Tissue-mimicking Material Research Articles

Reliable Method for Fabricating Tissue-Mimicking Materials With Designated Relative Permittivity and Conductivity at 128 MHz.

Artificial materials that can simultaneously mimic the relative permittivity and conductivity of various human tissues are usually used in medical applications. However, the method of precisely designing these materials with designated values of both relative permittivity and conductivity at 3 T MRI resonance frequency is lacking. In this study, a reliable method is established to determine the compositions of artificial dielectric materials with designated relative permittivity and conductivity at 128 MHz. Sixty dielectric materials were produced using oil, sodium chloride, gelatin, and deionized water as the main raw materials. The dielectric properties of these dielectric materials were measured using the open-ended coaxial line method at 128 MHz. Nonlinear least-squares Marquardt-Levenberg algorithm was used to obtain the formula, establishing the relationship between the compositions of the dielectric materials and their dielectric properties at 128 MHz. The dielectric properties of the blood, gall bladder, muscle, skin, lung, and bone at 128 MHz were selected to verify the reliability of the obtained formula. For the obtained formula, the coefficient of determination and the expanded uncertainties with a coverage factor of k = 2 were 0.991% and 4.9% for relative permittivity and 0.992% and 6.4% for conductivity. For the obtained artificial materials measured using the open-ended coaxial line method, the maximal difference of relative permittivity and conductivity were 1.0 and 0.02 S/m, respectively, with respect to the designated values. In conclusion, the compositions of tissue-mimicking material can be quickly determined after the establishment of the formulas with the expanded uncertainties of less than 10%. Bioelectromagnetics. 2021;42:86-94. © 2020 Bioelectromagnetics Society.

Radiological characteristics of a new experimental rubber elastomeric polymer used in three-dimensional printing with different infill densities and patterns

The objective of this study was to investigate new 3D printable materials, namely PORO-LAY series in both solid and flexible forms, on behalf of their radiological properties by measuring their Hounsfield units (HUs) values at varying infill densities, different infill structures and several kinds of fluids in order to assess their suitability as tissue mimicking materials (TMMs) for phantom applications. In this study, it was found that PORO-LAY materials can be used to achieve low and high values of HU ranges from −990 to +950 depending on their infill density and the filling fluids. In addition, PORO-LAY materials have an acceptable dimensional stability and dimensional accuracy in their solid and flexible forms. The results also indicate that the shape of infill pattern influences the values of HU with percentage difference ranges from 3 to 33% depending on the selected infill structure. The results of this study showed that PORO-LAY materials were feasible to be used as TMMs for CT and PET/CT applications.

Doppler Passive Acoustic Mapping.

In therapeutic ultrasound using microbubbles, it is essential to drive the microbubbles into the correct type of activity and the correct location to produce the desired biological response. Although passive acoustic mapping (PAM) is capable of locating where microbubble activities are generated, it is well known that microbubbles rapidly move within the ultrasound beam. We propose a technique that can image microbubble movement by estimating their velocities within the focal volume. Microbubbles embedded within a wall-less channel of a tissue-mimicking material were sonicated using 1-MHz focused ultrasound. The acoustic emissions generated by the microbubbles were captured with a linear array (L7-4). PAM with robust Capon beamforming was used to localize the microbubble acoustic emissions. We spectrally analyzed the time trace of each position and isolated the higher harmonics. Microbubble velocity maps were constructed from the position-dependent Doppler shifts at different time points during sonication. Microbubbles moved primarily away from the transducer at velocities on the order of 1 m/s due to primary acoustic radiation forces, producing a time-dependent velocity distribution. We detected microbubble motion both away and toward the receiving array, revealing the influence of acoustic radiation forces and fluid motion due to the ultrasound exposure. High-speed optical images confirmed the acoustically measured microbubble velocities. Doppler PAM enables passive estimation of microbubble motion and may be useful in therapeutic applications, such as drug delivery across the blood-brain barrier, sonoporation, sonothrombolysis, and drug release.

Evaluation of Pseudorandom Sonications for Reducing Cavitation With a Clinical Neurosurgery HIFU Device.

Transcranial high-intensity focused ultrasound is used in clinics for treating essential tremor (ET) and proposed for many other brain disorders. This promising treatment modality requires high energy resulting eventually in undesired cavitation and potential side effects. The goals of the present work were: 1) to evaluate the potential increase of the cavitation threshold using pseudorandom gated sonications and 2) to assess the heating capabilities with such sonications. The experiments were performed with the transcranial magnetic resonance (MR)-compatible ExAblate Neuro system (InSightec, Haifa, Israel) operating at a frequency of 670 kHz, either in continuous wave (CW) or with pseudorandom gated sonications of 50% duty cycle. Cavitation activity with the two types of sonications was compared using chemical dosimetry of hydroxyl radical production at the focus of the transducer, after propagation in water or through a human skull. Heating trials were performed in a hydrogel tissue-mimicking material embedded in a human skull to mimic a clinical situation. The temperature was measured by MR-thermometry when focusing at the geometrical focus and steering off focus up to 15 mm. Compared with CW sonications, the use of gated sonication did not affect the efficiency (60%) nor the steering abilities of the transducer. After propagation through a human skull, gated sonication required a higher pressure level (10 MPa) to initiate cavitation as compared with CW (5.8 MPa). Moreover, at equivalent acoustic power above the cavitation threshold, the level of cavitation activity initiated with gated sonications was much lower with gated sonication than with continuous sonications, almost half after propagation through water and one-third after propagation through a skull. This lowered cavitation activity may be attributed to a breaking of the dynamic of the bubbles moving from monochromatic to more broadband sonications and to the removal of residual cavitation nuclei between pulses with gated sonications. The heating capability was not affected by the gated sonications, and similar temperature increases were reached at focus with both types of sonications when sonicating at equivalent acoustic power, both in water or after propagation through a human skull (+15 °C at 325 W for 10 s). These data, acquired with a clinical system, suggest that gated sonication could be an alternative to continuous sonications when cavitation onset is an issue.

T 1-mapping and dielectric properties evaluation of a 3D printable rubber-elastomeric polymer as tissue mimicking materials for MRI phantoms

In this study, a new series of 3D printable rubber-elastomeric polymer called PORO-LAY materials have been investigated regarding their suitability to serve as tissue mimicking materials (TMMs) for MRI phantoms. PORO-LAY materials have been previously used in biofuel cell developments, particle filtrations and modeling elastic tissues. We evaluated the electrical permittivity, electrical conductivity, spin-lattice T 1-relaxation time and acquired the MRI contrast for simple and multi-material complex 3D printed shapes made of PORO-LAY materials at 3.0 T. The results showed a T 1 diversity within PORO-LAY materials, which reveals in different MR image contrasts. The outcome favors PORO-LAY as an appropriate candidate that can be used in multi-materials additive manufacturing to produce realistic shapes such as white/grey matter structures for MRI phantoms with visible clear contrast. Finally, this study could serve as a reference and guideline when using these materials as tissue mimicking materials for different types of human body tissues and provide a promising opportunity to design novel phantoms for a wide range of MRI applications.

Acoustic impedance measurement of tissue mimicking materials by using scanning acoustic microscopy

Tissue-mimicking materials (TMMs) play a key role in the quality assurance of ultrasound diagnostic equipment and should have acoustic properties similar to human tissues. We propose a method to quantify the acoustic properties of TMM samples through the use of an 80 MHz Scanning Acoustic Microscopy (SAM), which provides micrometer resolution and fast data recording. We produced breast TMM samples in varying compositions that resulted in acoustic impedance values in the range of 1.373 ± 0.031 and 1.707 ± 0.036 MRayl. Additionally, liver TMM and blood mimicking fluid (BMF) samples were prepared that had acoustic impedance values of 1.693 ± 0.085 MRayl and 1.624 ± 0.006 MRayl, respectively. The characterization of the TMMs by SAM may provide reproducible and uniform acoustic reference data for tissue substitutes in a single-run microscopy experiment.

Comparison between experimental and computational methods for the acoustic and thermal characterization of therapeutic ultrasound fields

For high intensity therapeutic ultrasound (HITU) devices, pre-clinical testing can include measurement of power, pressure/intensity and temperature distribution, acoustic and thermal simulations, and assessment of targeting accuracy and treatment monitoring. Though relevant standards are available, technical challenges remain because of the often focused, large amplitude pressure fields encountered. Measurement and modeling issues include using hydrophones and radiation force balances at HITU power levels, validation of simulation models, and a suitable tissue-mimicking material (TMM) for temperature measurements. To better understand these issues, a comparison study was undertaken between simulations and measurements of the HITU acoustic field distribution in water and TMM, and temperature rise in TMM. For the specific conditions of this study, the following results were obtained. In water, the simulated values for p + and p- were 3% lower and 10% higher, respectively, than those measured by hydrophone. In TMM, the simulated values for p + and p- were 2% and 10% higher than those measured by hydrophone, respectively. The simulated spatial-peak temporal-average intensity values in water and TMM were greater than those obtained by hydrophone by 3%. Simulated and measured end-of-sonication temperatures agreed to within their respective uncertainties (coefficients of variation of approximately 20% and 10%, respectively).

Quantification of the backscatter coefficient in heterogeneous media: Comparison of attenuation compensation methods

The estimation of backscatter coefficient, from in vivo tissue requires accurate compensation for intervening tissue attenuation. Current attenuation compensation methods (ACM) have been tested in fully or piecewise homogeneous phantoms. Thus, evidence of their performance in complex media remains scant. In this study we compare the performance of two ACM in a tissue-mimicking material with strong reflectors (SR). A gel-based phantom with SR was scanned with a L11-5v transducer on a Vantage 128 system (Verasonics). Estimates of from the phantom's background were obtained from the IQ echo signals using the known phantom attenuation (KA) or two ACM (a Constrained-Log-Difference (CLD) and a dynamic programming (DP) regularized method).The former method was used as gold standard. To analyze bias versus depth, a Pearson correlation coefficient was computed and the mean difference (MD) vs. depth was estimated. CLD-based showed a strong correlation (r = 0.96) with depth, while the correlation of DP-based estimates was weaker. DP-based values were closer to those obtained with KA (MD [0.9–1.15]) compared to the CLD-based estimates (MD [1.0–1.79]). The DP, regularized ACM show less sensitivity to the presence of SR compared to conventional ACM. We are currently using DP-based ACM to characterize breast lesions.

Dispersion curve calculation in viscoelastic tissue-mimicking materials using non-parametric, parametric, and high-resolution methods

Ultrasound shear wave elastography is a modality used for noninvasive, quantitative evaluation of soft tissue mechanical properties. A common way of exploring the tissue viscoelasticity is through analyzing the shear wave velocity dispersion curves. The variation of phase velocity with frequency or wavelength is called the dispersion curve. An increase of the available spectrum to be used for phase velocity estimation is meaningful for a tissue dispersion analysis in vivo. A number of available methods for dispersion relation estimation exist which can give diffuse results due the presence of noise in the measured data. In this work we compare six selected methods used for dispersion curve calculation in viscoelastic materials. Non-parametric, parametric and high-resolution methods were examined and compared. We tested selected methods on digital phantom data created using finite-difference-based method in tissue-mimicking viscoelastic media as well as on the experimental custom tissue-mimicking phantoms. In addition, we evaluated the algorithms with different levels of added white Gaussian noise to the shear wave particle velocity from numerical phantoms. Tests conducted showed that more advanced methods can offer better frequency resolution, and less variance than the fast Fourier transform. In addition, the non-parametric Blackman-Tukey approach exhibits similar performance and can be interchangeably used for shear wave phase velocity dispersion curves calculation.

Elastic Deformation of Soft Tissue-Mimicking Materials Using a Single Microbubble and Acoustic Radiation Force

Mechanical effects of microbubbles on tissues are central to many emerging ultrasound applications. Here, we investigated the acoustic radiation force a microbubble exerts on tissue at clinically relevant therapeutic ultrasound parameters. Individual microbubbles administered into a wall-less hydrogel channel (diameter: 25–100 µm, Young's modulus: 2–8.7 kPa) were exposed to an acoustic pulse (centre frequency: 1 MHz, pulse length: 10 ms, peak-rarefactional pressures: 0.6–1.0 MPa). Using high-speed microscopy, each microbubble was tracked as it pushed against the hydrogel wall. We found that a single microbubble can transiently deform a soft tissue-mimicking material by several micrometres, producing tissue loading–unloading curves that were similar to those produced using other indentation-based methods. Indentation depths were linked to gel stiffness. Using a mathematical model fitted to the deformation curves, we estimated the radiation force on each bubble (typically tens of nanonewtons) and the viscosity of the gels. These results provide insight into the forces exerted on tissues during ultrasound therapy and indicate a potential source of bio-effects.

Acoustic and ultrasonographic characterization of polychloroprene, beeswax, and carbomer-gel to mimic soft-tissue for diagnostic ultrasound.

Materials with acoustic properties similar to soft-tissue are essential as tissue-mimicking materials (TMMs) for diagnostic ultrasound (US). The velocity (cus), acoustic impedance (AI) and attenuation coefficient of US (µ) in a material collectively define its acoustic property. In this work, the acoustic properties of polychloroprene rubber, beeswax, and Carbomer-gel are determined. The pulse-echo technique is used to estimate cus and µ. The product of a sample density (ρ) and cus gives its AI. Using a reference based on the International Commission on Radiation Units and Measurements Report-61, Tissue Substitutes, Phantoms and Computational Modelling in Medical Ultrasound, the results are evaluated. The acceptance criteria are 1.043 ± 0.021g/cm3 (ρ), 1561 ± 31.22m/s (cus), 1.63 ± 0.065 MRayls (AI) and µ within 0.5-0.7dB/cm/MHz. Sample computerized tomography (CT) and US scanning are performed to evaluate their similarities (contrast and speckle pattern) with respective images of the human liver (a clinical soft-tissue). The average errors in measuring cus and µ were 0.14% and 1.2% respectively. From the present findings, acoustic properties of polychloroprene and beeswax are unacceptable. However, the results of Carbomer-gel ρ = 1.03g/cm3, cus = 1567m/s, AI = 1.61 MRayls are satisfactory and µ = 0.73dB/cm/MHz, is higher than the reference (4.3%). Carbomer-gel could produce CT and US images, efficiently mimicking the respective liver images. Carbomer-gel containing 95% water is a low-cost material with a simple formulation. Present results suggest, Carbomer- gel mimics soft-tissue and can be used as a TMM for diagnostic US.

3D printing tissue-mimicking materials for realistic transseptal puncture models

Applications of additive manufacturing (commonly referred to as 3D printing) in direct fabrication of models for pre-surgical planning, functional testing, and medical training are on the rise. However, one current limitation to the accuracy of models for cardiovascular procedural training is a lack of printable materials that accurately mimic human tissue. Most of the available elastomeric materials lack mechanical properties representative of human tissues. To address the gap, the authors explore the multi-material capability of material jetting additive manufacturing to combine non-curing and photo-curing inks to achieve material properties that more closely replicate human tissues. The authors explore the impact of relative material concentration on tissue-relevant properties from puncture and tensile testing under submerged conditions. Further, the authors demonstrate the ability to mimic the mechanical properties of the fossa ovalis, which proves beneficial for accurately simulating transseptal punctures. A fossa ovalis mimic was printed and assembled within a full patient-specific heart model for validation, where it exhibited accuracy in both mechanical properties and geometry. The explored material combination provides the opportunity to fabricate future medical models that are more realistic and better suited for pre-surgical planning and medical student training. This will ultimately guide safer, more efficient practices.

A generalized system of tissue-mimicking materials for computed tomography and magnetic resonance imaging

The advent of technologies such as magnetic resonance imaging (MRI)-guided radiation therapy has led to the need for phantom materials that are capable of producing tissue-like contrast on both MRI and computed tomography (CT) imaging modalities. The purpose of this work is to develop a system of easily made and formed materials with adjustable T1 and T2 relaxation times, and x-ray attenuation properties, for mimicking soft tissue and bone with both MRI and CT imaging modalities. The effects on T1/T2 relaxation times and CT numbers were quantified for a range of gadolinium contrast (0–25 μmol g-1), agarose (0%–8% w/w), glass microspheres (0%–10% w/w) and CaCO3 (0%–50% w/w) concentrations in a carrageenan-based gel. 105 gel samples were prepared with the additives, carrageenan and water. Samples were imaged in a 3D-printed holding structure to find the attainable range of T1/T2 relaxation time and CT number combinations. T1 and T2 relaxation time maps were generated using voxel-wise inversion-recovery and spin-echo techniques, respectively. A multivariate linear regression model was generated to allow the materials system to be generalized to semi-arbitrary T1/T2 relaxation times and CT numbers. Nine diverse tissue types were mimicked for fit model validation. The achievable T1/T2 relaxation times and CT numbers for the additive concentrations tested in this study spanned from 82 to 2180 ms, 12 to 475 ms, and −117 to +914 Hounsfield units (HU), respectively. The mean absolute error between the fit model predicted and measured T1/T2 relaxation times and CT numbers for the nine tested tissue types was 113 ± 64 ms, 16 ± 26 ms and 11 ± 14 HU, respectively. In conclusion, we have created a system of materials capable of producing tissue-like contrast for 3.0 T MRI and CT imaging modalities.

Effect of Temperature and Frequency on the Acoustic Properties of Konjac Glucomannan-Agar Gels as Tissue Mimicking Materials

In this study, we determine the effect of temperature and frequency on the acoustic properties of konjac glucomannan (KGM)-agar gels to confirm their compatibilities as tissue mimicking materials (TMMs). The acoustic properties of four samples; A (KGM-0.10 g agar), B (KGM-0.20 g agar), C (KGM-0.30 g agar) and D (KGM-0.40 g agar) were measured using pulse echo immersion technique. Findings indicated that the longitudinal velocities of all samples were increased while their attenuation coefficients were decreased as the temperature increased from 27.0 to 37.0°C. It also showed that the phase velocities of all samples were independent to frequency but their attenuation coefficients were increased as the frequency increased from 4.0 to 6.0 MHz. KGM-agar gels are compatible as soft TMMs since their acoustic properties are comparable with the acoustic properties of soft tissue.

Magnetorheological Gel Mimicking Cervical Ripening as a Potential Model for Evaluating Shear Wave Elastography

The mechanical characteristics of tissue can reflect its biochemical content and, therefore, be a powerful tool in the diagnosis of diseases. Many different methods have been developed for testing the mechanical properties of tissue, such as aspiration, indentation and shear wave elastography. Soft tissues are, however, more complex in behaviour than current commercial tissue-mimicking materials and the models used in measurement methods. Complex behaviours of the tissue include anisotropy and heterogeneous elasticity. The oversimplified models assumed in different measuring methods often neglect the effects of these behaviours, resulting in inaccuracies. The aim of this study was to develop a tissue-mimicking material able to capture the complexity of tissue mechanical behaviour. It will be used to improve mechanical property measuring methods by quantitatively determining how complexities in tissue behaviour affect the measurements made and evaluating the effectiveness of methods designed to overcome it, and will be used to train users for consistency in measurement. The tissue-mimicking material designed in this study focuses on the mechanical properties of the cervix as measured by shear wave elastography. The characteristic behaviours of cervical tissues highlighted are anisotropy, a wide range of elasticity that changes with gestational age and an elasticity gradient across the tissue. Magnetorheological gels were used as their elastic properties can be tuned with the application of magnetic fields. The sample was simulated with the finite-element software COMSOL before being tested by shear wave elastography and the INSTRON universal material testing machine. It had an elasticity range of 6.75–11.06 kPa, which is similar to that of cervical tissue. It was determined that a change in the orientation of the probe with respect to the orientation of anisotropy can cause up to a 30 % increase in measured elasticity. There was a 16% decrease in elasticity across the sample.

A simple, realistic walled phantom for intravascular and intracardiac applications

PurposeThis work aims to develop a simple, anatomically and haptically realistic vascular phantom, compatible with intravascular and intracardiac ultrasound. The low-cost, dual-layered phantom bridges the gap between traditional wall-only and wall-less phantoms by showing both the vessel wall and surrounding tissue in ultrasound imaging. This phantom can better assist clinical tool training, testing of intravascular devices, blood flow studies, and validation of algorithms for intravascular and intracardiac surgical systems.MethodsPolyvinyl alcohol cryogel (PVA-c) incorporating a scattering agent was used to obtain vessel and tissue-mimicking materials. Our specific design targeted the inferior vena cava and renal bifurcations which were modelled using CAD software. A custom mould and container were 3D-printed for shaping the desired vessel wall. Three phantoms were prepared by varying both the concentrations of scattering agent as well as the number of freeze–thaw cycles to which the phantom layers were subjected during the manufacturing process. Each phantom was evaluated using ultrasound imaging using the Foresight™ ICE probe. Geometrical validation was provided by comparing CAD design to a CT scan of the phantom.ResultsThe desired vascular phantom was constructed using 2.5% and 0.05% scattering agent concentration in the vessel and tissue-mimicking layers, respectively. Imaging of the three phantoms showed that increasing the number of freeze–thaw cycles did not significantly enhance the image contrast. Comparison of the US images with their CT equivalents resulted in an average error of 0.9{,mathrm{mm}} for the lumen diameter.ConclusionThe phantom is anatomically realistic when imaged with intracardiac ultrasound and provides a smooth lumen for the ultrasound probe and catheter to manoeuvre. The vascular phantom enables validation of intravascular and intracardiac image guidance systems. The simple construction technique also provides a workflow for designing complex, multi-layered arterial phantoms.

Bio Phantoms Mimicking the Dielectric and Mechanical Properties of Human Skin Tissue at Low-Frequency Ranges

Tissue phantoms are widely used as substitute materials for real tissue validation of various newly emerging biomedical technologies such as ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI). However, there is no specific recipe for fabricating skin-mimicking phantoms which can mimic both the mechanical and dielectric properties of human skin at lower frequency ranges. 
 
 The objective of this paper is to present a variety of tissue-mimicking materials for filling this research gap in the lower frequency range from 20 Hz to 300 kHz. The starting point of our experiments is based on the oil-in-gelatin based tissue-mimicking materials (TMMs) that have shown to mimic the dielectric properties of human skin in higher frequency ranges. This paper examines the mechanical and dielectric performance of five major classes of tissue-mimicking materials (1) Oil-in-gelatin, (2) lignin and graphene nanopowder in gelatin, (3) gelatin and distilled water, (4) mixed oil in gelatin and distilled water, and (5) lignin in gelatin and distilled water. 
 
 Mechanical and electrical testing was performed using compression testing and parallel plate method respectively. The effect of electrode polarization was considered in the measured data and the intrinsic impedance values were found to be following the Cole-Cole equation. The Young's modulus range of all tissue-mimicking materials was within the range of skin.

Cardiac Tissue-Mimicking Ballistic Gel Phantom for Ultrasound Imaging in Clinical and Research Applications

Ballistic gel was investigated as a tissue-mimicking material in an anthropomorphic cardiac phantom for ultrasound imaging. The gel was tested for its acoustic properties and its compatibility with conventional plastics molding techniques. Speed of sound and attenuation were evaluated in the range 2–12 MHz. The speed of sound was 1537 ± 39 m/s, close to typical values for cardiac tissue (∼1576 m/s). The attenuation coefficient was 1.07 dB/cm·MHz, within the range of values previously reported for cardiac tissue (0.81–1.81 dB/cm·MHz). A cardiac model based on human anatomy was developed using established image segmentation processes and conventional plastic molding techniques. Key anatomic features were observed, captured and identified in the model using an intracardiac ultrasound imaging system. These favorable results along with the material's durability and processes that allow for repetitive production of detailed whole-heart models at low cost are promising. There are numerous applications for geometrically complex phantoms in research, training, device development and clinical use.

Electric Field Probe Used for Gradient Coil-Induced Field Measurements During Medical Device Testing: Design, Calibration, and Validation

In this article, an ultralow-frequency electric field probe capable of measuring time-varying gradient coil induced electric fields in a tissue-mimicking material was constructed, calibrated, and validated. This probe consisted of a 2 cm short dipole antenna followed by an instrumentation amplifier. The designed amplifier had a gain of 100 and a cutoff frequency of 31 kHz. The probe was fabricated on a four-layer printed circuit board (PCB), FR4 of thickness 1.57 mm and a copper thickness of $35~\mu \text{m}$ . A waterproofed 3-D probe holder was used to cover the electronics and expose the dipole tip to the saline solution. To calibrate this probe in a known environment, a parallel-plate conductor was used. The calibrated probe was validated by measuring the electric field in a saline-filled cylindrical phantom placed within a cylindrically symmetric test coil. In order to do the validation, simulations were performed using an in-house tool developed in MATLAB and a validated tool in Sim4Life.

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.