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Robust spectroscopy method to evaluate chemical diffusion in biological tissues

20 Apr | By Luís Oliveira
Robust spectroscopy method to evaluate chemical diffusion in biological tissues
Figure: Representation of the diffusion time as a function of optical clearing agent concentration in the treating solution. The smooth splines used to fit the data present a maximum value that corresponds to the true diffusion time of the agent in the tissue. The minimum value observed for saturated solutions represents the diffusion time of water. Pathological tissues have different content for mobile water than normal tissues and show peaks for different agent concentrations.
By: Luís Oliveira; Valery V. Tuchin

Authors: Luís Oliveira (Polytechnic of Porto – School of Engineering, Portugal);

     Valery V. Tuchin (Saratov State University, Russian Federeation).

 

Introduction

Evaluation of the diffusion properties of chemicals in biological tissues has significant interest for various research fields. In tissue optical clearing, the transparency effect can be characterized by the diffusion time (tau) and the diffusion coefficient (D) of the optical clearing agent in the tissue. These properties are unique for any agent diffusing in a particular tissue.

 

Method

Considering an ex vivo experimental setup, thin slab-form tissues can be used to estimate the diffusion properties of agents. For example, by immersing a 0.5 mm thick tissue slab in an aqueous solution containing an osmotic agent, e.g., glucose, the spectral collimated transmittance (Tc) can be measured during the treatment. Selecting a wavelength band where the tissue has no absorption bands, we can calculate the time dependence for Tc at various wavelengths within that band. If those time dependencies are displaced to have Tc=0 at the beginning of the treatment and then normalized to the highest observed value, the data for each wavelength can be fitted with an equation as: [1 – exp(–t/tau)]. Since this equation mimics the time dependence for the agent concentration inside the tissue slab, Tmeasurements are suited to estimate tau. By averaging between the tau values obtained for the various wavelengths, we obtain the mean diffusion time for the agent in the tissue.

In a general optical clearing treatment, there are two fluxes: interstitial or intracellular mobile water goes out due to the osmotic pressure of the outside agent and the agent flows in to replace that water. These fluxes are the basis of the two clearing mechanisms – tissue dehydration and refractive index matching. The unique agent flux into the tissue occurs only when an equilibrium is established between the water in the solution and the mobile water in the tissue. For tissues with unknown mobile water content, a few treatments need to be made with different agent concentrations in the treating solution. By representing the mean tau as a function of the agent concentration in the treating solution, a spline can fit the data, showing a maximum at an intermediate agent concentration and a minimum for a highly saturated solution. The maximum in the spline corresponds to the true tau of the agent in the tissue and the minimum corresponds to the tau of water in the tissue. These values characterize the dehydration and the refractive index matching mechanisms of tissue clearing.

If we measure the thickness (d) time dependencies for similar tissue slabs under treatment with the solutions that contain the same amount of agent that originated the maximum and minimum for the diffusion time, we can calculate the diffusion coefficient for the agent (or water) in the tissue as: D=d²/(tau*pi²).



Results

Applying this method to various tissues, we were able to estimate tau and D for some clearing agents, such as glucose, ethylene glycol, glycerol, fructose or polypropylene glycol, in those tissues. This ex vivo method can also be used to discriminate the free water content between normal and pathological tissues. Using normal and cancer tissues from the human colorectal mucosa in our lab at the Polytechnic Institute of Porto – School of Engineering (Portugal), we were able to show that cancer tissues contain about 5% more free water than normal tissues. Data for human colorectal tissues are presented in the figure, showing that pathological mucosa has about 65% (100% ‒ 35%) mobile water, while normal tissues have about 60% (100% ‒ 40%). Also, a strong sensitivity of measured tau and D for different clearing agents to another terrible pathology, such as glycation of proteins in skin, myocardium, skeletal muscle and pancreas for rats with diabetes was discovered in our lab at the Saratov State University (Russian Federation).

 

Applications

Since this is a method that needs excised tissues it cannot be used in vivo directly, but the results it produces are good estimations for the in vivo situation. This method in a back reflectance mode can be used in vivo to estimate the diffusion properties of drugs, lotions, creams or oils in skin, or ex vivo for testing of cryoprotecting agents in various tissues or organs to be cryopreserved, or food industry, where similar agents are used for long term preservation of fruits, vegetables or meat.


More information can be found here: https://doi.org/10.1002/jbio.201800333





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