1.

Introduction

Recently, terahertz (THz) technology has attracted interest as an effective analysis method to characterize the molecular dynamics of biomaterials. This is because the majority of the conformational modes of water and biomolecules are contained in this frequency region.^1–7 The high sensitivity of THz waves allows diagnostic imaging of abnormal tissues, such as malignant tumors and burns, using differences in the concentrations of interstitial water or body fluid components in the tissues.^6–8 Studies of blood, tissue, and lymph fluids, as well as digestive juices, in the THz frequency range, therefore, have the potential to enhance the sensitivity of diagnostic images and expand the range of analyzable diseases.

Blood contains qualitative and quantitative information about essential substances and waste products in the body, and characterizing blood can facilitate early diagnosis of several diseases, including cancer.^9–12 For example, changes in the concentration of red blood cells (RBCs), which deliver oxygen and dioxide, can indicate the presence of anemia, blood cancer, or dengue fever. Various chemical, physical, and optical techniques have been exploited for blood analysis. Molecular information regarding, e.g., protein and nucleic acids in blood is acquired by molecular profiling methods, such as the western blot and the polymerase chain reaction.¹³ Flow cytometry has also been employed to count and sort cells. However, these invasive and time-consuming methods require blood sampling.¹³ Optical techniques, e.g., involving light-emitting diodes, have been used as noncontact, nondestructive analysis methods.¹⁴ However, they are limited to analysis of blood in vessels just below the skin owing to the high scattering rate of visible light and the low penetration depth of mid-infrared light in tissues.

THz waves have a scattering rate lower than that of infrared and visible light and hence may be used as a feasible probe for analyzing blood inside vessels.¹⁵ However, there are few reports of optical techniques utilizing the THz frequency range for blood characterization. In this study, we measured the optical constants of blood and its components using THz time-domain spectroscopy (TDS) and determined the correlation between the RBC concentration and the THz absorbance. These correlations are also present in a cylindrical tube containing blood with various RBC concentrations using THz imaging.

2.

Materials and Methods

3.

Results and Discussion

The measured transmitted THz time-domain waveforms of blood and its ingredients are presented in Fig. 1(a). The THz waveforms of water with 0.1 μL heparin per 1 mL and saline containing 0.9% NaCl, both of which were used to dilute the samples, were almost identical to pure water. The amplitude of the pulse signal passing through the RBCs was the highest, followed by those of blood, plasma, and water, in descending order. Identical results were observed in the frequency domain waveforms obtained by the fast Fourier transform, as shown in Fig. 1(b).

Fig. 1

Terahertz (THz) waveforms and complex optical constants of water, plasma, blood, and red blood cells (RBCs). (a) Time-domain waveforms and (b) frequency-domain waveforms from the same data.

The refractive index and absorption coefficients of the samples were obtained using the differences in amplitude and phase signals between the frequency domain signals with and without a sample, as shown in Fig. 2.³ These constants decreased in the order of water, plasma, blood, and RBCs. The absorption coefficients of water, plasma, blood, and RBCs at 1 THz were 237.4, 231.1, 210.8, and $186.0{\mathrm{cm}}^{-1}$, respectively. In other words, the absorbance of plasma, blood, and RBCs was 2.8%, 11.2%, and 21.6% lower than that of water, respectively. The differences could be explained by the different proportions of the water and biomaterials except water. The plasma, with an absorption coefficient close to that of water, was mainly water with only 8% of its composition being made up of various proteins, fats, sugars, inorganic salt, urea, amino acids, and uric acid.¹³ The RBCs had the smallest absorption coefficients, indicating that the water content of the RBCs was much lower than that of the plasma.

Fig. 2

THz complex optical constants of water, plasma, blood, and RBCs. (a) Refractive index and (b) absorption coefficients. The graphs in the insets show magnifications of the signal from 0.8 to 1.2 THz. The line and error bar represent the mean value and standard deviation of the optical constants of three blood samples, each obtained from a different rat.

The absorption coefficient of the blood, which was composed mainly of RBCs and plasma, was in between those of plasma and RBCs as shown by the red line in Fig. 2(b). This implies that the absorbance of blood can be represented by a simple linear equation

Table 1

Comparison of volume fraction of red blood cells (RBCs).

To reveal the correlation between the hematocrit and the absorption coefficients in the THz region and THz sensitivity in the concentration of RBCs, we diluted the RBCs to various concentrations and measured the absorption coefficients of the samples. The RBCs were diluted in saline (0.9% NaCl) from 100% to 6.25% using the half-dilution method, which diluted one of two divided samples to a half concentration repeatedly. As shown in Fig. 3, as the concentration of RBCs decreased, the THz absorption coefficients increased. To show the quantitative relationship between the RBC concentration and the absorption coefficients, we extracted the absorption coefficients at 0.5, 1.0, and 1.5 THz as shown in Fig. 3(b). The relationship between the absorption coefficient and the RBC concentration was fitted well by linear equations ($R2\approx 0.99$), indicating almost perfect linearity with their sensitivity of $\sim 3\%$ RBC concentration. Thus, the hematocrit test can be performed by employing THz technology.

Fig. 3

Quantification of the RBC concentration in saline. (a) The absorption coefficients with varying RBC concentrations diluted in saline in the THz region. The graphs in the inset show a magnification of the signal from 0.8 to 1.2 THz. The lines and error bars represent the mean values and standard deviations of the optical constants of the three blood samples ($N=3$). (b) The linear relation between the RBC concentration and the absorption coefficient at 0.5, 1.0, and 1.5 THz. The lines are linear fits at each frequency.

To demonstrate that the THz-hematocrit test can be realized clinically, the relation between the hematocrit and THz optical constants was verified in the blood. RBC-related diseases are generally suspected clinically when the RBC concentration is $>70\%$ or $<30\%$.^13,14 Therefore, blood samples with various RBC concentrations were prepared by adding or eliminating RBCs with various concentrations and separated from the centrifuged blood. As the RBC concentration decreases, the THz absorption increases as shown in Fig. 4. The absorption coefficients at 0.5, 1.0, and 1.5 THz decrease linearly with increasing RBC concentration. The values fitted well with linear functions ($R^2=1.00$, 0.98, and 0.97, respectively), similar to the results in saline. The result confirms the feasibility of the hematocrit test in blood using THz waves.

Fig. 4

Quantification of the RBC concentration in blood. (a) The absorption coefficients of various RBC concentrations. The added and eliminated volume rate are represented by the + and − symbols, ranging from $+20$ to $-20$. The graphs in the insets show magnifications of the signal from 0.8 to 1.2 THz. The lines and error bars represent the mean values and standard deviations of the optical constants of the three blood samples ($N=3$). (b) The linear relationship between the relative RBC concentration change and the absorption coefficient at 0.5, 1.0, and 1.5 THz. The lines are linear fits at each frequency.

Finally, we show that quantitative blood analysis imaging can be realized by THz imaging, using a cylindrical tube. Polyurethane resin tubes were positioned on a z-cut quartz window, as shown in Fig. 5(a). The tubes containing water and blood samples with RBC concentrations of 25%, 45%, and 65% were prepared. The time-domain signals were focused and reflected at different layers of the tube yielding the data presented in Fig. 5(b). The first (negative) peak of the pulse represents signals reflected at the interface between the quartz and the tube. The third (positive) peak (in the dashed box) indicates that the pulse was reflected at the interface between the tube and the samples in the tube. Thus, these third peaks include the optical properties of the samples and result in the image in Fig. 5(c).⁵ As the RBC concentrations increase, the color scale of the images shifts from red to blue; these results are similar to the ones implemented above. To quantify the linearity of the images, we chose values at the 5 points indicated by ($x$) marks and extracted the means and standard deviations of these. These mean values show an excellent linear correlation of 0.9982 with the RBC concentrations, as shown in Fig. 5(d). This linear correlation confirms that quantitative blood analysis is feasible using THz spectroscopic imaging.

Fig. 5

THz images of the blood samples with various RBC concentrations in the cylindrical tube. (a) The THz imaging experiment scheme and the THz beam optical pathway on the sample. (b) Time-domain pulses reflected at the interface between the tube and the various samples. The graph in the inset shows expanded magnification of the signals between 4.5 and 5.7 ps. (c) Blood images of the blood vessel at the maximum points in the inset figure in (b). (d) The average values of contrast at the ($x$) marks in (c). The red solid line is a linear fit.

4.

Conclusions

We have demonstrated that THz-TDS can be exploited as a quantitative blood analysis technique. The optical properties of blood and its ingredients, i.e., water, plasma, and RBCs, were characterized in the THz frequency range using a THz-TDS. The ratios of the optical constants of these components are identical to their volume fractions in nature. An excellent linear relationship between the THz absorbance and the RBC concentration was found in saline as well as in whole blood. Finally, we confirmed the feasibility of quantitative imaging of whole blood containing various concentrations of RBCs using the THz time-domain spectroscopic imaging system. This work has shown that THz imaging is potentially feasible for diagnosing blood-related diseases. In future work, we will extend our study to the noninvasive characterization of flowing blood.¹⁶