biomechanics. Biomedical imaging modalities such as ultrasound (US) and magnetic resonance (MR) imaging are the most used techniques. Although US imaging is relatively cheap and easy to carry out, it suffers from low contrast and is hard to measure organs such as the brain. Capable of imaging deep structures, MR Elastography (MRE) could measure a variety of soft tissues6,12, especially the brain13,14. With applied external vibration, MRE could measure the viscoelastic properties of soft tissues at a specific frequency.
Studies have shown that at 50-60 Hz, the shear modulus of the normal brain is ~1.5-2.5kPa5,6,13,14,15 and ~2-2.5 kPa for normal liver16. Therefore, gelatin phantoms that have similar biomechanical properties have been widely used for mimicking soft tissues for testing and validation17,18,19. In this protocol, gelatin phantoms with two different concentrations were prepared and tested. Viscoelastic properties of the gelatin phantoms were characterized using a custom-built electromagnetic MRE device14 and an indentation device1,3. The testing protocols could be used for testing many soft tissues such as the brain or liver.
1. Gelatin phantom preparation
2. MRE test
3. Indentation test
Following the MRE protocol, a clear shear wave propagation in the gelatin phantoms at 40 and 50 Hz were observed (Figure 3). The viscoelastic properties measured from MRE, and indentation tests are shown in Figure 4. The estimated G' and G" values at each testing for each phantom are summarized in Table 2. Following the indentation protocol, the viscoelastic properties of each phantom at each test point are summarized in Table 3.
As shown in Figure 4, for measurements using MRE, a comparison of G' and G" values measured at 40 and 50 Hz showed significant differences between the two gelatin phantoms (student's t-test, p < 0.05). In addition, significant differences were observed for both G' and G" values between 40 and 50 Hz measurements (student's t-test, p < 0.05). Similarly, for measurements using indentation test, significant differences between the two phantoms were observed for G0 and G∞ values (student's t-test, p < 0.05). Both MRE and indentation provided consistent results for distinguishing soft and stiff gelatin phantoms.
Figure 1: MRE test. (A) Put the vibration plate on top of the gelatin phantom. (B) Place the gelatin phantom inside the head coil and mount the electromagnetic actuator on top of the head coil. (C) An overview of the electromagnetic MRE system showing the connections between each component. Please click here to view a larger version of this figure.
Figure 2: Indentation test. (A) Put the gelatin phantom right under the indenter head in the tester. (B) Prepare the indentation using the Control Setup panel in the GUI. Input the indentation parameters in the GUI to set up the ramp-relaxation test. Observe the indentation curves in the Data Viewer window. Please click here to view a larger version of this figure.
Figure 3: Wave propagation images for the two gelatin phantoms at 40 and 50 Hz. The four phases correspond to the four temporal points at one sinusoidal cycle. Please click here to view a larger version of this figure.
Figure 4: Viscoelastic properties measured from MRE and indentationexperiments. (A) Typical estimated G' and G'' maps at 40 and 50 Hz for the two gelatin phantoms from MRE. (B) Mean and standard deviation of the G0 and G∞ values for the two phantoms from six repeated indentation tests. (C) Mean and standard deviation of the G' and G'' values at 40 and 50 Hz for the two phantoms from six repeated MRE tests. The asterisk symbol indicates a significant difference (student's t-test; p < 0.05). Please click here to view a larger version of this figure.
Gelatin | Water | Glycerol | Total | |
Phantom 1 | 100 (4.35%) | 1200 (52.17%) | 1000 (43.48%) | 2300 (100%) |
Phantom 2 | 160 (6.96%) | 1140 (49.56%) | 1000 (43.48%) | 2300 (100%) |
Table 1: The mass and mass concentration of the gelatin, glycerol, and water used for preparing the two gelatin phantoms. The mass unit is grams.
Modulus(Pa) | Test 1 | Test 2 | Test 3 | Test 4 | Test 5 | Test 6 | Mean | Std | ||
Phantom 1 | 40 Hz | G' | 2978 | 2976 | 2976 | 2974 | 2971 | 2972 | 2975 | 3 |
G'' | 198 | 197 | 197 | 198 | 199 | 199 | 198 | 1 | ||
50 Hz | G' | 2854 | 2852 | 2852 | 2851 | 2850 | 2848 | 2851 | 2 | |
G'' | 341 | 342 | 342 | 342 | 341 | 341 | 341 | 1 | ||
Phantom 2 | 40 Hz | G' | 5603 | 5589 | 5596 | 5590 | 5586 | 5588 | 5592 | 7 |
G'' | 419 | 412 | 419 | 413 | 408 | 408 | 413 | 5 | ||
50 Hz | G' | 5343 | 5341 | 5336 | 5336 | 5329 | 5331 | 5336 | 6 | |
G'' | 317 | 317 | 318 | 324 | 321 | 323 | 320 | 3 |
Table 2: Storage modulus (G') and loss modulus (G") of the two gelatin phantoms measured by MRE. Each phantom was tested six times at an actuation frequency of 40 and 50 Hz.
Test 1 | Test 2 | Test 3 | Test 4 | Test 5 | Test 6 | Mean | Std | ||
Phantom 1 | C0 | 1.54 | 1.88 | 1.81 | 1.71 | 1.65 | 1.60 | 1.70 | 0.13 |
C1 | 0.64 | 0.16 | 0.09 | 0.16 | 0.16 | 0.21 | 0.23 | 0.20 | |
C2 | 0.10 | 0.12 | 0.15 | 0.11 | 0.13 | 0.11 | 0.12 | 0.02 | |
τ1 (s) | 459.71 | 177.52 | 114.14 | 7.32 | 6.1 | 3.73 | 128.09 | 177.51 | |
τ2 (s) | 9.83 | 6.38 | 5.83 | 199.28 | 200.2 | 55.78 | 79.55 | 94.98 | |
R2 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.99 | 1.00 | 0.00 | |
G0 (Pa) | 2273 | 2145 | 2040 | 1991 | 1935 | 1920 | 2051 | 136 | |
G∞ (Pa) | 1535 | 1875 | 1808 | 1714 | 1650 | 1601 | 1697 | 128 | |
Phantom 2 | C0 | 5.97 | 6.29 | 6.16 | 6.20 | 6.14 | 6.11 | 6.14 | 0.11 |
C1 | 0.29 | 0.30 | 0.43 | 0.38 | 0.18 | 0.48 | 0.34 | 0.11 | |
C2 | 0.64 | 0.24 | 0.24 | 0.17 | 0.39 | 0.18 | 0.31 | 0.18 | |
τ1 (s) | 5.99 | 3.50 | 2.46 | 2.71 | 69.34 | 2.36 | 14.39 | 26.95 | |
τ2 (s) | 96.28 | 124.98 | 123.87 | 88.01 | 2.34 | 63.35 | 83.14 | 45.88 | |
R2 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.00 | |
G0 (Pa) | 6899 | 6827 | 6825 | 6751 | 6710 | 6771 | 6797 | 67 | |
G∞ (Pa) | 5967 | 6286 | 6160 | 6197 | 6144 | 6113 | 6145 | 105 |
Table 3: Viscoelastic parameters estimated from indentation tests for the two gelatin phantoms. Each phantom was tested six times.
Gelatin phantoms are commonly used as tissue-mimicking materials for testing and validation of algorithms and devices17,19,22,23,24,25,26,27. One of the pioneering studies using the gelatin phantom to compare MRE and dynamic shear testing was presented by Okamoto et al. (2011)17. In their study, the mass concentration of the gelatin was ~2.8%, and the estimated G' and G'' values after correction were in the ranges of 1.06-1.15 kPa and 0.11-0.27 kPa, respectively. Zeng et al. (2020)19 also used gelatin phantom to validate the inversion algorithm for MRE. With a gelatin mass concentration of ~3.5%, the estimated G' value was ~2.5 kPa. Since the shear modulus increases with the concentration of gelatin, these values were all consistent with the estimation in this study.
To make gelatin phantoms, it is noted that a complete and thorough stirring is required when mixing a large amount of gelatin powder with water. This is necessary for full dissolution to make homogenized phantoms. To increase the melting temperature and shear modulus, glycerol was added to the mixture17. The water bath at around 60 °C will help accelerate the mixing and is recommended during the stirring process. Usually, the gelatin will be formed in a container with a specific shape, e.g., cube or cylinder. Therefore, it is important to filter out the bubbles before pouring the mixed solution into the container.
When preparing for the MRE test, a stable transmission of the shear wave is crucial. Therefore, it is necessary to make sure the vibrating plate is firmly pressed on top of the phantom. This is to avoid any possible slipping between the plate and the phantom. However, this will potentially bring a certain level of local pre-stress. Thus, it is important not to over-press the plate on the phantom. When setting up the actuation frequency, it is noted that the damping of the wave propagation increases with the frequency.
It is suggested to place the indentation device on a vibration isolation table. This is because even a small vibration will affect the ramp-hold recording process. In addition, re-calibration of the sensors is needed if the device has not been used for more than 1 month.
To have the best measurement performance of MRE, it is suggested to keep the frequency within 100 Hz. This is because the higher the frequency, the more dissipation of the vibration, thus inducing a lower SNR of the images acquired. The indentation test mainly measures the sample at a frequency range lower than that of MRE. For a discussion of the parameter conversions between the two methods, one can refer to Chen et al. (2020)11. The MRE and indentation can be used to measure many soft biological tissues to investigate the biomechanical properties and explore the potential biomechanics-based biomarkers for disease diagnosis or treatment evaluation.
The authors have nothing to disclose.
Funding support from the National Natural Science Foundation of China (grant 31870941), Natural Science Foundation of Shanghai (grant 22ZR1429600), and the Science and Technology Commission of Shanghai Municipality (grant 19441907700) is acknowledged.
24-channel head & Neck coil | United Imaging Healthcare | 100120 | Equipment |
3T MR Scanner | United Imaging Healthcare | uMR 790 | Equipment |
Acquisition board | Advantech Co | PCI-1706U | Equipment |
Computer-Windows | HP | 790-07 | Equipment |
Electromagnetic actuator | Shanghai Jiao Tong University | Equipment | |
Function generator | RIGOL | DG1022Z | Equipment |
Gelatin | CARTE D’OR | Reagent | |
Glycerol | Vance Bioenergy Sdn.Bhd | Reagent | |
Indenter control program | custom-designed | Software; accessed via: https://github.com/aaronfeng369/FengLab_indentation_code. | |
Laser sensor | Panasonic | HG-C1050 | Equipment |
Load cell | Transducer Technique | GSO-10 | Equipment |
MATLAB | Mathworks | Software | |
Power amplifier | Yamaha | A-S201 | Equipment |
Voice coil electric motor | SMAC Corporation | DB2583 | Equipment |