Many proposed clinical applications of photoacoustic imaging (PAI) rely on relative or absolute measurements of blood oxygen saturation (sO2), and evaluation of oximetry measurement accuracy is crucial for assessing device performance. Available bench test methods use phantoms connected to blood flow circuits with tunable oxygenation, but these methods are complex, costly, and pose biohazard safety risks. To address these issues, we have developed stable and tunable blood-mimicking solutions using binary mixtures of commercially available near-infrared organic dyes (NIR746A and IRA980) to enable non-biological phantom-based PAI oximetry test methods. We used spectrophotometry and a custom PA spectroscopy system to characterize dye extinction and PA response at 750 nm and 850 nm, then formulated various dye recipes mimicking sO2 levels from 40 % to 100 %. We then used a custom PAI system to image breast-mimicking polyacrylamide hydrogel phantoms with embedded tubes injected with static volumes of either dye solutions or bovine blood deoxygenated using sodium dithionite. Phantom testing with dyes produced similar performance metrics to blood, with root-mean-squared difference (RMSD) values between photoacoustic sO2 and reference sO2 of 6-17 % for blood and 4-18 % for dyes, sensitivity (slope of the regression line) ranged from 0.4 to 0.7 for blood and 0.4-0.9 for dyes, and depth-averaged bias ranged from 4 % to 17 % for blood and 3-10 % for dyes. These blood-mimicking dyes may offer a simpler, cheaper, safer, and more stable approach to evaluate PAI oximetry accuracy compared to traditional blood flow phantoms. This tool could facilitate establishment of less burdensome and more reproducible phantom-based PAI test methods, ultimately expediting clinical adoption of PAI technology.

In this study, we propose a quantitative technique to analyze and evaluate microstructures of skin hair follicles based on Mueller Matrix transmission microscopy. We measure the Mueller matrix polar decomposition (MMPD) parameter images to reveal the characteristic linear birefringence distribution induced by hair follicles in mouse skin tissue samples. The results indicate that the Mueller matrix-derived parameters can be used to reveal the location and structural integrity of hair follicles. For accurate hair follicle location identification and quantitative structural evaluations, we use the image segmentation method, sliding window algorithm, and image texture analysis methods together to process the Mueller matrix-derived images. It is demonstrated that the hair follicle regions can be more accurately recognized, and their locations can be precisely identified based on the Mueller matrix-derived texture parameters. Moreover, comparisons between manual size measurement and polarimetric calculation results confirm that the Mueller matrix parameters have good performance for follicle size estimation. The results shown in this study suggest that the technique based on Mueller matrix microscopy can realize automatically hair follicle identification, detection, and quantitative evaluation. It has great potential in skin structure-related studies and clinical dermatological applications.

In this paper, we propose a smartphone-based biosensor for detecting human total hemoglobin concentration in vivo with high accuracy. Compared to the existing biosensors used to measure hemoglobin concentration, the smartphone-based sensor utilizes the camera, memory, and computing power of the phone. Thus, the cost is largely reduced. Compared to existing smartphone-based sensors, we developed a highly integrated multi-wavelength LED module and a specially designed phone fixture to reduce spatial errors and motion artifacts, respectively. In addition, we embedded a new algorithm into our smartphone-based sensor to improve the measurement accuracy; an L*a*b* color space transformation and the "a" parameter were used to perform the final quantification. We collected 24 blood samples from normal and anemic populations. The adjusted R2 of the prediction results obtained from the multiple linear regression method reached 0.880, and the RMSE reached 9.04, which met the accuracy requirements of non-invasive detection of hemoglobin concentration.

Photoacoustic imaging (PAI), combining the advantages of optical excitation and of acoustic detection, has been widely used for both structural and functional imaging with scalable resolution and penetration in biological tissues. However, due to the strong light scattering in tissue, the maximum penetration depth of PAI, especially microscopy, is limited. Meanwhile, strong light scattering also introduces strong spatial resolution degradation along the depth direction in optical resolution PAI. As an effective method to reduce optical scattering, the optical clearing technique has been widely used in optical imaging modalities. In this chapter, the status of applying optical clearing in PAI is summarized. Examples of both optical resolution and acoustic resolution PAI are illustrated in detail. Future directions in the use of optical clearing in PAI are also discussed in this chapter.

Since the industrial production is becoming more and more large-scale, producing a large number of signal data. Which make it difficult for traditional methods to effectively and accurately extract and analyze the signals. In this paper, a novel Term and phase invariant neural network (TPINN) based on one-dimensional convolution neural network combined and timing analysis is proposed, which can solve the problem effectively. In the first stage, the improved one-dimensional big kernel convolution network is used to extract the phase invariant features of the original signal. In the second stage, the collected features are analyzed in time domain combined with timing analysis layer. This model can extract the phase invariant features when facing the random initial sampling point, and then combine the timing analysis to get the final classification result. Compared with the traditional methods, this method can accurately extract the signal features without deliberately selecting the sampling points, improve the efficiency, and analyze the characteristics of the signal in the time domain, improve the final classification accuracy.

When a clinically suspicious pigmented lesion is biopsied and histologic examination shows melanoma, tumor depth, or Breslow’s depth (BD), is the key parameter that both determines surgical margins for definitive excision and serves as an indication to perform sentinel lymph node biopsy (Smith and MacNeil, 2011Smith L. MacNeil S. State of the art in non-invasive imaging of cutaneous melanoma.Skin Res Technol. 2011; 17: 257Crossref PubMed Scopus (76) Google Scholar). Optimally, BD is measured during an initial excisional biopsy that includes the entire lesion. However, in many cases an incisional biopsy is performed that takes a sample of only a portion of the tumor, possibly resulting in an inaccurate measurement (Guitera and Menzies, 2011Guitera P. Menzies S.W. State of the art of diagnostic technology for early-stage melanoma.Expert Rev Anticanc. 2011; 11: 715Crossref PubMed Scopus (20) Google Scholar, Sellheyer et al., 2010Sellheyer K. Nelson P. Bergfeld W.F. Inadequate biopsy technique and specimen size: an alarming tread that compromises patient care and an appeal to our clinical colleagues.Arch Dermatol. 2010; 146: 1180-1181Crossref PubMed Scopus (10) Google Scholar). Wide local excision based on a potentially inaccurate BD may be insufficient to adequately treat the tumor, necessitating a second definitive surgery. Thus, a precise in vivo measurement of tumor depth would facilitate appropriate surgical treatment at the time of wide local excision when only a partial biopsy sample has been obtained. In this work, we propose applying photoacoustic tomography (PAT) to address this problem. Compared with traditional optical imaging methods, PAT uses ultrasonic waves, which give approximately 1,000 times less scattering than optical waves, breaking through the optical diffusion limit (∼1 mm in the skin) for penetration (Wang and Hu, 2012Wang L.V. Hu S. Photoacoustic tomography: in vivo imaging from organelles to organs.Science. 2012; 335: 1458-1462Crossref PubMed Scopus (3187) Google Scholar, Wang and Gao, 2014Wang L.V. Gao L. Photoacoustic microscopy and computed tomography: from bench to bedside.Annu Rev Biomed Eng. 2014; 16: 155-185Crossref PubMed Scopus (184) Google Scholar). In addition, by using optical excitation of acoustic waves due to light absorption, PAT provides improved contrast of melanin, which is deficient in ultrasonographic imaging. In previous mouse models, we measured a melanoma greater than 7 mm in depth (Zhou et al., 2015Zhou Y. Li G. Zhu L. Li C. Cornelius L.A. Wang L.V. Handheld photoacoustic probe to detect both melanoma depth and volume at high speed in vivo.J Biophotonics. 2015; 8: 961-967Crossref PubMed Scopus (49) Google Scholar). Here we extend our work to patients with melanoma, with the aim of determining the accuracy of PAT-measured melanoma depth compared with the actual BD based on excisional biopsy results. In addition, we also compare our PAT measurement with the histologic measurement obtained from partial incisional biopsy to ultimately determine the predictability and application of PAT measurements in a clinical setting. We applied a linear array-based handheld photoacoustic probe (Figure 1) (LZ250, Visualsonics Inc., Toronto, Ontario, Canada) for melanoma imaging in patients (Hai et al., 2015Hai P. Zhou Y. Liang J. Li C. Wang L.V. Photoacoustic tomography of vascular compliance in humans.J Biomed Opt. 2015; 20: 126008Crossref PubMed Scopus (19) Google Scholar, Needles et al., 2013Needles A. Heinmiller A. Sun J. Theodoropoulos C. Bates D. Hirson D. Yin M. Foster F.S. Development and initial application of a fully integrated photoacoustic micro-ultrasound system.IEEE Trans Ultrason Ferroelectr Freq Control. 2013; 60: 888-897Crossref PubMed Scopus (152) Google Scholar). The transducer array has 256 elements with a size of 23 mm × 3 mm. Each element in the array is cylindrically focused at 15 mm distance. With a central frequency of 21 MHz and a 70% one-way bandwidth, the probe provides spatial resolutions of 1,237 μm, 119 μm, and 86 μm in the elevational, lateral, and axial directions, respectively (Zhou et al., 2015Zhou Y. Li G. Zhu L. Li C. Cornelius L.A. Wang L.V. Handheld photoacoustic probe to detect both melanoma depth and volume at high speed in vivo.J Biophotonics. 2015; 8: 961-967Crossref PubMed Scopus (49) Google Scholar). For the highest melanoma detection sensitivity, pulse-light at 680 nm illuminates the target, where blood has low absorption (Yao and Wang, 2014Yao J. Wang L.V. Sensitivity of photoacoustic microscopy.Photoacoustics. 2014; 2: 87-101Crossref PubMed Scopus (252) Google Scholar). The optical fluence on the skin surface is approximately 10 mJ/cm2, which is less than the safety limit set by the American National Standards Institute (20 mJ/cm2) at this wavelength. Our study was approved by the Institutional Review Board of Washington University in St. Louis (IRB ID#: 201410125). Ten patients were recruited, and 13 lesions were imaged. Benefits and risks of the study were explained to the patients in detail, and written consent was obtained. We initially performed PAT imaging of the melanomas using our handheld probe followed by an excisional biopsy to determine the BD of each melanoma. Of the 13 lesions that were imaged, six presented after an initial incisional biopsy, providing a provisional BD (pBD) before PAT imaging (See Supplementary Table S1 and S2 online). After imaging and histologic examination, we determined that one lesion was composed entirely of melanophages with no tumor cells, and another amelanotic tumor extended to a depth (>10 mm) that was beyond the maximum imaging capacity of the PAT probe, although the most superficial portion of the tumor could be detected. A third patient who presented with widely metastatic disease received systemic therapy at an outside institution before wide local excision of his primary lesion. Thus, we excluded the lesions from these three patients from our final data analysis. PAT images from a cutaneous melanoma metastasis on the left lower extremity (Figure 2a) and a primary cutaneous acral lentiginous melanoma (Figure 2d) clearly show the ability of PAT to detect dermal and epidermal melanin-containing components of the tumors, allowing for direct in vivo measurement of melanoma depth. (PAT measurement was adjusted by a factor of 0.88 to account for ex vivo tissue fixation and processing [Winsor, 1994Winsor L. Tissue processing.in: Woods A. Ellis R. Laboratory histopathology. Churchill Livingstone, New York1994: 1-39Google Scholar], and this is indicated as corrected [cPA] depth.) Immediately after imaging, an excisional biopsy was performed and actual BD was determined (Figure 2c and f). As shown in Figure 2a–c, the cutaneous melanoma metastasis PAT depth in vivo (cPA) is concordant with the actual BD. Similarly, the primary tumor cPA measurment more closely represented the acutal BD than the pBD obtained from a partial biopsy (Figure 2d–f). The depth of all of the tumors that were successfully imaged ranged between 0.2 mm and 6.0 mm (Figure 2g). cPA depths are concordant with actual BD obtained after excisional biopsy (mean absolute error (MAE) = 0.18 mm; R2 = 0.97). When comparing cPA obtained by PAT with the actual BD in the five patients with primary melanomas who had incisional biopsy before PAT, PAT achieved accurate measurements (MAE = 0.14 mm). Comparing cPA measurement with the incisional biopsy pBD is less concordant, as might be expected (MAE = 0.21 mm). Thus, in our pilot study, PAT was accurate in determining BD in melanoma and was an effective imaging tool that accurately determined melanoma depth to potentially guide definitive surgical intervention. In this pilot study with a limited number of patients, PAT did not alter but rather confirmed current surgical treatment based on pBD, particularly for standard pigmented melanomas compared with amelanotic melanomas. PAT provided an accurate measurement of melanoma depth as determined by BD measurement of the residual tumor on excisional biopsy. We acknowledge that melanomas with a depth of 10 mm or greater were beyond the detection limit of our current system because of strong light attenuation resulting in weak signal. Although we have addressed this technical problem in previous systems (Zhou et al., 2014Zhou Y. Xing W. Maslov K.I. Cornelius L.A. Wang L.V. Handheld photoacoustic microscopy to detect melanoma depth in vivo.Opt Lett. 2014; 39: 4731-4734Crossref PubMed Scopus (92) Google Scholar), this technology was not incorporated into our non-handheld prototype because of the technical design of the probe. In this current handheld system, we conclude that PAT technology is noninvasive, provides high contrast for melanoma imaging with detection capabilities, and adapts easily to bedside monitoring, providing information that may potentially facilitate early and effective surgical intervention. The authors state no conflict of interest. LVW has a financial interest in Microphotoacoustics, Inc., which, however, did not support this work. The authors would like to thank James Ballard for manuscript editing. This work was sponsored in part by National Institutes of Health grants DP1 EB016986 (NIH Director’s Pioneer Award), R01 CA186567 (NIH Director’s Transformative Research Award), and S10 RR026922. Download .pdf (.17 MB) Help with pdf files Supplementary Material and Supplementary Tables S1 and S2

While lasers have been commonly used as illumination sources in photoacoustic (PA) imaging, their high purchase and maintenance costs, as well as their bulkiness, have hindered the rapid clinical dissemination of PA imaging. With this in mind, we explore an alternative illumination source for PA tomography—a xenon flash lamp with high pulse energy and a microsecond pulse width. We demonstrate that, by using a single xenon flash lamp, we can image both a black latex cord placed in chicken breast tissue at a depth of up to 3.5 cm ex vivo and an entire mouse body in vivo. Our findings indicate that the xenon flash lamp, producing optical illumination that is safe for humans, can be potentially applied to human tissue imaging.

Elastography can noninvasively map the elasticity distribution of biological tissue, which is often altered in pathological states. In this work, we report quantitative photoacoustic elastography (QPAE), capable of measuring Young's modulus of human tissue in vivo. By combining photoacoustic elastography with a stress sensor having known stress-strain behavior, QPAE can simultaneously measure strain and stress, from which Young's modulus is calculated. We first applied QPAE to quantify the Young's modulus of tissue-mimicking agar phantoms with different concentrations. The measured values fitted well with both the empirical expectations based on the agar concentrations and those measured in independent standard compression tests. We then demonstrated the feasibility of QPAE by measuring the Young's modulus of human skeletal muscle in vivo. The data showed a linear relationship between muscle stiffness and loading. The results proved that QPAE can noninvasively quantify the absolute elasticity of biological tissue, thus enabling longitudinal imaging of tissue elasticity. QPAE can be exploited for both preclinical biomechanics studies and clinical applications.

We report photoacoustic microscopy (PAM) of arteriovenous (AV) shunts in early stage tumors in vivo, and develop a pattern recognition framework for computerized tumor detection. Here, using a high-resolution photoacoustic microscope, we implement a new blood oxygenation (sO(2))-based disease marker induced by the AV shunt effect in tumor angiogenesis. We discovered a striking biological phenomenon: There can be two dramatically different sO(2) values in bloodstreams flowing side-by-side in a single vessel. By tracing abnormal sO(2) values in the blood vessels, we can identify a tumor region at an early stage. To further automate tumor detection based on our findings, we adopt widely used pattern recognition methods and develop an efficient computerized classification framework. The test result shows over 80% averaged detection accuracy with false positive contributing 18.52% of error test samples on a 50 PAM image dataset.

Circulating tumor cell (CTC) clusters arise from multicellular grouping in the primary tumor and elevate the metastatic potential by 23 to 50 fold compared to single CTCs. High throughout detection and quantification of CTC clusters is critical for understanding the tumor metastasis process and improving cancer therapy. In this work, we report a lineararray- based photoacoustic tomography (LA-PAT) system capable of label-free high-throughput CTC cluster detection and quantification in vivo. LA-PAT detects CTC clusters and quantifies the number of cells in them based on the contrast-to-noise ratios (CNRs) of photoacoustic signals. The feasibility of LA-PAT was first demonstrated by imaging CTC clusters ex vivo. LA-PAT detected CTC clusters in the blood-filled microtubes and computed the number of cells in the clusters. The size distribution of the CTC clusters measured by LA-PAT agreed well with that obtained by optical microscopy. We demonstrated the ability of LA-PAT to detect and quantify CTC clusters in vivo by imaging injected CTC clusters in rat tail veins. LA-PAT detected CTC clusters immediately after injection as well as when they were circulating in the rat bloodstreams. Similarly, the numbers of cells in the clusters were computed based on the CNRs of the photoacoustic signals. The data showed that larger CTC clusters disappear faster than the smaller ones. The results prove the potential of LA-PAT as a promising tool for both preclinical tumor metastasis studies and clinical cancer therapy evaluation.

Quantification of vascular elasticity can help detect thrombosis and prevent life-threatening conditions such as acute myocardial infarction or stroke. Here, we propose vascular elastic photoacoustic tomography (VE-PAT) to measure vascular elasticity in humans. VE-PAT was developed by incorporating a linear-array-based photoacoustic computed tomography system with a customized compression stage. By measuring the deformation of blood vessels under uniaxial loading, VE-PAT was able to quantify the vascular compliance. We first demonstrated the feasibility of VE-PAT in blood vessel phantoms. In large vessel phantoms, VE-PAT detected a decrease in vascular compliance due to simulated thrombosis, which was validated by a standard compression test. In small blood vessel phantoms embedded 3 mm deep in gelatin, VE-PAT detected elasticity changes at depths that are difficult to image using other elasticity imaging techniques. We then applied VE-PAT to assess vascular compliance in a human subject and detected a decrease in vascular compliance when an occlusion occurred downstream from the measurement point, demonstrating the potential of VE-PAT in clinical applications such as detection of deep venous thrombosis.

We report quantitative photoacoustic elastography (QPAE) capable of measuring Young’s modulus of biological tissue in vivo in humans. By combining conventional PAE with a stress sensor having known stress–strain behavior, QPAE can simultaneously measure strain and stress, from which Young’s modulus is calculated. We first demonstrate the feasibility of QPAE in agar phantoms with different concentrations. The measured Young’s modulus values fit well with both the empirical expectation based on the agar concentrations and those measured in an independent standard compression test. Next, QPAE was applied to quantify the Young’s modulus of skeletal muscle in vivo in humans, showing a linear relationship between muscle stiffness and loading. The results demonstrated the capability of QPAE to assess the absolute elasticity of biological tissue noninvasively in vivo in humans, indicating its potential for tissue biomechanics studies and clinical applications.

Measuring blood flow speed in the optical diffusive regime in humans has been a long standing challenge for photoacoustic tomography. In this work, we proposed a cuffing‐based method to quantify blood flow speed in humans with a handheld photoacoustic probe. By cuffing and releasing the blood vessel, we can measure the blood flow speed downstream. In phantom experiments, we demonstrated that the minimum and maximum measurable flow speeds were 0.035 mm/s and 42 mm/s, respectively. In human experiments, flow speeds were measured in three different blood vessels: a radial artery in the right forearm, a radial artery in the index finger of the right hand, and a radial vein in the right forearm. Taking advantage of the handheld probe, our method can potentially be used to monitor blood flow speed in the clinic and at the bedside.

With high sensitivity to melanin and high resolution at depths, photoacoustic tomography (PAT) has been studied for the diagnosis of primary and metastatic melanoma for more than a decade. In this chapter, we summarize the most recent advances in this field, including measuring primary tumor thicknesses and rates of growth, locating sentinel lymph nodes (SLNs), label-free screening for SLNs, and detecting circulating melanoma cells. We also discuss the potential applications of PAT in the diagnosis of primary and metastatic melanoma, such as noninvasive primary tumor examination, label-free histology, and metastatic tumor detection. Results from current studies suggest PAT is a promising tool for facilitating the diagnosis of both primary and metastatic melanoma in clinics.

Optical-resolution photoacoustic microscopy (OR-PAM) can achieve submicron lateral resolution by tightly focusing the excitation light, while the axial resolution is still limited by the frequency bandwidth of the ultrasonic transducer. The Grueneisen relaxation effect, in which the Grueneisen parameter changes within the thermal relaxation time following a laser impulse heating, can provide excellent axial resolution due to its optical sectioning property. Based on this effect, Grueneisen relaxation photoacoustic microscopy (GR-PAM) was developed and demonstrated ex vivo. Here, we present for the first time in vivo imaging of mouse brains with improved axial resolution based on GR-PAM. An intensity-modulated continuous-wave (CW) 532 nm laser thermally heated the in-focus absorber. Another 532 nm pulsed laser, which is aligned confocally with the CW laser, generated the photoacoustic (PA) signal from the absorber. The difference between the amplitudes of the photoacoustic signals with and without heating was used for image reconstruction. The achieved axial resolution is ~12.5 µm, which is fivefold better than the acoustically determined value for a 20 MHz-bandwidth ultrasound transducer. The system was demonstrated by imaging a blood-filled tube ex vivo and blood vessels of mouse brains in vivo. The blood-filled tube diameter obtained from the PA image by GR-PAM is 105 µm, which is much closer to its actual diameter (100 µm) than the value from conventional OR-PAM (160 µm). This axial resolution improvement was further validated in imaging mouse brains in vivo, and yielded significantly narrower axial profiles of the vessels. This in vivo demonstration of imaging by GR-PAM might inspire more applications in PA biomedical imaging and sensing.

Using a handheld photoacoustic probe, we proposed a cuffing-based method to quantify blood flow speed in humans By cuffing and releasing the blood vessel, we can measure the blood flow speed downstream. In phantom experiments, we demonstrated that the minimum and maximum measurable flow speeds were 0.035 mm/s and 42 mm/s, respectively. In human experiments, flow speeds were measured in three different blood vessels: a radial artery in the right forearm, a radial artery in the index finger of the right hand, and a radial vein in the right forearm.

Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT’s basic principles, major implementations, imaging contrasts, and recent applications.

Microwave-based thermoacoustic tomography (TAT), based on the measurement of ultrasonic waves induced by microwave pulses, can reveal tissue dielectric properties that may be closely related to the physiological and pathological status of the tissues. Using microwaves as the excitation source improved imaging depth because of their deep penetration into biological tissues. We demonstrate, for the first time, in vivo microwave-based thermoacoustic imaging in rats. The transducer is rotated around the rat in a full circle, providing a full two-dimensional view. Instead of a flat ultrasonic transducer, we used a virtual line detector based on a cylindrically focused transducer. A 3 GHz microwave source with 0.6 µs pulse width and an electromagnetically shielded transducer with 2.25 MHz central frequency provided clear cross-sectional images of the rat's body. The high imaging contrast, based on the tissue's rate of absorption, and the ultrasonically defined spatial resolution combine to reveal the spine, kidney, muscle, and other deeply seated anatomical features in the rat's abdominal cavity. This non-invasive and non-ionizing imaging modality achieved an imaging depth beyond 6 cm in the rat's tissue. Cancer diagnosis based on information about tissue properties from microwave band TAT can potentially be more accurate than has previously been achievable.

We applied a linear-array-based photoacoustic probe to detect the tumor depth and volume of melanin-containing melanoma in nude mice in vivo. We demonstrated the ability of this linear-array-based system to measure both the depth and volume of melanoma through phantom, ex vivo, and in vivo experiments. The volume detection ability also enables us to accurately calculate the rate of growth of the tumor, which is important in quantifying tumor activity. Our results show that this system can be used for clinical melanoma diagnosis and treatment at the bedside.