1. Introduction

Terahertz (THz) electromagnetic waves are highly sensitive to water molecules, and hence, they are being investigated for use in emerging medical imaging techniques for distinguishing minute differences in interstitial water contents between normal and abnormal tissues [1–9]. However, owing to this high sensitivity, which is attributed to the high absorbance of water in the THz frequency region, THz waves have a limited penetration depth in fresh tissue, up to a few hundred micrometers [10,11]. Thus far, only a few methods to enhance the penetration depth of THz waves have been reported. These methods decrease the effect of water in tissues by replacing water with paraffin or by freezing the water [12–14]. However, these methods requiring a complicated and time-consuming pre-process and do not apply to in vivo conditions. The noise reduction algorithms can be used to improve the penetration depth and image contrast of THz waves in vivo but this methods is not the perfect solution to overcome the obstacles of nature due to water [15,16]. These issues can be solved by using biocompatible chemical agents that have been reported to enhance the imaging depth and the resolution of infrared, near-infrared and visible ranges [17–19]. The biocompatible chemical agents absorbed into the tissues affect the optical properties of the extracellular fluid in the tissue, thus reducing the absorbance and the mismatch in the refractive index between cells. Thus, a THz penetration-enhancing agent (THz-PEA) must be a biocompatible material that is easily absorbed into tissues and that has an absorption coefficient lower than that of water in the THz frequency range. In this study, we used glycerol as the THz-PEA. The absorption coefficient of glycerol at 1 THz is three times smaller than that of water. Glycerol which has generally been used as the major ingredient of lotions and creams for personal care and medicine, can be used safely in the human body. As the THz-PEA applied to a tissue is absorbed, the volume fraction of the interstitial water contents in the tissue decreases and, finally, the THz waves penetrate deeper into the tissue. In this paper, we demonstrate the enhancement in the penetration depth of THz waves in fresh tissues by using glycerol as the THz-PEA. We also examine the permeation characteristics of the THz-PEA into the tissue by using THz time-of-flight imaging (B-scan) [20]. Moreover, using an artificial tumor model, we show that the THz-PEA technique facilitates the diagnostic imaging of a tumor below the skin.

2. Experimental method

A reflection-mode THz time-domain imaging system with a mode-locked Ti:sapphire laser with 80-fs pulses at a central wavelength of 800 nm was used in our experiments. The laser pulses were divided into pump and probe THz pulses, and were focused on the generation and detection photoconductive dipole antennas, respectively. A Ti/Au dipole antenna fabricated on a semi-insulated GaAs wafer was used as the generator. A similar dipole antenna with an emitter on a low-temperature-grown GaAs detector was used as the detector. We used four metal parabolic mirrors to focus the THz pulses on a sample and guide the pulses to the detector. The incident and reflection angles of the focused beams were 32° [5,8]. THz time-domain waveforms were measured by sampling cross-correlated signals between the THz transient current and the THz pulse at the detector via a linear optical delay. A fast scanner, operating at a frequency of 20 Hz and amplitude of 37 ps, was used for fast acquisition of THz pulses. The sample was located at the focal point of the THz beam. The THz image was obtained by using a two-dimensional sample scanning system, and the acquisition time per image was approximately 10 min for a 40 × 20 mm area with a 0.25-mm scanning resolution. To avoid interference caused by water vapor, the whole reflection system, except the samples, was sealed in a dry, air-tight box.

Fresh tissues were extracted surgically from the abdomen of 5- to 8-week-old male BALB/c-nude mice after euthanasia. The glycerol was spread on one of the tissue samples as shown in Fig. 1. After applying glycerol, the fresh tissues with and without glycerol were placed on a z-cut quartz window that had a diameter of 3 inches and a thickness of 3 mm. Pure petroleum jelly was spread across the surface of the sample side of the quartz window to remove the void between the surface of the sample window and the tissues. All fresh tissues on the sample stage were wrapped in a plastic wrap to avoid drying during the sample scan. All animal experiments were conducted with the approval of the institutional animal care and use committee, Yonsei University Health System.

 

Fig. 1 Experimental scheme.

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3. Results and discussion

Typical biomaterials such as ethanol, petroleum jelly, and glycerol were evaluated as THz-PEA candidates using the transmission mode THz time-domain spectroscopy. The absorption coefficients of ethanol, pure petroleum jelly, and glycerol were very small compared to that of water, as shown in Fig. 2. We selected glycerol as the THz-PEA even though the absorption coefficients of pure petroleum jelly and the refractive index of ethanol were very small compared to those of glycerol. Ethanol evaporates easily before it is absorbed into tissues, and petroleum jelly, which is hydrophobic, is not absorbed in tissues.

 

Fig. 2 THz spectrum of water, ethanol, petroleum jelly, and glycerol. (a) shows absorption coefficients and (b) shows the refractive indices.

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To verify the enhancement of the THz wave penetration depth by glycerol used as a THz-PEA, we obtained THz images of a metal target below the tissues with and without glycerol, as shown in Fig. 3. Glycerol was applied on half of the abdomen skin extracted surgically from the mouse, as shown in Fig. 3(a). The thickness of skin was 224 μm. THz images of the tissues were acquired 30 min after applying glycerol, as shown in Figs. 3(b) and 3(c). The experimental scheme is displayed in Fig. 3(d). The effect of glycerol on the tissue was not found on the THz image, which was obtained using the peak-to-peak values of the THz time-domain waveforms, as shown in Fig. 3(b). However, when the peak-to-peak values of the 2nd pulses were used, the THz image differences produced by applying glycerol were displayed, as shown in Fig. 3(c). The THz image of the 2nd pulses was obtained using the peak-to-peak values of the THz time-domain waveforms from 7.5 ps to end. The THz image of the metal target below the skin tissue without glycerol was blurred, whereas the contrast of the THz image of the region of the tissue with glycerol applied was considerably clearer. This result was confirmed by the THz time-domain waveforms at the same point in the tissues with and without glycerol, represented by *1 and *2 in Fig. 3(e). The 1st peak values of the THz reflected pulses on the tissues without and with glycerol were identical. However, when glycerol was applied, the intensity of 2nd peak, which represents the THz pulse passing through the tissue and reflecting on the metal target, was almost doubled compared to that without glycerol. The time difference between the 1st and 2nd peaks decreased. These results indicated that the THz optical properties of tissues, such as absorbance and refractivity, were changed by replacing the interstitial water content in the tissue to a mixture of water and glycerol. The increase in the 2nd peak and the decrease in the time difference imply a reduction in the absorption coefficients and the refractive indices.

 

Fig. 3 Visual and THz images of a knife below a tissue without and a tissue with glycerol. (a) shows the visual image of a knife below the tissues. (b) shows THz image from (a) using peak-to-peak values of the whole range of the THz time-domain waveforms in (e). (c) shows the THz image from (a) using peak-to-peak values of time-domain waveforms from 7.5 ps to end in (e). (d) shows the experimental scheme. (e) shows THz time-domain waveforms of asterisks from (a), with the blue and red line indicating the waveforms in *1 and *2, respectively. The size of THz image was 5 × 3 cm².

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To investigate the sequential changes in the THz optical properties of the tissues, the THz time-domain waveforms were measured while glycerol was penetrating the tissue, as shown in Fig. 4. The glycerol drop was on the tissue that was placed on the quartz window. The reflected THz time-domain waveforms were measured at the center of the tissue. After applying glycerol, the intensity of peak-to-peak THz pulses decreased and the position of the 2nd positive peak was closer to the 1st peak, that is, the pulse width became narrow. These results implied that, as glycerol permeated into the tissue, the index mismatch between the quartz window and the tissue was reduced, and phase difference between the 1st positive peak and the 2nd positive peak decreased. The frequency dependent properties were investigated using the frequency-domain waveforms, which were obtained using fast Fourier transform, as shown in Fig. 4(b). As time passed after applying glycerol, the THz frequency range was cut off in order of frequency. This result can be explained by the frequency-dependent absorption properties of skin and glycerol [11]. The THz absorption coefficients of skin at low frequencies are smaller than those at higher frequencies. As the glycerol was penetrated into the skin, the absorption coefficients decreased to a lower value at low frequencies. Therefore, reflected THz waves barely observed at the low frequency region, because these were easily transmitted out of the tissue. The high frequency region was reflected on the boundary between tissue with and without the penetrated glycerol.

 

Fig. 4 Sequential THz waveforms in the tissue with glycerol. (a) shows time-dependent THz time-domain waveforms in the tissue after applying glycerol. (b) shows the frequency-domain waveforms from (a). The glycerol drop was placed on the tissue that was on the quartz window. The THz waveforms were reflected from the surface between the tissue and the quartz window.

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The sequential THz permeation enhancement and glycerol delivery into the tissues was verified by the THz B-scan images, as shown in Fig. 5. A drop of glycerol was kept on the petroleum jelly wall to prevent an outward diffusion of the glycerol drop during acquisition of the image, as shown in Fig. 5(a). The THz waveforms were reflected from the surface between the tissue and the quartz window, as shown in Fig. 5(b). As time passed, glycerol, which was transmitted into the tissue, was observed on the THz peak-to-peak 2-dimensional images of the opposite surface of the tissue. These results were confirmed by the THz B-scan images of the dotted line in Fig. 5(c), as shown in Fig. 5(d). The 1st bright region corresponded with the 1st positive peak of the reflected THz pulse at the interface between the tissue and the quartz window. As time passed after application of the glycerol drop, this region became narrow and the 2nd bright region, which corresponded with the 2nd positive peak of the THz pulses, appeared on the 1st bright region. Thus, using the THz B-scan imaging, we demonstrated that glycerol enhanced the penetration depth of THz waves.

 

Fig. 5 Time-dependent THz two-dimension and B-scan images of the tissue after application of glycerol. (a) shows the top view photograph of the tissue with glycerol. (b) shows the scheme of the side view of tissue from (a). (c) shows THz images from the surface of the tissues. (d) shows the THz B scan images of the dotted line from (c).

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Finally, we also demonstrated that a THz-PEA could potentially be used to diagnose a tumor below the skin that is barely observed using conventional THz imaging techniques due to the penetration limit of THz in the skin. To implement this experiment, two abdomen skins extracted surgically from a 9-week-old male Sprague-Dawley rat were prepared. Because the skin of the rat was very thick, THz waves barely penetrated through the skin. Glycerol was applied for 30 min on one of the skins. Matrigel drops, which are normally in gel form except at a temperature of 4 °C, were used as the artificial tumor. The matrigel drops were placed on the center of the tissues with and without the application of glycerol, as shown in Fig. 6(a). When using the peak-to-peak values of whole-time domain signals, no differences in the tumor images were found in the THz images with and without glycerol, as shown in Fig. 6(b). However, when the main peak-to-peak range was cut off, the THz image of the artificial tumor was observed only in the tissue with glycerol, whereas the artificial tumor image was barely observable in the region without glycerol, as shown in Fig. 6(c).

 

Fig. 6 THz image of artificial tumors on tissues without and with glycerol. (a) shows a top view visible image of the tissues and the artificial tumor. (b) & (c) shows a THz image of the tissues with the artificial tumor when the peak-to-peak values of the whole-time domain signal was used and when the main peak-to-peak range was cut off, respectively. The size of the THz image was 5 × 3 cm².

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THz B-scan images of Fig. 6 were used to confirm that glycerol caused an enhancement in the THz wave penetration depth, as shown in Fig. 7. The THz B-scan images on the red dotted lines of Fig. 7(a) are displayed in Fig. 7(b). Even though penetrated THz signals are barely found in the B-scan images of the skin without glycerol, the post-pulses, which occurred between skin and matrigel drop, are as shown Fig. 7(b) 4, 5, and air at the edge of the matrigel drop was found in Fig. 7(b) 2. These results demonstrate that THz-PEA can be potentially used to detect tumors and vessels below the skin.

 

Fig. 7 THz B-scan image of artificial tumors on the tissues without and with glycerol. (a) shows a THz image of a tissue with an artificial tumor. (b) shows THz B scan images of the red dotted line from (a). The green arrows indicate the 2nd pulses occurring between the tissue and the artificial tumor, while the red arrow indicates the post-pulses due to air.

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4. Conclusion

We have demonstrated enhancement of the penetration depth of THz waves by using a penetration-enhancing agent (PEA). Glycerol was validated as a THz-PEA candidate. The intensity of the penetrated signal through fresh tissues on application of glycerol was two times larger than the regular THz signal. The reduction of the THz optical constants that occurred by replacing water with glycerol was shown by the sequential time- and frequency-domain waveforms. The THz B-scan images showed the sequential distribution of delivery of glycerol into the tissues. In addition, by using an artificial tumor model we also showed that glycerol could be potentially used as a penetration depth-enhancement agent in the diagnosis of cancer. In conclusion, a THz-PEA can potentially enable us to obtain diagnostic images of a tumor or abnormal lesions below the skin and this expands the feasibility of THz wave applications in clinical fields.