Real-time study of spatio-temporal dynamics (4D) of physiological activities in alive biological specimens with different FOVs and resolutions simultaneously.

This article reports the development of a microscopy imaging system that gives feasibility for studying spatio-temporal dynamics of physiological activities of alive biological specimens (over entire volume not only for a particular section, i.e., in 4D). The imaging technology facilitates to obtain two image frames of a section of the larger specimen ([Formula: see text]) with different FOVs at different resolutions or magnifications simultaneously in real-time (in addition to recovery of 3D (volume) information). Again, this imaging system addresses the longstanding challenges of housing multiple light sources (6 at the maximum till date) in microscopy (in general) and light sheet fluorescence microscopy (LSFM) (in particular), by using a tuneable pulsed laser source (with an operating wavelength in the range [Formula: see text]-670 nm) in contrast to the conventional CW laser source being adopted for inducing photo-excitation of tagged fluorophores. In the present study, we employ four wavelengths ([Formula: see text] 488 nm, 585 nm, 590 nm, and 594 nm). Our study also demonstrates quantitative characterization of spatio-temporal dynamics (velocity-both amplitude and direction) of organelles (mitochondria) and their mutual correlationships. Mitochondria close to the nucleus (or in clustered cells) are observed to possess a lower degree of freedom in comparison to that at the cellular periphery (or isolated cells). In addition, the study demonstrates real-time observation and recording of the development and growth of all tracheal branches during the entire period ([Formula: see text] min) of embryonic development (Drosophila). The experimental results-with experiments being conducted in various and diversified biological specimens (Drosophila melanogaster, mouse embryo, and HeLa cells)-demonstrate that the study is of great scientific impact both from the aspects of technology and biological sciences.

Improvement of LED-based photoacoustic imaging using lag-coherence factor (LCF) beamforming.

BACKGROUND: Owing to its portability, affordability, and energy-efficiency, LED-based photoacoustic (PA) imaging is increasingly becoming popular when compared to its laser-based alternative, mainly for superficial vascular imaging applications. However, this technique suffers from low SNR and thereby limited imaging depth. As a result, visual image quality of LED-based PA imaging is not optimal, especially in sub-surface vascular imaging applications. PURPOSE: Combination of linear ultrasound (US) probes and LED arrays are the most common implementation in LED-based PA imaging, which is currently being explored for different clinical imaging applications. Traditional delay-and-sum (DAS) is the most common beamforming algorithm in linear array-based PA detection. Side-lobes and reconstruction-related artifacts make the DAS performance unsatisfactory and poor for a clinical-implementation. In this work, we explored a new weighting-based image processing technique for LED-based PAs to yield improved image quality when compared to the traditional methods. METHODS: We are proposing a lag-coherence factor (LCF), which is fundamentally based on the combination of the spatial auto-correlation of the detected PA signals. In LCF, the numerator contains lag-delay-multiply-and-sum (DMAS) beamformer instead of a conventional DAS beamformer. A spatial auto-correlation operation is performed between the detected US array signals before using DMAS beamformer. We evaluated the new method on both tissue-mimicking phantom (2D) and human volunteer imaging (3D) data acquired using a commercial LED-based PA imaging system. RESULTS: Our novel correlation-based weighting technique showed LED-based PA image quality improvement when it is combined with conventional DAS beamformer. Both phantom and human volunteer imaging results gave a direct confirmation that by introducing LCF, image quality was improved and this method could reduce side-lobes and artifacts when compared to the DAS and coherence-factor (CF) approaches. Signal-to-noise ratio, generalized contrast-to-noise ratio, contrast ratio and spatial resolution were evaluated and compared with conventional beamformers to assess the reconstruction performance in a quantitative way. Results show that our approach offered image quality enhancement with an average signal-to-noise ratio and spatial resolution improvement of around 20% and 25% respectively, when compared with conventional CF based DAS algorithm. CONCLUSIONS: Our results demonstrate that the proposed LCF based algorithm performs better than the conventional DAS and CF algorithms by improving signal-to-noise ratio and spatial resolution. Therefore, our new weighting technique could be a promising tool to improve the performance of LED-based PA imaging and thus accelerate its clinical translation.

Energy compensated synthetic aperture focusing technique for photoacoustic microscopy.

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 ∼ 5 % ), lateral resolution (up to ∼ 5 % ), SNR (up to ∼ 6 % ) and CR (up to ∼ 8 % ).

Higher-order correlation based real-time beamforming in photoacoustic imaging.

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.

Delay-and-sum-to-delay-standard-deviation factor: a promising adaptive beamformer.

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).

Enhancement of Photoacoustic Signal Strength with Continuous Wave Optical Pre-Illumination: A Non-Invasive Technique.

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 ~ 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).

Photoacoustic elastography imaging: a review.

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.

Elastic property attributes to photoacoustic signals: an experimental phantom study.

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.

Quantitative estimation of mechanical and optical properties from ultrasound assisted optical tomography data.

We demonstrate quantitative optical property and elastic property imaging from ultrasound assisted optical tomography data. The measurements, which are modulation depth M and phase ϕ 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 ω (the US frequency) plots and optical absorption (µ(a)) and scattering (µ(s)) coefficients from the measured differential phase changes. All experimental observations are verified also using Monte Carlo simulations.