1. Luís Oliveira a,b, Maria Inês Carvalho c, Elisabete Nogueira a, Valery V. Tuchin d,e,f a DFI – Polytechnic of Porto, School of Engineering, Rua Dr. António Bernardino de Almeida, 431, 4200-072 Porto, Portugal; b PhD student at FEUP – University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal c DEEC/FEUP and INESC TEC, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal d Research-Educational Institute of Optics and Biophotonics, Saratov State University, 83 Astrakhanskaya str., Saratov 410012, Russia; e Laboratory of Laser Diagnostics of Technical and Living Systems, Institute of Precise Mechanics and Control RAS, Saratov 410028, Russia; f Optoelectronics and Measurement Techniques Laboratory, P. O. Box 4500, University of Oulu, FIN-90014, Oulu, Finland Saratov Fall Meeting – 2012September 25 – 28, 2012Saratov, Russia

2. 1. Introduction 2 The importance of performing optical measurements from biological tissues is very high to evaluate how the tissues respond to light stimulation. Several classes of optical measurements can be obtained from ex vivo tissue samples: transmittance, absorbance, reflection, etc. If those measurements are to be made while tissue undergoes optical clearing treatments, the optical measurements can be performed in a manner to evaluate the time-dependence of the optical response of that tissue sample. With the objective of studying the time-dependence of the optical response of muscle to light stimulation and to lead in the near future to the determination of the time-dependence of the optical properties of that tissue class under treatment with ethylene glycol and glucose, we have performed a set of measurements.

3. 3 Due to the use (or not) of an integrating sphere, we have two classes of measurements: integrated – that include total transmittance and total reflectance and non-integrated – that include collimated transmittance and specular reflectance. In all cases we have used a Tungsten Halogen lamp with a broad spectrum and a spectrometer to measure the spectra. The lamp is the HL-2000 model and the spectrometer is the AvaSpec-2048-USB2 model with UA grating set for 200-1100 nm and 50 μm slit, both from Avantes. We will present and discuss here the measurements of total transmittance, collimated transmittance, specular reflectance and total reflectance measured from muscle samples under treatment with these optical clearing agents. Additionally we will present also the calculated absorbance and diffuse reflectance. 2. Experimental method

4. 4 To measure total reflectance illumination is now made through the illuminating port at 8° with the normal direction to the sample (right side of the sphere in figure 2). Total reflected light is collected at the exit port of the sphere (lower hole in figure 2). 2.1 Integrated measurements – Total transmittance Figure 1: Total transmittance measuring assembly. To measure total transmittance the sample is placed at the sample port of the integrating sphere (left entrance in figure 1) and light is introduced into the sphere through the sample. Total transmitted light is collected at the exit port (lower hole in figure 1). 2.2 Integrated measurements – Total reflectance Figure 2: Total reflectance measuring assembly.

5. 5 2.3 Non integrated measurements – Collimated transmittance Figure 3: Collimated transmittance measuring assembly. Figure 3 shows the simple assembly used to measure collimated transmittance: A collimated beam (Ф=6 mm) is directed normally to the sample. Immediately before and after the sample two pinholes (Ф=1 mm) were placed to reduce the beam diameter. The transmitted beam was measured on the opposite side of the sample. 2.4 Non integrated measurements – Specular reflectance Figure 4: Specular reflectance measuring assembly. The measurement assembly for specular reflectance (figure 4) is accordingly with the total reflectance measurement assembly in angles and dimensions (figure 2). it uses an incident beam at 8° with the normal direction to the sample surface and the reflected beam is also measured at the same angle on the other side.

6. 6 2.5 Tissue samples The tissue samples used in this experimental study were obtained from the abdominal wall muscle from rat (Species Wistar Han, see figure 5). All samples used in these studies (both agents) were obtained from a single animal to guarantee the maximum similarity in the physiology of all samples. Figure 5: Whistar Han rat with abdominal wall muscle at the center. Abdominal wall muscle Figure 6: Muscle block, excised from the abdominal wall of animal. After animal sacrifice, the entire abdominal wall muscle was dissected from the animal and a muscle block (like the one seen in figure 6) was available to prepare samples to be studied.

7. 7 2.6 Optical clearing agents The optical clearing agents used in this study were ethylene glycol and glucose. The solution of ethylene glycol had 99% of this agent and 1% of water and for the glucose solution we have dissolved glucose in water to produce a solution containing 40% of glucose. We have measured the refractive index of these solutions, obtaining 1.4280 for ethylene glycol and 1.3850 for glucose 40% [1]. These solutions are recognized as useful optical clearing agents as we have already observed in our previous studies [1] [2] and also as verified by other researchers [3] [4] [5 ] [6]. In the present study, instead of measuring only the collimated transmittance as before [1], we have selected 4 types of measurements for later use in the determination of the optical properties of the muscle and their variation while in optical clearing.

8. 8 3. Experimental results We have performed measurements to obtain total transmittance, total reflectance, collimated transmittance and specular reflectance. These measurements also allow to calculate absorbance and diffuse reflectance from the sample. These are the results that we will present in subsections 3.1 and 3.2 below and their definition is presented here. In each particular measurement, we measured the light reference spectrum and the spectra for natural tissue and while under optical clearing treatment using the Scan mode of the spectrometer. Considering that the light reference is 100% of light used in each experiment, we have calculated from the measurements the total transmittance, total reflectance, collimated transmittance and specular reflectance spectra by using equations 1, 2, 3 and 4 below. The results obtained with these equations are presented in percentage of the light reference used in each case.

9. 9 (1) (2) For the case of total transmittance, we consider S tt (λ) as the spectrum of the light reference and R tt (λ,t) as the total transmitted spectrum measured from the sample at a time t of the treatment. Using these symbols we can present equation 1 that calculates the total transmittance spectrum at that time t: Similarly, for the case of collimated transmittance, we consider S tc (λ) as the spectrum of the light reference and R tc (λ,t) as the collimated transmitted spectrum measured from the sample at a time t of the treatment. In equation 2 we show the calculation of the collimated transmittance spectrum of the sample at time t:

10. (3) To calculate the total reflectance spectrum of the sample at a time t of the treatment, we consider S rt (λ) as the spectrum of the light reference and R rt (λ,t) as the total reflected spectrum measured from the sample at a time t of the treatment. Equation 3 shows this calculation: In a similar manner, equation 4 shows the calculation of the specular reflectance spectrum at a time t that uses the measurements of the source spectrum (S sr (λ)) and the specular reflected spectrum (R sr (λ,t) ) from the sample at time t: (4) 10 Using the experimental measurements we can also calculate the absorbance and diffuse reflectance of the tissue.

11. On the other hand, we know that total reflectance contains both specular and diffuse terms. This way, using the measurements of total and specular reflectance (performed in the same conditions), we can use equation 6 to calculate diffuse reflection of the sample at time t: Considering the integrated measurements of total transmittance and total reflectance presented in equations 1 and 3 and assuming once again that the light spectrum that is used in the integrated measurements is 100%, the absorbance of the sample can be determined as the difference between 100% of light used and the integrated measurements [7]. Equation 5 shows the calculation of sample’s absorption spectrum at a time t during the optical clearing treatment: (5) (6) 11

12. 12 The results from the study with ethylene glycol are presented in sub-section 3.1 and the results from the study with glucose are presented in sub-section 3.2. In each study we will begin by presenting the results for total transmittance, followed by the results of collimated transmittance, total reflectance, specular reflectance, absorbance and diffuse reflectance. Each case is well identified. For each of these particular cases, we present 5 figures: In the first figure we present the corresponding spectrum for the natural sample. The second figure contains the spectral evolution in the first 2 minutes of treatment. The starting reference (natural spectrum) is seen as a thicker line. Between 5 and 40 seconds, spectra are presented at each 5 seconds and after that spectra are presented at each 10 seconds. The viewing point for this figure varies from case to case to optimize the identification of variations. The third figure shows the time dependence lines for some particular wavelengths during the first minute of treatment. The chosen wavelengths were 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm and 1000 nm.

13. 13 The fourth figure shows the spectral variation during 15 minutes of treatment. It is similar to figure 2, but now spectra are separated by 1 minute. Natural spectrum is again presented as a thicker line and viewing point may vary from case to case to optimize the identification of time variations. Similarly to figure 3, figure 5 shows the time dependence curves for the same particular wavelengths (400 nm ….. 1000 nm), but now for 30 minutes of treatment and with one minute resolution. This set of figures allows immediate identification of the time dependence of the sample spectrum with the optical clearing treatment applied. The analysis of these figures and comparison between the two studies allows to identify differences between the two treatments and recognize some particularities in each treatment that allow to detect the variation of the optical responses of the muscle sample with the treatment. Additionally, we can take some conclusions regarding the variation of the optical properties of the tissue, even without making their calculation.

14. 3.1 Experimental results – Ethylene Glycol 3.1.1 Total transmittance Figure 7: Total transmittance spectrum from natural muscle. 14 In this first study, we used a solution of ethylene glycol 99%. The results obtained from the different measurement assemblies and calculations with equations 1 to 6 are presented below: Figure 7 shows the natural spectrum of the muscle measured with the total transmittance assembly. Typical absorption bands. Total transmittance rises with wavelength

15. 15 Figure 8: Total transmittance spectral evolution in the first two minutes. Figure 9: Total transmittance evolution in the first minute of treatment for some wavelengths. Figure 10: Total transmittance spectral evolution in the first 15 minutes. Figure 11: Total transmittance evolution during the treatment for some particular wavelengths. Saturation regime begins at ~12 minutes. Total transmittance spectrum retains its form and rises in the first 2 minutes of treatment.

16. 3.1.2 Collimated transmittance Figure 12: Collimated transmittance spectrum from natural muscle. 16 Figure 12 shows the collimated transmittance spectrum of the natural muscle. Collimated transmittance rises with wavelength Typical absorption band. Absorption bands of Hemoglobin are not well seen due to tissue blood-washout after animal sacrifice

17. 17 Figure 13: Collimated transmittance spectral evolution in the first two minutes. Figure 14: Collimated transmittance evolution in the first minute of treatment for some particular wavelengths. Figure 15: Collimated transmittance spectral evolution in the first 15 minutes. Figure 16: Collimated transmittance evolution during the treatment for some particular wavelengths. Variations are more evident in the present case, due that collimated transmittance is a non integrated measurement.

18. 3.1.3 Total reflectance Figure 17: Total reflectance spectrum from natural muscle. 18 The total reflectance spectrum of the natural muscle is represented in figure 17: The spectral form is very similar to the one seen for total transmittance. The difference is that total reflectance shows much smaller values than total transmittance.

19. 19 Figure 18: Total reflectance spectral evolution in the first two minutes. Figure 19: Total reflectance evolution during in the first minute of treatment for some particular wavelengths. Figure 20: Total reflectance spectral evolution in the first 15 minutes. Figure 21: Total reflectance evolution during the treatment for some particular wavelengths. Total reflectance lowers with treatment (majorly within the first minute). Some oscilations are evident in this measurement. They tend to lower their magnitude with time.

20. 3.1.4 Specular reflectance Figure 22: Specular reflectance spectrum from natural muscle. 20 Figure 22 shows the specular reflectance spectrum of the natural muscle: In opposition to the total reflectance spectrum and apart from the absorption band seen near 400 nm, the specular reflectance decreases with wavelength until the upper limit of the visible range.

21. 21 Figure 23: Specular reflectance spectral evolution in the first two minutes. Figure 24: Specular reflectance evolution in the first minute of treatment for some particular wavelengths. Figure 25: Specular reflectance spectral evolution in the first 15 minutes. Figure 26: Specular reflectance evolution during the treatment for some particular wavelengths. Like in the case of total reflectance, the spectrum of specular reflectance decreases significantly in the first few seconds. Osmotic pressure of agent causes initial increase in specular reflectance. Saturation regime shows almost linearly increasing specular reflectance.

22. 3.1.5 Absorbance Figure 27: Absorbance spectrum from natural muscle. 22 Using the measurements of total transmittance and total reflectance in equation 5 we have determined the absorbance spectra during the treatment with ethylene glycol. The Absorbance spectrum for natural tissue is represented in figure 27: We verify that the muscle presents major absorbance at lower wavelengths.

23. Figure 31: Absorbance evolution during the treatment for some particular wavelengths. 23 Figure 28: Absorbance spectral evolution in the first two minutes. Figure 29: Absorbance evolution in the first minute of treatment for some particular wavelengths. Figure 30: Absorbance spectral evolution in the first 15 minutes. From these figures we verify a decreasing behavior for absorbance. Such behavior suggests also a decrease in the absorption coefficient of the muscle.

24. 3.1.6 Diffuse reflectance Figure 32: Diffuse reflectance spectrum from natural muscle. 24 The last results for this study are for diffuse reflectance. They were calculated by using total and specular reflectances in equation 6. For natural tissue, we can see the diffuse reflectance spectrum in figure 32:

25. 25 Figure 33: Diffuse reflectance spectral evolution in the first two minutes. Figure 34: Diffuse reflectance evolution in the first minute of treatment for some particular wavelengths. Figure 35: Diffuse reflectance spectral evolution in the first 15 minutes. Figure 36: Diffuse reflectance evolution during the treatment for some particular wavelengths. Agent insertion into the muscle and muscle fiber bundle re-arrangement produce na initial boost in diffuse reflectance which tends to a long-time decrease.

26. Figure 37: Total transmittance spectrum from natural muscle. 26 In this second study, we will present the results obtained with equations 1, 2, 3 and 4 from measurements performed from muscle under treatment with a glucose 40% solution. We will also present the absorbance and diffuse reflectance by calculations with equations 5 and 6. The sequence of figures in each case is the same as in the previous case. 3.2 Experimental results – Glucose 40% 3.2.1 Total transmittance Figure 7 shows the natural spectrum of the muscle sample used in the present study. This spectrum is very similar to the one obtained in the study with ethylene glycol. Now, transmittance is a little higher for longer wavelengths. The differences observed between the two natural samples are imposed by the physiology (which varies between samples, even from the same animal, but from different areas of muscle block).

27. Figure 41: Total transmittance evolution during the treatment for some particular wavelengths. 27 Figure 38: Total transmittance spectral evolution in the first two minutes. Figure 39: Total transmittance evolution in the first minute of treatment for some particular wavelengths. Figure 40: Total transmittance spectral evolution in the first 15 minutes. The increase in total transmittance verified in the case of glucose 40% for the first 2 minutes is much smaller than the one verified in the case of ethylene glycol. The differences observed between the two studies are justified by agent concentration in solution and also the different magnitudes of the correspondent diffusion coefficients (probably greater for ethylene glycol in muscle, as we have already observed in our previous studies [1] ).

28. 28 3.2.2 Collimated transmittance Figure 42: Collimated transmittance spectrum from natural muscle. The first result from the collimated transmittance measurements is the spectrum for natural muscle sample, which is presented in figure 42: Once more, the similarity between this spectrum and the one obtained from the sample used with ethylene glycol (figure 12) is high. In this case, collimated transmittance is a little higher for lower wavelengths and smaller for longer wavelengths.

29. Figure 46: Collimated transmittance evolution during the treatment for some particular wavelengths. 29 Figure 43: Collimated transmittance spectral evolution in the first two minutes. Figure 44: Collimated transmittance evolution in the first minute of treatment for some particular wavelengths. Figure 45: Collimated transmittance spectral evolution in the first 15 minutes. We can see that the magnitude of the variations in the first 2 minutes is similar between studies (compare figures 13 and 14 with figure 43 and 44). Considering the long-time representations, the treatment with ethylene glycol produces higher magnitude variations (see also figures 15 and 16). Comparing figures 16 and 46 we see that the major increase occurs within the first 10 minutes for ethylene glycol, while in the case of glucose 40% it occurs within the first minute.

30. 3.2.3 Total reflectance Figure 47: Total reflectance spectrum from natural muscle. 30 The total reflectance spectrum was obtained from measurement on the natural sample before the solution was added. Figure 47 shows this spectrum: Once more, no significant differences are observed in the spectral form registered from the two samples used in both studies. In the present case, total reflectance is smaller for longer wavelengths that in the case of the study with ethylene glycol.

31. Figure 51: Total reflectance evolution during the treatment for some particular wavelengths. 31 Figure 48: Total reflectance spectral evolution in the first two minutes. Figure 49: Total reflectance evolution during the treatment for some particular wavelengths. Figure 50: Total reflectance spectral evolution in the first 15 minutes. Once more, similar variations are observed between the two studies in the first two minutes (in form and magnitude). Again, total reflectance shows some oscillations during the first minute. This is certainly caused by the positioning of agent in-between the muscle fiber bundles of the muscle, forcing them to oscillate from their central position during the process.

32. 32 3.2.4 Specular reflectance Figure 52: Specular reflectance spectrum from natural muscle. The specular reflectance spectrum of the natural sample is also very similar to the one obtained in the study with ethylene glycol. Comparing between figures 22 and 52, we see the same spectral form. One of the two differences is a less intense absorption band at 410 nm in this case. The other difference is that in the present case we see lower level specular reflectance at longer wavelengths.

33. 33 Figure 53: Specular reflectance spectral evolution in the first two minutes. Figure 54: Specular reflectance evolution in the first minute of treatment for some particular wavelengths. Figure 55: Specular reflectance spectral evolution in the first 15 minutes. Figure 56: Specular reflectance evolution during the treatment for some particular wavelengths. In figure 53 we see an oscillation in the first seconds of treatment. Specular reflectance lowers from natural state until 10 seconds and then it rises smooth until 20 seconds. After that it tends to a constant value. Specular reflectance lowers in different stages as we can see from figure 55. This fact indicates that the diffusion of agent into the muscle sample is also in stages due to the small diffusion power of glucose. The oscillations seen in this case do not exist in the case of ethylene glycol since its concentration in solution is very high.

34. 34 3.2.5 Absorbance Figure 57: Absorbance spectrum from natural muscle. Like in the previous cases, the absorbance spectrum is very similar between the two studies. Using total transmittance and total reflectance measurements, we have calculated absorbance through equation 5. The absorbance spectrum for natural muscle is represented in figure 57: For longer wavelengths we verify a lower absorption than in the case presented in the previous study.

35. 35 Figure 58: Absorbance spectral evolution in the first two minutes. Figure 59: Absorbance evolution in the first minute of treatment for some particular wavelengths. Figure 60: Absorbance spectral evolution in the first 15 minutes. Figure 61: Absorbance evolution during the treatment for some particular wavelengths. By comparing between the two studies, we see that ethylene glycol reduces absorbance significantly, while glucose produces modest reduction (compare figures 28, 29, 30 and 31 with 58, 59, 60 and 61).

36. 36 Figure 62: Diffuse reflectance spectrum from natural muscle. 3.2.6 Diffuse reflectance Finally, we present the results for diffuse reflectance. Once more these spectra were calculated with equation 6 and from the measurements of total reflectance and specular reflectance. Figure 62 shows the spectrum for natural muscle: Again the diffuse reflectance obtained in the present case is very similar in form and magnitude to the one obtained from the sample used in the previous study (compare with figure 32). The absorption band at lower wavelengths is not so evident in the present case. Higher level of diffuse reflectance for longer wavelengths.

37. 37 Figure 63: Diffuse reflectance spectral evolution in the first two minutes. Figure 64: Diffuse reflectance evolution in the first minute of treatment for some particular wavelengths. Figure 65: Diffuse reflectance spectral evolution in the first 15 minutes. Figure 66: Diffuse reflectance evolution during the treatment for some particular wavelengths. In opposition to the case of ethylene glycol (figure 33) witch presents a strong rise in the first 5 seconds and then remains constant, glucose shows a rise in the first 10 seconds, then lowers a little before entering the saturation regime (figure 63). The existence of oscillations in the time dependence verified in figures 64 and 66 are caused by the oscillations measured in total reflectance (see figures 49 and 51).

38. 4. Conclusions We will use these results later to estimate the variations of the optical properties of muscle under treatment with these solutions. From the increase in collimated transmittance and decrease in specular reflectance that we have observed in both cases we can estimate that the scattering coefficient will decrease with the applied treatments. Also as a consequence of these variations, the g factor will probably increase to improve directionality of the incident beam, which was observed by the increase in collimated transmittance. On the other hand, the decrease in sample's absorbance that we have verified in both cases indicates that the absorption coefficient has also decreased during the treatment with these solutions. 38 We have measured and calculated the optical response variations of muscle during the treatments with ethylene glycol and glucose in aqueous solution. By analyzing and comparing between measurements in the two cases, we verify that ethylene glycol creates a higher magnitude optical clearing effect than the solution of glucose 40%. An interesting case was observed in the measurements of specular reflectance for the treatment of glucose 40%, where some initial oscillations were detected. These oscillations indicate that this agent presents a small diffusion coefficient in muscular tissue. An additional explanation for this fact might be the lower concentration of glucose (40%) in solution when compared to the concentration of ethylene glycol used (99%). When determining the variations of the optical properties of the muscle for these optical clearing effects we will be able to confirm these assumptions and will complement the results here presented.

39. Acknowledgements The authors would like to thank the following institutions for all the help in preparation of the samples and resources made available to perform the measurements:  CIETI – Centro de Inovação em Engenharia e Tecnologia Industrial, ISEP – Instituto Superior de Engenharia do Porto, Portugal.  LAIMM – Laboratório de Apoio à Investigação em Medicina Molecular, Departamento de Biologia Experimental, Faculdade de Medicina da Universidade do Porto, Portugal. 39 This work was also supported in part by grants: 224014 PHOTONICS4LIFE of FP7-ICT-2007-2, 1.4.09 of RF Ministry of Education and Science; RF Governmental contracts 02.740.11.0770, 02.740.11.0879, and 11.519.11.2035; FiDiPro, TEKES Program (40111/11), Finland; SCOPES EC, Uzb/Switz/RF, Swiss NSF, IZ74ZO_137423/1; RF President’s grant “Scientific Schools”, 1177.2012.2.

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