Chromatic-aberration-free multispectral optical-resolution photoacoustic microscopy using reflective optics and a supercontinuum light source.

A supercontinuum (SC) light source enables multispectral photoacoustic imaging at excitation wavelengths in the visible-to-near-infrared range. However, for such a broad optical wavelength range, chromatic aberration is non-negligible. We developed a multispectral optical-resolution photoacoustic microscopy (MS-OR-PAM) setup with a nanosecond pulsed SC light source and a reflective objective lens to avoid chromatic aberration. Chromatic aberrations generated by reflective and conventional objective lenses were compared, and the images acquired using the reflective objective were not affected by chromatic aberration. Hence, MS-OR-PAM with the reflective objective was used to distinguish red blood cells from melanoma cells via spectral subtraction processing.

Measurement of blood-oxygen saturation using a photoacoustic technique in the rabbit hypoxemia model.

The golden standard method to obtain accurate blood oxygen saturation is blood gas analysis that needs invasive procedure of blood sampling. Photoacoustic technique enables us to measure real-time blood oxygen saturation without invasive procedure. The aim of this study is to use the photoacoustic technique, an optical method, for accurately determining oxygen saturation in vivo. We measured induced photoacoustic signals of arterial blood in the rabbit model of stable hypoxemia after irradiation at 750 and 800 nm. Oxygen saturation was calculated from the photoacoustic signals using two calibration curves. Calibration curve 1 is a conventional curve derived from the absorbance coefficient of hemoglobin, whereas calibration curve 2 is derived from the photoacoustic signals obtained from the original blood vessel model. Simultaneously, blood-gas analysis was performed to obtain the reference standard of oxygen saturation. Regression analysis and Bland-Altman analysis were performed to assess the accuracy of oxygen saturation obtained using the two methods. The oxygen saturation calculated using calibration curves 1 and 2 showed strong correlations with the reference standard in regression analysis (R = 0.965, 0.964, respectively). The Bland-Altman analysis revealed better agreement and precision with calibration curve 2, whereas there was significant underestimation of values obtained using calibration curve 1. Photoacoustic measurement of oxygen saturation using calibration curve 2 provided an accurate estimate of oxygen saturation, which was similar to that obtained using a portable blood-gas analyzer.

Spectral-differential-based unmixing for multispectral photoacoustic imaging.

We propose the use of a spectral differential method (SDM) to emphasize the spectral peaks of multispectral photoacoustic images. Because contrast agent signals have spectral peaks at the contrast agent absorption peak, the SDM can selectively emphasize contrast agent signals. Unlike the conventional spectral fitting method (SFM), the SDM does not require reference background spectra and, consequently, does not suffer from separation error caused by the deviation of reference spectra from the measured spectra. We performed multispectral photoacoustic imaging of tissue-mimicking phantoms and subcutaneous tumors of mice injected with small organic molecule-based contrast agents. Contrast agent images obtained by the SDM were clearer than those obtained by SFM.

Appropriate timing of blood sampling for blood gas analysis in the ventilated rabbit.

BACKGROUND: Arterial and venous blood gas analyses (BGAs) are essential to evaluate devices that measure biological oxygenation. The appropriate timing of blood sampling for BGA after respiratory rate (RR) change in animal experiments has not been reported. This study investigated the appropriate timing of blood sampling for BGA in ventilated rabbits and whether venous samples are an alternative to arterial samples. MATERIALS AND METHODS: Under general anesthesia, 14 rabbits (body weight, 3.02 ± 0.09 kg) were ventilated and their RR was changed (40/min, 30/min, and 20/min). Blood was sampled through cervical arterial and venous catheters. Experiment 1: in seven rabbits, arterial BGA was measured at 0, 0.5, 1, 2, 3, 5, 10, 15, and 20 min after the RR change. Experiment 2: in seven different rabbits, simultaneous arterial and venous BGA were measured at 0, 2, 5, 10, 15, and 20 min after the RR change. RESULTS: Oxygen partial pressure (PO2) and saturation (SO2) of the arterial blood stabilized 0.5 min after the RR changed. In venous BGA, no index stabilized during observation. The arterial and venous values of the carbon dioxide partial pressure (PCO2) and pH had significant correlations (arterial PCO2 = 0.9316 × venous PCO2-4.4425 [r = 0.9178]; arterial pH = 1.0835 × venous pH-0.5795 [r = 0.9453]). CONCLUSIONS: In ventilated rabbits, arterial PO2 and SO2 stabilized in 0.5 min. No venous value stabilized after the RR change. Only the PCO2 and pH of venous samples may be an alternative to arterial samples under the defined formula.

Thermoelastic wave generation and its longitudinal wave propagation measurement by a microscopic optical interferometer.

Laser ultrasonics is a noncontact measurement method that uses a laser-induced elastic wave source in combination with an optical surface displacement-tracking system. This study compared the performances of two optical interferometers with different characteristics when applied to measurement of pulsed thermoelastic waves. The surface displacement-tracking system was designed to measure the center of the microscopic view. A pulsed laser beam irradiated a black ink layer to generate the thermoelastic waves. The out-of-plane displacement on the axially opposite side was then measured using either a Michelson interferometer or a Sagnac interferometer. The objective lens of the system was of a type commonly used in biological observations. The Michelson interferometer estimated a maximum displacement of 0.43 nm and a maximum sound pressure of 24.7 kPa. The signal-to-noise ratios from 16 averages were 14.9 dB (Michelson interferometer) and 19.2 dB (Sagnac interferometer). Furthermore, this paper compares the performance of the numerically estimated Sagnac interferometer outputs calculated from the measured Michelson interferometer outputs with the experimentally obtained Sagnac interferometer outputs. The numerically estimated Sagnac interferometer's output was shown to be identical to the experimentally acquired output. The Michelson interferometer requires a higher average operating frequency (i.e., it needs a longer data acquisition time), although this interferometer does offer superior displacement output linearity. This property enables calculation of the sound pressure from the displacement amplitude. These findings indicated that combination of the measurement points of the Sagnac interferometer with those of the sparsely distributed Michelson interferometer reduced the measurement time when compared with a single use of the Michelson interferometer while also maintaining the data acquisition quality.

Real-time fetal monitoring using photoacoustic measurement of placental oxygen saturation in a rabbit hypoxia model.

INTRODUCTION: Ensuring adequate fetal oxygenation is an essential aim of fetal monitoring. The purpose of this study was to establish a basic technique for real-time measurement of blood oxygen saturation of the placenta by photoacoustic (PA) technique as a new fetal monitoring method. METHODS: The hypoxia model established in our previous study was applied to 7 pregnant rabbits. Three phases were induced: normal phase, hypoxia phase, and recovery phase. Three methods were simultaneously used for real-time fetal monitoring: fetal heat rate (FHR) monitoring, oxygen saturation (SO2) measurement by near-infrared spectroscopy (SNO2), and placenta SO2 measured by PA technique (SplO2). The maternal hypoxia was assessed by skin SO2 measured by PA technique (SsO2), and arterial blood SO2 by blood gas analysis (SaO2). RESULTS: The average of SplO2 in normal phase was 52.6 ± 13.9 %. The averages of SNO2, SSO2, and SplO2 in the seven rabbits changed in parallel from the normal phase to hypoxia phase. In the recovery phase, the SplO2 rose in parallel with recovery of SaO2. There was lag in increase of the FHR compared to the change in the other values. In the detailed analysis of PA signals from the labyrinth and decidua, a unique change in oxygen saturation was seen in one case. DISCUSSION: Results of this study showed that sensitivity of our novel PA technique in detecting tissue hypoxia was similar to near-infrared spectroscopy (NIRS). As an advantage, unlike NIRS, monitoring with PA technique was unaffected by ischemia and surface changes in oxygen saturation because of its higher spatial resolution. We conclude that PA technique provides more accurate information about fetal blood placenta than NIRS. Ultrasound imaging, combined with oxygen saturation monitoring by PA technique, would improve fetal monitoring and fetal diagnosis in the future.

Quantification of effective attenuation coefficients using continuous wavelet transform of photoacoustic signals.

A method for quantifying the effective attenuation coefficients of optical absorbers by using the continuous wavelet transform (CWT) to calculate the time-resolved frequency spectra of photoacoustic signals is proposed. Because the coefficients can be quantified according to the relative intensity of the frequency content of the signals, it is unnecessary to determine the fluences. A computational simulation reveals that the time-resolved frequency spectra exhibit better correlation with the coefficients than do power spectra calculated using a Fourier transformation. The CWT-based method was experimentally verified, and the coefficients were quantified with mean square error of 2.0 cm(-1).

3D analysis of intracortical microvasculature during chronic hypoxia in mouse brains.

The purpose of this study is to determine when and where the brain microvasculature changes its network in response to chronic hypoxia. To identify the hypoxia-induced structural adaptation, we longitudinally imaged cortical microvasculature at the same location within a mouse somatosensory cortex with two-photon microscopy repeatedly for up to 1 month during continuous exposure to hypoxia (either 8 or 10% oxygen conditions). The two-photon microscopy approach made it possible to track a 3D pathway from a cortical surface arteriole to a venule up to a depth of 0.8 mm from the cortical surface. The network pathway was then divided into individual vessel segments at the branches, and their diameters and lengths were measured. We observed 3-11 vessel segments between the penetrating arteriole and the emerging vein over the depths of 20-460 µm within the 3D reconstructed image (0.46 × 0.46 × 0.80 mm(3)). The average length of the individual capillaries (<7 µm in diameter) was 67 ± 46 µm, which was not influenced by hypoxia. In contrast, 1.4 ± 0.3 and 1.2 ± 0.2 fold increases of the capillary diameter were observed 1 week after exposure to 8 % and 10% hypoxia, respectively. At 3 weeks from the exposure, the capillary diameter reached 8.5 ± 1.9 and 6.7 ± 1.8 µm in 8% and 10 % hypoxic conditions, respectively, which accounted for the 1.8 ± 0.5 and 1.4 ± 0.3 fold increases relative to those of the prehypoxic condition. The vasodilation of penetrating arterioles (1.4 ± 0.2 and 1.2 ± 0.2 fold increases) and emerging veins (1.3 ± 0.2 and 1.3 ± 0.2 fold increases) showed relatively small diameter changes compared with the parenchymal capillaries. These findings indicate that parenchymal capillaries are the major site responding to the oxygen environment during chronic hypoxia.

Regulation of miRNA expression by low-level laser therapy (LLLT) and photodynamic therapy (PDT).

Applications of laser therapy, including low-level laser therapy (LLLT), phototherapy and photodynamic therapy (PDT), have been proven to be beneficial and relatively less invasive therapeutic modalities for numerous diseases and disease conditions. Using specific types of laser irradiation, specific cellular activities can be induced. Because multiple cellular signaling cascades are simultaneously activated in cells exposed to lasers, understanding the molecular responses within cells will aid in the development of laser therapies. In order to understand in detail the molecular mechanisms of LLLT and PDT-related responses, it will be useful to characterize the specific expression of miRNAs and proteins. Such analyses will provide an important source for new applications of laser therapy, as well as for the development of individualized treatments. Although several miRNAs should be up- or down-regulated upon stimulation by LLLT, phototherapy and PDT, very few published studies address the effect of laser therapy on miRNA expression. In this review, we focus on LLLT, phototherapy and PDT as representative laser therapies and discuss the effects of these therapies on miRNA expression.

Spectroscopic photoacoustic microscopic imaging during single spatial scan using broadband excitation light pulses with wavelength-dependent time delay.

In most multispectral optical-resolution photoacoustic microscopy (OR-PAM), spatial scanning is repeated for each excitation wavelength, which decreases throughput and causes motion artifacts during spectral processing. This study proposes a new spectroscopic OR-PAM technique to acquire information on the photoacoustic signal intensity and excitation wavelength from single spatial scans. The technique involves irradiating an imaging target with two broadband optical pulses with and without wavelength-dependent time delays. The excitation wavelength of the sample is then calculated by measuring the time delay between the photoacoustic signals generated by the two optical pulses. This technique is validated by measuring the excitation wavelengths of dyes in tubes. Furthermore, we demonstrate the three-dimensional spectroscopic OR-PAM of cells stained with suitable dyes. Although the tradeoff between excitation efficiency and excitation bandwidth must be adjusted based on the application, combining the proposed technique with fast spatial scanning methods can significantly contribute to recent OR-PAM applications, such as monitoring quick biological events and microscale tracking of moving materials.

Photoacoustic imaging of fresh human surgically and endoscopically resected gastrointestinal specimens.

Objective: Photoacoustic (PA) imaging is a novel noninvasive technique that offers high-contrast tomographic imaging with ultrasound-like resolution at depths of centimeters, enabling visualization of deep small vessels. The aim of this pilot study was to survey the characteristics of deep vessel networks in the mucosa of neoplastic gastrointestinal (GI) lesions using PA imaging. Methods: Specimens of patients who had undergone surgical and endoscopic resection for GI lesions were included in this study. The PA/ultrasound imaging system for clinical research is characterized by a technology that can superimpose a PA image over an ultrasound image. Three-dimensional PA images were acquired for the resected specimen before fixation. The stomach and colon of live pigs were incised, and the walls were scanned from the mucosa. Results: A total of 32 specimens (nine esophageal, 12 gastric, 11 colorectal) were scanned. The pathological diagnoses were adenomas (n = 2), intramucosal cancers (n = 14), and invasive cancers (n = 16). The deep vessel networks of all lesions could be visualized. In the intramucosal lesions, the deep vessel network was similar to that of a normal tissue. In invasive cancers, the thick and prominent vessel network was visible in the surface layer of esophageal cancers, infiltrated area of gastric cancers, and surface layer and infiltrated area of colorectal cancers. In the images of living pigs, visualizing the vascular network deeper than the submucosa in both the stomach and large intestine was possible. Conclusion: Our study confirmed that the deep vessel networks of neoplastic GI lesions were visible by PA imaging.

Reconstruction of fluorescence/bioluminescence sources in biological medium with spatial filter.

We propose a new method for reconstruction of emitting source distributions by use of a spatial filter and a successive updating process of the forward model for fluorescence/bioluminescence diffuse optical tomography. The spatial filter transforms a set of the measurement data to a single source strength at a position of interest, and the forward model is updated by use of the estimated source strengths. This updating process ignores the dispensable source positions from reconstruction according to the reconstructed source distribution, and the spatial resolution of the reconstructed image is improved. The estimated sources are also used for the reduction of artifacts induced by noises based on the singular value decomposition. Some numerical experiments show the advantages of the proposed method by comparing the present results with those obtained by the conventional methods of the least squares method nd Algebraic Reconstruction Technique. Finally the criteria for practical use of the method are quantitatively presented by the simulations for 2D and 3D geometries.

Total light approach of time-domain fluorescence diffuse optical tomography.

In this study, time-domain fluorescence diffuse optical tomography in biological tissue is numerically investigated using a total light approach. Total light is a summation of excitation light and zero-lifetime emission light divided by quantum yield. The zero-lifetime emission light is an emitted fluorescence light calculated by assuming that the fluorescence lifetime is zero. The zero-lifetime emission light is calculated by deconvolving the actually measured emission light with a lifetime function, an exponential function for fluorescence decay. The object for numerical simulation is a 2-D 10 mm-radius circle with the optical properties simulating biological tissues for near infrared light, and contains regions with fluorophore. The inverse problem of fluorescence diffuse optical tomography is solved using time-resolved simulated measurement data of the excitation and total lights for reconstructing the bsorption coefficient and fluorophore concentration simultaneously. The mean time of flight is used as the featured data-type extracted from the time-resolved data. The reconstructed images of fluorophore concentration show good quantitativeness and spatial reproducibility. By use of the total light approach, computation is performed much faster than the conventional ones.

Low Reactive Level Laser Therapy for Mesenchymal Stromal Cells Therapies.

Low reactive level laser therapy (LLLT) is mainly focused on the activation of intracellular or extracellular chromophore and the initiation of cellular signaling by using low power lasers. Over the past forty years, it was realized that the laser therapy had the potential to improve wound healing and reduce pain and inflammation. In recent years, the term LLLT has become widely recognized in the field of regenerative medicine. In this review, we will describe the mechanisms of action of LLLT at a cellular level and introduce the application to mesenchymal stem cells and mesenchymal stromal cells (MSCs) therapies. Finally, our recent research results that LLLT enhanced the MSCs differentiation to osteoblast will also be described.

Image reconstruction of the absorption coefficients with l 1-norm minimization from photoacoustic measurements.

BACKGROUND: The photoacoustic (PA) imaging by considering light propagation into image reconstruction process can provide quantitative information of photon absorbers, such as hemoglobin and exogenous dyes, and to improve their imaging contrasts. METHODS: A 2D image reconstruction of the distribution of the light absorption coefficient from the PA measurements with light source and ultrasound transducer placed at the identical position was tested. The PA pressures were formulated with the PA wave equation and the photon diffusion equation. The relation between the PA pressure and the absorption coefficient was linearized. The image reconstruction was carried out by minimizing the squared error between the measured and calculated PA signals. The l 1-norm of the reconstructed image was simultaneously minimized to improve the localization of the reconstructed target in the image. The image reconstruction with the l 1-norm minimization was compared to that with the Tikhonov regularization by numerical simulation and phantom experiment. In phantom experiment, an aqueous solution of the intralipid and the indocyanine green was used as the measured object. The PA probe had optical fiber for illumination and piezoelectric film for detection placed at the identical position. RESULTS: The l 1-norm minimization reconstructed more localized target than the Tikhonov regularization. CONCLUSIONS: The l 1-norm minimization is useful for the sparse PA image reconstruction.

Reconstruction of localized fluorescent target from multi-view continuous-wave surface images of small animal with lp sparsity regularization.

Fluorescence diffuse optical tomography using a multi-view continuous-wave and non-contact measurement system and an algorithm incorporating the lp (0 < p ≤ 1) sparsity regularization reconstructs a localized fluorescent target in a small animal. The measurement system provides a total of 25 fluorescence surface 2D-images of an object, which are acquired by a CCD camera from five different angles of view with excitation from five different angles. Fluorescence surface emissions from five different angles of view are simultaneously imaged on the CCD sensor, thus leading to fast acquisition of the 25 images within three minutes. The distributions of the fluorophore are reconstructed by solving the inverse problem based on the photon diffusion equations. In the reconstruction process incorporating the lp sparsity regularization, the regularization term is reformulated as a differentiable function for gradient-based non-linear optimization. Numerical simulations and phantom experiments show that the use of the lp sparsity regularization improves the localization of the target and quantitativeness of the fluorophore concentration. A mouse experiment demonstrates that a localized fluorescent target in a mouse is successfully reconstructed.

Blue laser irradiation generates intracellular reactive oxygen species in various types of cells.

UNLABELLED: Abstract Objective: The purpose of this study was to measure intracellular reactive oxygen species (ROS) production after laser irradiation in various types of cells. BACKGROUND DATA: ROS are considered to be the key secondary messengers produced by low-level laser therapy (LLLT). Although various mechanisms for the effects of LLLT have been proposed, and intracellular ROS were indicated as the one of the key factors, direct measurement of intracellular ROS of several types of cells after different wavelength lasers irradiation has not been reported. MATERIALS AND METHODS: Various types of cells were used in this study: mouse preadipocytes (3T3-L1), prechondrocytes (ATDC5), myoblasts (C2C12), mesenchymal stromal cells (KUSA-A1), lung cancer cells (LLC), insulinoma cells (MIN6), fibroblasts (NIH-3T3), human cervix adenocarcinoma cells (HeLa), macrophages differentiated from lymphocytes (THP-1) after treatment with phorbol ester, and rat basophilic leukemia cells (RBL-2H3). Cells were irradiated with a blue laser (wavelength: 405 nm), a red laser (wavelength: 664 nm) or a near infrared laser (wavelength: 808 nm) at 100 mW/cm(2) for 60 or 120 sec. Intracellular ROS levels were measured by fluorometric assay using the intracellular ROS probe, CM-H2DCFDA in a flow cytometer. RESULTS: After a blue laser irradiation, intracellular ROS levels were increased in all types of cells. In contrast, intracellular ROS generation was not observed after irradiation with a red laser or near-infrared laser. CONCLUSIONS: Potential sources of intracellular ROS were excited by blue laser irradiation, resulting in ROS production within cells. Although the low-level intracellular ROS should be generated after a red or a near-infrared laser irradiation, the only high level intracellular ROS were detected by the ROS probe used in this study. As ROS are considered to be key secondary messengers, the specific functional regulation of cells by laser irradiation will be studied in a future study.

Responses of cancer cells induced by photodynamic therapy.

Photodynamic therapy (PDT) involves the administration of a photosensitizer, followed by local irradiation of tumor tissues using a laser of an appropriate wavelength to activate the photosensitizer. Since multiple cellular signaling cascades are concomitantly activated in cancer cells exposed to the photodynamic effect, understanding the responses of cancer cells to PDT will aid in the development of new interventions. This review describes the possible cell-death signaling pathways initiated by PDT. In addition, we describe our latest findings regarding the induction of expression of miRNAs specific to apoptosis in cancer cells and the induction of antitumor immunity following PDT against cancer cells. A more detailed understanding of the molecular mechanisms related to PDT will potentially improve long-term survival of PDT treated patients.

Phantom and mouse experiments of time-domain fluorescence tomography using total light approach.

Phantom and mouse experiments of time-domain fluorescence tomography were conducted to demonstrate the total light approach which was previously proposed by authors. The total light approach reduces the computation time to solve the forward model for light propagation. Time-resolved temporal profiles were acquired for cylindrical phantoms having single or double targets containing indocyanine green (ICG) solutions. The reconstructed images of ICG concentration reflected the true distributions of ICG concentration with a spatial resolution of about 10 mm. In vivo experiments were conducted using a mouse in which an ICG capsule was embedded beneath the skin in the abdomen. The reconstructed image of the ICG concentration again reflected the true distribution of ICG although artifacts due to autofluorescence appeared in the vicinity of the skin. The effectiveness of the total light approach was demonstrated by the phantom and mouse experiments.

Improvement of image quality of time-domain diffuse optical tomography with l sparsity regularization.

An l(p) (0 < p ≤ 1) sparsity regularization is applied to time-domain diffuse optical tomography with a gradient-based nonlinear optimization scheme to improve the spatial resolution and the robustness to noise. The expression of the l(p) sparsity regularization is reformulated as a differentiable function of a parameter to avoid the difficulty in calculating its gradient in the optimization process. The regularization parameter is selected by the L-curve method. Numerical experiments show that the l(p) sparsity regularization improves the spatial resolution and recovers the difference in the absorption coefficients between two targets, although a target with a small absorption coefficient may disappear due to the strong effect of the l(p) sparsity regularization when the value of p is too small. The l(p) sparsity regularization with small p values strongly localizes the target, and the reconstructed region of the target becomes smaller as the value of p decreases. A phantom experiment validates the numerical simulations.