Akio Yasuda

Terahertz imaging applied to cancer diagnosis

We report on terahertz (THz) time-domain spectroscopy imaging of 10 microm thick histological sections. The sections are prepared according to standard pathological procedures and deposited on a quartz window for measurements in reflection geometry. Simultaneous acquisition of visible images enables registration of THz images and thus the use of digital pathology tools to investigate the links between the underlying cellular structure and specific THz information. An analytic model taking into account the polarization of the THz beam, its incidence angle, the beam shift between the reference and sample pulses as well as multiple reflections within the sample is employed to determine the frequency-dependent complex refractive index. Spectral images are produced through segmentation of the extracted refractive index data using clustering methods. Comparisons of visible and THz images demonstrate spectral differences not only between tumor and healthy tissues but also within tumors. Further visualization using principal component analysis suggests different mechanisms as to the origin of image contrast.

Dielectric coagulometry: a new approach to estimate venous thrombosis risk.

We present dielectric coagulometry as a new technique to estimate the risk of venous thrombosis by measuring the permittivity change associated with the blood coagulation process. The method was first tested for a simple system of animal erythrocytes suspended in fibrinogen solution, where the coagulation rate was controlled by changing the amount of thrombin added to the suspension. Second, the method was applied to a more realistic system of human whole blood, and the inherent coagulation process was monitored without artificial acceleration by a coagulation initiator. The time dependence of the permittivity at a frequency around 1 MHz showed a distinct peak at a time that corresponds to the clotting time. Our theoretical modeling revealed that the evolution of heterogeneity and the sedimentation in the system cause the peak of the permittivity.

Point-of-care testing system enabling 30 min detection of influenza genes

We developed a portable and easy-to-use nucleic acid amplification test (NAT) system for use in point-of-care testing (POCT). The system shows sensitivity that is sufficiently higher than that of the currently available rapid diagnostic kit and is comparable to that of real-time reverse transcription polymerase chain reaction (RT-PCR) for influenza testing.

Dielectric inspection of erythrocyte morphology

We performed a systematic study of the sensitivity of dielectric spectroscopy to erythrocyte morphology. Namely, rabbit erythrocytes of four different shapes were prepared by precisely controlling the pH of the suspending medium, and their complex permittivities over the frequency range from 0.1 to 110 MHz were measured and analyzed. Their quantitative analysis shows that the characteristic frequency and the broadening parameter of the dielectric relaxation of interfacial polarization are highly specific to the erythrocyte shape, while they are insensitive to the cell volume fraction. Therefore, these two dielectric parameters can be used to differentiate erythrocytes of different shapes, if dielectric spectroscopy is applied to flow-cytometric inspection of single blood cells. In addition, we revealed the applicability and limitations of the analytical theory of interfacial polarization to explain the experimental permittivities of non-spherical erythrocytes.

Temporal variation of dielectric properties of preserved blood

Rabbit blood was preserved at 277 K in Alsevers solution for 37 days, and its dielectric permittivity was monitored in a frequency range from 0.05 to 110 MHz throughout the period. The relaxation time and Cole-Cole parameter of the interfacial polarization process for erythrocytes remained nearly constant during the first 20 days and then started to increase and decrease, respectively. On the other hand, the relaxation strength and the cell volume fraction continued to decrease for 37 days, but the decrease rates of both changed discontinuously on about the 20th day. Microscope observation showed that approximately 90% of the erythrocytes were spinous echinocytes at the beginning of preservation and started to be transformed into microspherocytes around the 20th day. Therefore, dielectric spectroscopy is a sensitive tool to monitor the deterioration of preserved blood accompanied by morphological transition of erythrocytes through the temporal variation of their dielectric properties.

Dielectric Cytometry with Three-Dimensional Cellular Modeling

We have developed what we believe is an efficient method to determine the electric parameters (the specific membrane capacitance C(m) and the cytoplasm conductivity kappa(i)) of cells from their dielectric dispersion. First, a limited number of dispersion curves are numerically calculated for a three-dimensional cell model by changing C(m) and kappa(i), and their amplitudes Deltaepsilon and relaxation times tau are determined by assuming a Cole-Cole function. Second, regression formulas are obtained from the values of Deltaepsilon and tau and then used for the determination of C(m) and kappa(i) from the experimental Deltaepsilon and tau. This method was applied to the dielectric dispersion measured for rabbit erythrocytes (discocytes and echinocytes) and human erythrocytes (normocytes), and provided reasonable C(m) and kappa(i) of the erythrocytes and excellent agreement between the theoretical and experimental dispersion curves.

Aspheric silicon lenses for terahertz photoconductive antennas

We report on the design and characterization of aspheric focusing silicon lenses for terahertz photoconductive antennas. The lenses are engineered using ray-tracing software and characterized using an optical fiber terahertz time-domain spectroscopy system. We find that using aspheric lenses improves terahertz radiation coupling from the emitter and enables improved collection by the detector. The signal-to-noise ratio and the cutoff frequency of measured terahertz spectra are improved. Minimized aberrations also reduce the focal spot size. Simulations based on Fresnel–Kirchhoff diffraction theory, taking into account the radiation pattern of the emitter and aberrations of the lenses, show good agreement with our measurements.

Contrast improvement of terahertz images of thin histopathologic sections

We present terahertz images of 10 μm thick histopathologic sections obtained in reflection geometry with a time-domain spectrometer, and demonstrate improved contrast for sections measured in paraffin with water. Automated segmentation is applied to the complex refractive index data to generate clustered terahertz images distinguishing cancer from healthy tissues. The degree of classification of pixels is then evaluated using registered visible microscope images. Principal component analysis and propagation simulations are employed to investigate the origin and the gain of image contrast.

The effects of erythrocyte deformability upon hematocrit assessed by the conductance method

A comparative study of centrifugation and conductance methods for the estimation of cell volume fraction (phi) was performed to examine whether the strong forces exerted upon erythrocytes during centrifugation affect their volume, and the results are discussed in terms of erythrocyte deformability. Rabbit erythrocytes of four shapes (spherocytes, echinocytes, stomatocyte-like enlarged erythrocytes and discocytes) were prepared by controlling the pH of the suspending media. The packed cell volumes of the suspensions were measured by standard hematocrit determination methods using centrifugation in capillary tubes. Simultaneously, the same suspensions and their supernatants were used in dielectric spectroscopy measurements, and the low-frequency limits of their conductivities were used for the numerical estimation of phi. The hematocrit values of spherocytes and echinocytes were markedly less than the volume fractions obtained by the conductance method. Namely, the centrifugation reduced the cell volume. For enlarged erythrocytes and discocytes, however, the reduction of cell volume was not observed. These findings showed that phi obtained by the centrifugation method can be greatly affected by the deformability of the cells, but the level of the effect depends on the cell types. Consequently, phi obtained by the centrifugation method should be carefully interpreted.

Detection of the CLOCK/BMAL1 heterodimer using a nucleic acid probe with cycling probe technology

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms.