A simple mixture to enhance muscle transmittance

PROCEEDINGS OF SPIE SPIEDigitalLibrary.org/conference-proceedings-of-spie

A simple mixture to enhance muscle transmittance

Luís Oliveira, Armindo Lage, Manuel Pais Clemente, Valery V. Tuchin

Luís Oliveira, Armindo Lage, Manuel Pais Clemente, Valery V. Tuchin, "A simple mixture to enhance muscle transmittance," Proc. SPIE 6791, Saratov Fall Meeting 2007: Optical Technologies in Biophysics and Medicine IX, 67910J (9 June 2008); doi: 10.1117/12.803978 Event: Saratov Fall Meeting 2007: Optical Technologies in Biophysics and Medicine IX, 2006, Saratov, Russian Federation Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 6/12/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use

A simple mixture to enhance muscle transmittance Luís Oliveira ∗a, Armindo Lage b, Manuel Pais Clemente a and Valery V. Tuchin c a CETO – Centro de Ciências e Tecnologias Ópticas, Rua Caldas Xavier, Nº 38 – 6º E, 4150 Porto, Portugal b Electro-technical and Computers Department, Porto University – School of Engineering, Rua Dr. Roberto Frias, 4200 – 465 Porto, Portugal c Institute of Optics and Biophotonics, Saratov State University 83 Astrakhanskaya str., Saratov 410012, Russia ABSTRACT Skeletal muscle is a fibrous tissue composed by muscle fibers and interstitial fluid. Due to this constitution, the muscle presents a non uniform refractive index profile that origins strong light scattering. One way to improve tissue transmittance is to reduce this refractive index mismatch by immersing the muscle in an optical clearing agent. As a consequence of such immersion tissue also suffers dehydration. The study of the optical clearing effect created by a simple mixture composed by ethanol, glycerol and distilled water has proven its effectiveness according to the variations observed in the parameters under study. The effect was characterized in terms of its magnitude, time duration and histological variations. The applied treatment has created a small reduction of the global sample refractive index that is justified by the long time rehydration caused by water in the immersing solution. From the reduction in sample pH we could also identify the dehydration process created in the sample. The immersion treatment has originated fiber bundle contraction and a spread distribution of the muscle fiber bundles inside. New studies with the mixture used, or with other combinations of its constituents might be interesting to perform with the objective to develop new clinical procedures.

Keywords: Tissue optical clearing; muscle; controlling optical properties; optical clearing agent; refractive index matching

1. INTRODUCTION Optical immersion techniques have proven their potential in clinical diagnosis and treatment where light is used as a tool. Current research is directed to develop new immersion agents that effectively enhance optical clearing of tissues without harming their biological components. The enhancement degree, duration of the optical clearing effect created, reversibility of the process and harmful effects are the principal subjects under study of the current agents in a variety of biological tissues 1. Recently, we studied optical clearing effects created by different osmotic agents in muscle samples collected from the abdominal wall of rats (Species Wistar Han) 2.

∗

E-mail: luí[email protected]; phone: +351 226002471; fax: +351 226007002 Saratov Fall Meeting 2007: Optical Technologies in Biophysics and Medicine IX, edited by Valery V. Tuchin, Proc. of SPIE Vol. 6791, 67910J, (2008) 1605-7422/08/$18 doi: 10.1117/12.803978

Proc. of SPIE Vol. 6791 67910J-1 Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 6/12/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use

Fig. 1: Wistar Han rat

2. THE SAMPLE CHOICE The choice of skeletal muscle is related to its great abundance throughout the human and animal bodies. Muscular tissue is a biological tissue known to be composed by muscular fibers through which interstitial fluid percolates 3. This interstitial fluid is mainly composed by water and it contains a small percentage of salts and minerals 2. In figure 2, we present a cross-section photograph of the abdominal wall muscle from rat:

Fig. 2: Cross-section of abdominal wall muscle from rat (2x microscope magnification)

As we can see from figure 2, muscle fiber bundles are densely packed. On the lower part of figure 2, we can also observe some areas where this packing is not so dense, revealing the presence of interstitial fluid between the muscle fiber bundles.

3. OPTICAL SCATTERING Due to the heterogeneous composition of the abdominal wall muscle, any optical radiation introduced into this tissue will suffer optical attenuation 1. Such attenuation will be mainly due to scattering that occurs at the interfaces between solid part of tissue and interstitial fluid due to the existence of localized refractive index mismatches 1. In our experimental studies, we have calculated the refractive index of the muscle dry matter to be 1.52 2. This dry matter is composed by elastin and collagen fibers that in natural state of tissue are hydrated and grouped as muscle fiber bundles surrounded by interstitial fluid. Due to this arrangement, the muscle fiber bundles must have a refractive index a little lower than the dry matter, something like 1.47-1.48. The interstitial fluid is mainly composed by water that presents a refractive index of 1.33 for visible wavelengths 3. In addition to water, the interstitial fluid has a small content of organic compounds and dissolved salts. This way, its refractive index must be a bit higher than pure water, something like 1.35. This way we observe a refractive index discrepancy of about 1.35 to 1.47-1.48 which is directly responsible for the scattering of light inside the muscle 1.


1.35 Li&fl Scatteitg

Step Refractive index prorne 1.48

Fig. 3: Refractive index mismatch originates light scattering

The refractive index mismatch can be reduced or minimized by immersing the tissue in an optical clearing agent with a refractive index higher than 1.35.

4. OPTICAL IMMERSION By immersing the tissue in an innocuous hyper osmotic agent, we obtain an optical clearing effect by the combination of two mechanisms: tissue dehydration and refractive index matching 1. Figure 4 shows the two mechanisms that occur when the tissue is immersed.

/ Agent

Fig. 4: Tissue immersion in optical clearing agent

As the agent diffuses into the inter-fiber spaces the osmotic pressure created and directed to the inside of the tissue forces water to come out the sample. This way dehydration occurs (see previous figure) 2. On the other hand, the exiting water is replaced by the agent that presents a higher refractive index, more approximated to the refractive index of the tissue fiber bundles 2. High Step Refractive index profile

Low Step Refractive index profile

Fig. 5: Refractive index mismatch reduction

With the decrease of the refractive index mismatch, optical scattering is also reduced, permitting higher beam transmittance and collimation trough the biological tissue.


5. EXPERIMENTAL STUDY We have studied the time evolution of the optical clearing effects created in muscle samples by some well known optical clearing agents like Glucose and Glycerol 2,3. Additionally, we have repeated such study using as optical clearing agent a simple innocuous mixture to evaluate its optical clearing potential. Such mixture was prepared in our laboratory and is composed by Ethanol, Glycerol and distilled water in the following proportions 2: 50%

25%

25%

Fig. 6: Mixture preparation

Such mixture composition was motivated by the introduction of low content glycerol that makes it more biocompatible and the usage of ethanol provides a higher biological membrane permeation due to the interaction of this chemical with the intercellular lipids, as it was verified in other studies 1.

5.1 Sample preparation and characterization With this agent we have performed 5 independent experimental studies to permit statistical evaluation of the optical clearing effect created. These five studies were performed in different days. To prepare samples with desired dimensions for study, we have used a cryostat. To perform muscle slicing in cryostat it is necessary to freeze muscle block at -20ºC. After slicing the various samples for one study, they defreeze naturally at room temperature becoming then ready to perform the optical treatment study with the acquisition of transmitted spectra trough time. The freezing and defreezing process causes volume changes in the muscle samples, though it is possible that the final tissue samples to be studied might not have the desired thickness. In every experimental study two samples were prepared with a square form with superficial areas of approximately 1.5 cm x 1.5 cm and then sliced in a cryostat with a calibrated thickness of 0.5 mm 2. One of these samples was submitted to chemical immersion in the agent while being crossed by a powerful beam of white light. The light transmitted trough the sample was collected by a spectrometer for spectral evolution analysis during optical immersion. The other sample was kept in a physiological solution to be analyzed in natural state 2. After the optical study, both samples were postprocessed to acquire cross-section photographs at microscope. Such prost-processing takes several hours to be made. Cross-sections of samples are only ready for microscope photography 24 hours after optical treatment. To avoid contamination between different samples treated with different optical clearing agents, we have introduced each sample inside two microscope glasses. We are aware that these glasses cause mechanical stress on the samples, and though when released from the glasses the samples might tend to expand in the thickness direction. We will be alert to these possible variations in further analysis. In figure 7, we present the cross-section photographs obtained with microscope from one natural sample used in the experimental study:


-V

—-

1mm

. V/1

tf

0.5 mm

a) 2x optical magnification

b) 10x optical magnification

Fig. 7: Natural tissue sample in cross-section

First, we wanted to verify if temperature variations and mechanical stress applied to the samples during their preparation and post-processing has caused a significant change in the mean cross-section dimension. We have calibrated cryostat to slice samples with 0.5 mm thickness. From each of the 2x optical magnification photographs, we took 10 measurements of the tissue cross section along the cut. The mean thickness, from all 5 natural samples was determined as 0.55±0.01 mm. This value indicates volume changes of the samples caused by temperature variations and mechanical stress applied. Also, from the 2x optical magnification photographs, we verified that the slices are not homogeneous: cross-sections are thicker in some places than in others. See figure 7.a, as an example. From each 10x optical magnification photograph, we counted the number of muscle fiber bundles per photograph area (photograph area was considered unitary since it does not change from one photograph to another). The calculated mean number of fiber bundles for the natural state of the samples was 43. Also, in natural state, fiber bundles are homogeneously distributed trough the photograph area, as we can verify in figure 7 b. We can see in table 1 the information acquired from the cross-section photographs of natural tissue samples: Table 1: Mean parameters for natural tissue Natural muscle Mean thickness (mm)

Mean number of muscle fiber bundles

0.55±0.01

43

The natural transmittance of the muscle was also measured, to establish a natural reference. The mean natural transmittance from the 5 samples is represented in figure 8:


12

11-75

11.5 11-25 11

a7 10-5

Ia am 9.5

am 9

a76 AM

•25 A

7-75

7.6 7-25 7

370 490 439 4 490 620 659 600 010 640 670 700 730 700 790 WIt E0 AB000 W,veIenfli(rrn)

Fig. 8: Natural transmittance of abdominal wall muscle

From figure 8, we observe that the muscle shows low transmittance at lower wavelengths and higher transmittance at higher wavelengths. Such transmittance spectrum is typical for fibrous tissue 1. We also observe the existence of two absorption bands localized approximately at 415 and 525 nm. The first is the Soret band and the second is a combination of the two hemoglobin absorption bands 1. The natural refractive index of the muscle was measured with a Abbe refractometer (using red contrasting illumination). Since Abbe refractometer was not available at our study location, we have prepared several muscle samples with the same dimensions indicated above to measure refractive index on a single day. All the samples used to measure refractive index were sliced at cryostat and immersed in optical clearing agents or physiological solution (natural samples) for 2 hours before performing measurements. To measure refractive index, we first dried tissue samples with absorbing paper to avoid false readings. From the two samples immersed in physiological solution, we verify that muscle refractive index is 1.3980. In every experimental study, the natural tissue samples were also evaluated in terms of pH. We have measured samples´ pH with a pH contact probe that also controls temperature in contact point. The mean pH value for the 5 samples considered was 6.34 at a mean temperature of 24.1±0.9 ºC.

5.2 Mixture characterization When preparing the immersing liquid, we have carefully controlled its composition. We have prepared 200 ml with 50 ml of Ethanol, 50 ml of Glycerol and 100 ml of distilled water 2. Apart from controlling the composition of this mixture, we also took some measurements from it in a way to characterize it. In our laboratory, we measured the pH of the mixture with our contact probe. The solution presented a pH of 7.61 at a measuring temperature of 25.2ºC. Independently, we have measured the solution’s refractive index with the Abbe refractometer. Solution presented a refractive index of 1.3805.


5.3 Transmittance studies In each one of the 5 studies and after measuring the natural pH of the samples, we initiated the transmittance spectra acquisition during the immersion treatment. Samples were maintained between source and collecting fibers without any mechanical stress applied. The immersing liquid was applied around samples inside a black box to prevent the insertion of undesirable light. Such study is maintained until we verify stabilization in the transmittance evolution regime. In the first 2 minutes of treatment, we have acquired transmittance spectra every 5 seconds so we could correctly represent eventual spectral oscillations known to occur at the beginning of the sample immersion in some agents 2,3. After that time interval, spectral evolution does not present fast variations, so we have adopted measuring intervals of 30 seconds or 1 minute. At the end of the 5 studies, we have calculated mean spectra for every measuring instant and normalized to the natural transmittance spectrum for easiness of calculations and for better detection of eventual changes in the spectral form during the immersion treatment. In all the representations below, we have adopted 10 different wavelengths within the spectrometer acquisition band. In figure 9, we have a short-time evolution representation of the mean sample transmittance with a resolution of 5 seconds to identify eventual oscillations in transmittance evolution. We have observed such oscillations in studies with other immersion agents, like glucose or glycerol 2,3.

Fig. 9: Mean transmittance evolution in the first 2 minutes at some wavelengths

From figure 9 we observe that in this case no transmittance oscillations occur at the beginning of treatment, although a little change of behavior can be observed at 5 and 10 seconds. Transmittance rises fast in the first 30 seconds, showing that the agent diffuses quickly into the tissue sample. On the other hand, after 30 seconds, transmittance tends to stabilize. This fact shows that the tissue samples are saturated with agent and equilibrium of concentrations of agent is achieved between the inside and the outside of the tissue sample 1 : The major flux of the agent to the inside of the sample has ended after 30 seconds. Considering the normalized transmittance units represented, we verify that the samples almost doubled their transmittance in this short period of time. Also, the transmittance behavior for all wavelengths considered is the same.


This means that the spectral form is maintained. A full spectral representation of the transmittance evolution in this period is made in figure 10:

Fig. 10: Mean spectral transmittance evolution in the first 2 minutes

The representation made in the previous figure is accordingly to what was said for the fast initial rise in transmittance and spectral form maintenance. In figure 10 we can observe the existence of some absorption bands in the infrared for the measurements performed at 5, 10, 15, 20 and 25 seconds. It is curious that these bands only exist for these initial measuring instants and then they disappear. The occurrence of these bands at the first instants of measurement might be related to the dehydration process 2, 3. Since the osmotic pressure caused by the agent forces water to come out the tissue, considerable amounts of water might localize temporarily in front of the spectrometer acquisition optics, and though contaminating the acquired spectrum. Also, from the previous figure, we can verify the stabilization of the spectral evolution after the initial 30 seconds. This fact translates the achievement of agent concentration equilibrium between the inside and the outside of the sample. In these first 2 minutes of treatment, we can also verify that transmittance has almost doubled for all wavelengths within the band represented. Apart from the temporary occurrence of the infrared absorption bands, no other spectral changes are verified in this period. In figure 11, we have represented the long-time transmittance evolution for the selected wavelengths with a resolution of 1 minute:


Fig. 11: Mean transmittance evolution during treatment at some wavelengths

We observe from figure 11, that tissue transmittance is very high even after 20 minutes of treatment. Such fact indicates that the tissue sample takes some time to expel the glycerol and ethanol present in the mixture. The long-period decaying transmittance indicates that the water in the mixture is kept inside, reverting the optical clearing effect slowly. Since no rehydration was artificially applied to the sample, transmittance is approximately 65% higher at the end of 20 minutes. From figure 11 we verify the same temporal behavior for all wavelengths selected. Due to normalization at the beginning of the time scale, all curves have the same origin, diverging until the first minute. After that, they have the same behavior until the end of the study. This fact shows that the spectral form is kept unchanged during the treatment. At the end of each immersion treatment, we have dried the tissue samples carefully with absorbing paper to proceed with the measurement of sample’s pH value. The mean pH value from all samples treated with the mixture was calculated to be 6.01 at a mean measuring temperature of 24.5±0.9ºC. As we previously mentioned, the refractive index measurements were performed on all the tissue samples in an independent study. In that study, we have measured a refractive index of 1.3855 from the tissue sample treated with the mixture. By comparing the pH and refractive index values between natural and treated samples, we observe that the treatment has produced a change in both parameters. The mean pH value has lowered by 0.33, while the refractive index has decreased by 0.0125. The variation in pH value translates the chemical change inside the tissue samples: interstitial fluid replaced by the mixture. As we have observed in other experimental studies, the refractive index matching created in biological samples by immersing agents, originates a sample’s refractive index rise 2. In this particular case, we have observed a decrease in this parameter. Such decrease can be justified by the great amount of water in the solution. During the optical clearing process, water in the solution substitutes the water in the interstitial fluid. On the other hand, when the treated sample was submitted to refractive index measurement, the refractometer sensing depth was a little below sample surface, where the refractive index is a little bit smaller than deep inside the sample. Such refractive index variation inside the sample is due to the water flux from the inside of the tissue sample to the outside, since in the inside of natural tissue we have approximately 70% of water and in the surrounding solution we have 50%.


The final step in the experimental study consisted in processing the treated samples for microscope analysis. This way we could identify any volume or internal composition changes caused by the treatment. Once again we have analyzed the cross-section photographs with 2x and 10x optical magnification to determine the mean sample thickness and count the number of muscle fiber bundles per photograph area. As an example, we have represented in figure 12 the two magnification photographs of one treated tissue:

V

'T4'4 I 1mm

a) 2x magnification

0.5 mm

:

b) 10x magnification

Figure 12: Photographs of a tissue treated with the Mixture

By comparing the photographs represented in figures 7 and 12, we can identify that the treated sample presents a higher mean thickness and less muscle fiber bundles in the photograph area. From the 2x optical magnification photographs taken from the treated samples, we have calculated the mean sample thickness to be 0.82±0.01 mm. The apparent mean thickness increase was not caused by the applied treatment. In fact, during the long period of post-treatment and as a consequence of long-time rehydration, the tissue sample could be expanded. Also, we must account for the expansion that the sample suffers after it is released from the microscope glasses used in post-processing. On the other hand, regarding the muscle fiber bundles, we verify a smaller concentrated distribution in figure 12.b. By analyzing all photographs of treated tissues with 10x optical magnification we obtain a mean number of muscle fiber bundles of 31, which is less than the mean number observed in natural state. We have resumed the information acquired from the photographs of treated tissue samples in table 2: Table 2: Mean tissue parameters after treatment Treatment with the mixture Mean thickness (mm)

Mean number of muscle fiber bundles

0.82±0.01

31

By comparing the mean number of fiber bundles between natural and treated states, we observe a decrease of 12. This observed reduction in the mean number of fiber bundles indicates that the treatment has caused a more densely packed distribution of elastin and collagen fibers inside the fiber bundles: dehydration has occurred inside the bundles.

6. CONCLUSIONS We have created and quantified an optical clearing effect in muscle samples from the abdominal wall of rats by immersing them in a simple mixture composed by ethanol, glycerol and distilled water. Such immersion treatment has raised tissue transparency rapidly due to the presence of glycerol in the mixture, as we could observe from figures 9, 10 and 11. We have obtained this good and fast clearing of the muscle samples as a consequence of the refractive index matching and the dehydration inside the muscle fiber bundles that forces elastin and collagen fibers to form a more densely packed distribution. Tissue transmittance almost doubled its spectral values after 1


minute of treatment, being verified after that a decrease due to the strong presence of water in the immersion solution. This decreasing tendency tends to diminish after 10 minutes of treatment, as we can verify from figure 11. After 20 minutes of tissue treatment, the transmittance values are approximately 65% higher that the ones verified in natural state. No significant and irreversible spectral changes were observed, although we could verify the occurrence of some absorption bands localized in the infrared that are visible only before the first 30 seconds of treatment. This occurrence of absorption bands in the first few seconds might be related to the dehydration process that the tissue samples suffer. The dehydration of tissue samples is also confirmed by the variation of sample pH value. Regarding the variations observed in treated samples, we can conclude that the applied treatment has caused a rise in density of fibers inside the fiber bundles, forcing these to compact and consequently providing more space for interstitial fluid whose refractive index is continuously corrected by the inserting agent. It is important here to point out the applicability of this agent in clinical applications in which it is necessary to quickly create a transparency effect and in cases where only a short time for that effect is necessary. Some diagnosis applications, like for instance optical diagnosis can benefit with the creation of a transparency effect of this type. It is interesting to study further new combinations of this mixture’s components to compare the effects created. A particular case that might be motivating to study is the one where the water part in the mixture is reduced and compensated with an increase of Glycerol. Such solution might create a more intense optical clearing effect that remains for longer time. On the other hand the study of this kind of mixtures might be interesting to be done in different biological tissues where the creation of such an effect could be important.

REFERENCES 1.

V. V. Tuchin. Optical Clearing of Tissues and Blood. Bellingham: SPIE Press, 2006.

2.

Luis Oliveira, Study of the spectral transmission response of biological tissues under the influence of different osmotic agents, MsC thesis, FEUP – Faculdade de Engenharia da Universidade do Porto, Porto, 2007.

3.

Luis Oliveira, Armindo Lage, M. Pais Clemente and Valery Tuchin. Concentration dependence of the optical clearing effect created in muscle immersed in glycerol and ethylene glycol. Proceedings of SPIE - Volume 6535, Saratov Fall Meeting 2006: Optical Technologies in Biophysics and Medicine VIII, Valery V. Tuchin, Editor, 2007.
