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We report an adaptive energy-compensated synthetic aperture focusing technique (eC-SAFT) for improving the imaging performance of photoacoustic microscopy (PAM) in terms of depth of field (DOF), spatial resolution (both axial and lateral), and SNR. In addition to coherency and time-delay (in conventional SAFT), our beamforming-based reconstruction algorithm takes into account acoustic energy loss-a primary physical parameter in acoustic wave propagation-following Beer-Lambert's law. Experimental validation studies were performed in tissue-mimicking (Agar) phantoms, complex leaf veins, and chicken breast tissues. Results demonstrate that our proposed eC-SAFT+CF outperforms conventional SAFT+CF to improve axial resolution (up to

Although a delay-and-sum (DAS) beamformer is best suited for real-time photoacoustic (PA) image formation, the reconstructed images are often afflicted by noises, sidelobes, and other intense artifacts due to inaccurate assumptions in PA signal correlation. The present work aims to develop a reconstruction method that reduces the occurrence of sidelobes and artifacts and thus improves the reconstructed image quality or imaging performance. This beamformer is fundamentally based on higher-order signal correlation wherein a higher number of delayed PA signals—compared to conventional delay-multiply-and-sum (DMAS)—are combined and summed up. The proposed technique provides significant improvements in resolution, contrast, and signal-to-noise ratio (SNR) compared to traditional beamformers. For real-time implementation, the proposed algorithms were simplified, and their computational complexities were shrunk to the order of DAS [ O ( N ) ]. A GPU based study was also performed to validate the real-time capability of the proposed beamformers. For validation studies, both numerical simulation and experiments were conducted. Quantitative evaluation studies involving SNR, contrast ratio, generalized contrast-to-noise ratio, and FWHM demonstrate that the proposed higher-order DMAS beamformer is superior in PA image reconstruction. Conclusively, the proposed beamformer uniquely facilitates real-time PA image reconstruction with an achievable frame rate close to DAS and DMAS but with better imaging performance, which holds promise for real-time PA imaging and its clinical applications.

A new adaptive weighting method [delay-and-sum-to-delay-standard-deviation factor (DASDSF)] combined with minimum variance (MV) beamforming is introduced in photoacoustic imaging (PAI). Existing MV-based beamformers improve photoacoustic image quality in terms of achieving narrow main lobes and, thus, improving spatial resolution. But, the beamformers give a strong side-lobe signal strength that greatly degrades the reconstructed image contrast. As a feedback weighting factor, DASDSF addresses the persisting side-lobe issue present in MV-beamformed images, i.e., our proposed method is robust against reduction in noises as well as side lobes, and it outperforms MV and MV combined with coherence factor beamformers. Validation studies—being carried out both in numerical simulation and experiments employing a low-cost (16 elements) linear transducer array in a home-built PAI system—demonstrate an excellent performance of the proposed weighting approach in improving SNR, while reducing main-lobe width (i.e., FWHM) and side-lobe signal strength. The study demonstrates that the proposed algorithm holds promise for development of a cost-effective PAI system using a low-cost linear transducer ( ∼ 16 elements against ∼ 128 generally used).

Processes like single-cell isolation demand an immensely sterile environment and a remarkably magnified plat-form, such as the optical microscope, to analyze the growth of the cell lines and in the precise selection of cells from them. State-of-the-art technologies fail to provide microscopes compatible inside the biosafety cabinets, and therefore the samples are constantly moved from the safety cabinets to the microscope repeatedly for analysis. This, in turn, increases the risk of contamination of the sample as well as the laboratory surroundings. We report the design and development of an automated optical microscope adaptable in the safety cabinet and can be employed for cell isolation processes. Experiments were carried out using the developed imaging system, and results reveal the system’s reliability in cell biology applications.

Being the largest organelle in a cell, the nucleus houses the genetic code of an organism and primarily serves as the cell’s information center. Proper implementation and consolidation of physiological stress are intrinsic for proper growth and development. Studies reveal that in animals, under chronic stress, the nuclear size shows morphological plasticity. This paper proposes the chronic stress study on plant specimens, namely, Allium cepa root, upon induction on Rhodamine B fluorophore, using our in-house developed dual arm multi-level magnification light sheet microscopy (DMx-LSFM) system. Employing our home-built microscope, we can analyze the difference in girth of the nuclear membrane under different magnification levels simultaneously, which helps to inspect the minute details and structural changes occurring in the organelle. The study was then further extended to investigate the role of exposure time and concentration of the stress agent on the sample.

Temperature dependence of photoacoustic (PA) signal is a crucial factor in PA temperature sensing, imaging, and monitoring of thermal therapies. In this study, we report theoretical and simulation studies to describe the temperature dependence of PA-signal and hence the Gruneisen parameter. A light-absorbing organic dye (Methylene blue) is taken as the sample, and the variation of PA-signal against change in temperature is plotted from the simulated data. Experiments were conducted, employing a home-built photoacoustic microscopy imaging system to measure PA signal, for the validation study. A CW-laser beam (of wavelength, 642nm) is used to vary the temperature of the organic dye during the experiment, and the average PA-signal is measured from the acquired PA image. The change in temperature of the sample due to the laser irradiation is noted separately. PA-signal strength and its amplification against temperature change is plotted from the experimental data. It is observed that the experimental results are well-matched with the theory and simulated data.

Use of portable and affordable pulse light sources (light emitting diodes (LED) and laser diodes) for tissue illumination offers an opportunity to accelerate the clinical translation of photoacoustic imaging (PAI) technology. However, imaging depth in this case is limited because of low output (optical) power of these light sources. In this work, we developed a noninvasive technique for enhancing strength (amplitude) of photoacoustic (PA) signal. This is a photothermal-based technique in which a continuous wave (CW) optical beam, in addition to short-pulse ~&nbsp, nsec laser beam, is employed to irradiate and, thus, raise the temperature of sample material selectively over a pre-specified region of interest (we call the process as pre-illumination). The increase in temperature, in turn enhances the PA-signal strength. Experiments were conducted in methylene blue, which is one of the commonly used contrast agents in laboratory research studies, to validate change in temperature and subsequent enhancement of PA-signal strength for the following cases: (1) concentration or optical absorption coefficient of sample, (2) optical power of CW-optical beam, and (3) time duration of pre-illumination. A theoretical hypothesis, being validated by numerical simulation, is presented. To validate the proposed technique for clinical and/or pre-clinical applications (diagnosis and treatments of cancer, pressure ulcers, and minimally invasive procedures including vascular access and fetal surgery), experiments were conducted in tissue-mimicking Agar phantom and ex-vivo animal tissue (chicken breast). Results demonstrate that pre-illumination significantly enhances PA-signal strength (up to ~70% (methylene blue), ~48% (Agar phantom), and ~40% (chicken tissue)). The proposed technique addresses one of the primary challenges in the clinical translation of LED-based PAI systems (more specifically, to obtain a detectable PA-signal from deep-seated tissue targets).

Delay-and-sum (DAS) algorithms are widely used for beamforming in linear array photoacoustic imaging systems and are characterized by fast execution. However, these algorithms suffer from various drawbacks like low resolution, low contrast, high sidelobe artifacts and lack of visual coherence. More recently, adaptive weighting was introduced to improve the reconstruction image quality. Unfortunately, the existing state-of-the-art adaptive beamforming algorithms are computationally expensive and do not consider the specific noise characteristics of the acquired ultrasonic signal. In this article, we present a new adaptive weighting factor named the variational coherence factor (VCF), which takes into account the noise level variations of radio-frequency data. The proposed technique provides superior results in terms of image resolution, sidelobe reduction, signal-to-noise and contrast level improvement. The quantitative results of the numerical simulations and phantom imaging show that the proposed VCF assisted DAS method leads to 55% and 25% improvement in FWHM, 57% and 32% improvement in SNR, respectively, compared to the state-of-the-art DAS-based methods. The results demonstrate that the proposed method can effectively improve the reconstructed image quality and deliver satisfactory imaging performance even with a limited number of sensor elements. The proposed method can potentially reduce the instrumentation cost of the photoacoustic imaging system and contribute toward the clinical translation of the modality., Accepted for publications in IEEE Transactions on Instrumentation and Measurement

We report a study on theoretical aspects of the generation of initial photoacoustic (PA) pressure waves and its propagation in a mechanical medium, with detailed derivations of associated equations, which is the basis of all PA imaging modalities (both microscopy and tomography). We consider the tissue sample for imaging as a hydrostatic (PVT) thermodynamic system. The phenomenon of the generation of initial pressure wave, due to transient (\({\sim }\text {ns}\)) illumination by electromagnetic (EM) waves and subsequent rapid heating, is assumed as a thermodynamic process. For the propagation of PA wave, tissue sample is considered as a bio-mechanical system that supports the propagation of mechanical disturbances from one point to another. The derived equations are in agreement with standard equations that are commonly employed in PA imaging systems, therefore our assumptions of considering the system as a hydrostatic thermodynamic system and PA effect as a thermodynamic process are validated. This chapter will be of great value to the PA imaging research community for an in-depth theoretical understanding of the subject.

We report an optical-based microscopy imaging technology—simultaneous multiple-level magnification selective plane illumination microscopy (sMx-SPIM) imaging system—that addresses a longstanding technological challenge of obtaining images, specifically of biological specimen non-destructively, at different field of views (FOVs) and spatial resolutions (or magnification powers) simultaneously in real-time. This imaging system provides not only 3D images but also time-resolved sequential images with temporal resolution ∼ msec. Magnification powers (or FOVs) of the individual images can be controlled independently that is achieved by housing two separate detection arms, in SPIM imaging system, fitted with objective lenses of different magnification powers. This unique feature holds promises to observe and study: (a) sub-microscopic details and entire structure of biological specimen side-by-side simultaneously and (b) spatio-temporal dynamics of functional activities of biological specimen. For validation study of robustness of the proposed sMx-SPIM imaging system, experiments are conducted in various biological samples including Danio rerio (zebrafish) embryo, Drosophila melanogaster, Allium cepa root, and A549 cell line. Experimental results demonstrate that the study is of significant impacts from two aspects, viz., technological implication and biological applications.

Elastography imaging is a promising tool—in both research and clinical settings—for diagnosis, staging, and therapeutic treatments of various life-threatening diseases (including brain tumors, breast cancers, prostate cancers, and Alzheimer’s disease). Large variation in the physical (elastic) properties of tissue, from normal to diseased stages, enables highly sensitive characterization of pathophysiological states of the diseases. On the other hand, over the last decade or so, photoacoustic (PA) imaging—an imaging modality that combines the advantageous features of two separate imaging modalities, i.e., high spatial resolution and high contrast obtainable, respectively, from ultrasound- and optical-based modalities—has been emerging and widely studied. Recently, recovery of elastic properties of soft biological tissues—in addition to prior reported recovery of vital tissue physiological information (Hb, HbO2, SO, and total Hb), noninvasively and nondestructively, with unprecedented spatial resolution (μm) at penetration depth (cm)—has been reported. Studies demonstrating that combined recovery of mechanical tissue properties and physiological information—by a single (PA) imaging unit—pave a promising platform in clinical diagnosis and therapeutic treatments. We offer a comprehensive review of PA imaging technology, focusing on recent advances in relation to elastography. Our review draws out technological challenges pertaining to PA elastography (PAE) imaging, and viable approaches. Currently, PAE imaging is in the nurture stage of its development, where the technology is limited to qualitative study. The prevailing challenges (specifically, quantitative measurements) may be addressed in a similar way by which ultrasound elastography and optical coherence elastography were accredited for quantitative measurements.

Tissue elasticity is an important biomarker for early prediction of diseases, and elastography methods have been developed for several modalities. In practice, photoacoustic imaging (PAI) provides information about the optical properties of deep-seated tissue based on an ultrasonic readout. In this study, we demonstrate the use of PAI to measure tissue elasticity through high frequency acquisitions. The method was tested using tissue mimicking agar phantoms and transducers of different frequencies. The results show that elastic contrast can be recovered from PA signals only using high-frequency transducers, given significant differences of elasticity between the imaging target and background.

We report our study that shows selection in operating frequency of US-transducer used for boundary detection of PA-signals, which result due to the contrast in elastic property distribution ( E ( r → ) ) in sample material other than that of optical absorption coefficient (μa). Studies were carried out, experimentally, in tissue-mimicking Agar phantoms employing acoustic resolution photoacoustic microscopy (AR-PAM) system as an imaging unit. In the experiments, various transducers having different operating frequencies, ranging from 1MHz to 50MHz, were employed for studying frequency response of the photoacoustic signals. The study shows that, for detecting photoacoustic signals due to the contrast in elastic property, ultrasound transducer with higher operating frequency (∼50MHz) is demanded.

We demonstrate quantitative optical property and elastic property imaging from ultrasound assisted optical tomography data. The measurements, which are modulation depth M and phase phi of the speckle pattern, are shown to be sensitively dependent on these properties of the object in the insonified focal region of the ultrasound (US) transducer. We demonstrate that Young's modulus (E) can be recovered from the resonance observed in M versus omega (the US frequency) plots and optical absorption (mu(a)) and scattering (mu(s)) coefficients from the measured differential phase changes. All experimental observations are verified also using Monte Carlo simulations. (c) 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). DOI: 10.1117/1.JBO.17.10.101507]

We demonstrate the phase fluctuation introduced by oscillation of scattering centers in the focal volume of an ultrasound transducer in an optical tomography experiment has a nonzero mean. The conditions to be met for the above are: (i) the frequency of the ultrasound should be in the vicinity of the most dominant natural frequency of vibration of the ultrasound focal volume, (ii) the corresponding acoustic wavelength should be much larger than [Formula: see text], a modified transport mean-free-path applicable for phase decorrelation and (iii) the focal volume of the ultrasound transducer should not be larger than 4 - 5 times [Formula: see text]. We demonstrate through simulations that as the ratio of the ultrasound focal volume to [Formula: see text] increases, the average of the phase fluctuation decreases and becomes zero when the focal volume becomes greater than around [Formula: see text]; and through simulations and experiments that as the acoustic frequency increases from 100 Hz to 1 MHz, the average phase decreases to zero. Through experiments done in chicken breast we show that the average phase increases from around 110° to 130° when the background medium is changed from water to glycerol, indicating that the average of the phase fluctuation can be used to sense changes in refractive index deep within tissue.

We report an experimental study that shows the attribution of the elastic property of light absorbing targets to the generation of ultrasound signals induced due to the photoacoustic effect. We investigated the variation in strength of the detected photoacoustic signals for various targets embedded in a background phantom with: (1) different elastic coefficients (94-346 kPa) and (2) various sizes (0.25-1.5 mm²). The results show that photoacoustic signals increase with an increase in elastic coefficient (i.e., showing to contrast in elastic property) while it is independent of variation in target sizes. Quantitative (analysis) study, and 2D and 3D reconstructed images are also presented. This study demonstrates the feasibility of imaging elastic property using photoacoustic technique.

This report studies absolute estimation of both the direction and magnitude of flow velocity, at microscopic scale, with acoustic resolution photoacoustic microscopy. Fundamentally, it is based on the quantitative measurement of Doppler frequency shift in the short laser pulse induced photoacoustic (ultrasound) signals. Experiments were carried out in a transparent plastic tube through which light absorbing liquid solution (as imaging target) flows. We investigated various cases of flow velocity: (i) variation in direction (0°–90°) and (ii) variation in magnitude (0.5–5 mm/s). This study demonstrates simultaneous recovery of both direction and magnitude from a set of data noninvasively and nondestructively.