Development of a cost-effective compact diode-laser-based photoacoustic sensing instrument for breast tissue diagnosis.

Significance: The photoacoustic (PA) technique, a noninvasive pump-probe technique, has found interesting applications in biomedical tissue diagnosis over the last decade. To take it a step further to clinical applications, the PA technique needs to be designed as an instrument focusing on a compact design, reducing the cost, and quickly providing a quantitative diagnosis. Aim: This work presents a design and characterization of a cost-effective, compact PA sensing instrument for biomedical tissue diagnosis. Approach: A compact laser diode case design is developed to house several laser diodes for PA excitation, and a pulsed current supply unit is also developed in-house to power the laser diodes to generate a 25 ns current pulse at a frequency of 20 kHz. After PA experimental data acquisition, the signal's frequency spectra were calculated to characterize the tissue quantitatively and correlated with their mechanobiological properties. Results: The corresponding dominant frequency peak in the PA spectral response (PASR) study was low in the fibrofatty normal breast tissue 0.26±0.03 MHz, compared to the dominant frequency peak of 1.60±0.016 MHz in the fibrocystic disease tissue, which had increased glandular and stromal elements, thereby increased tissue density. The histopathological findings correlated with the PASR results, and the fibrocystic breast disease tissue exhibited a higher dominant frequency peak and energy compared to the normal breast tissue. Conclusions: We experimented with an in vitro PASR study of fibrocystic human breast tissues and successfully differentiated different tissue types using quantitative spectral parameters peak frequency, mean frequency, and spectral energy. This gives the potential to take this technique further for cost-effective and quick clinical applications.

Photoacoustic Spectral Response using Ultrasound and Interferometric Sensors: A Correlation Study for a High Bandwidth Real-Time Blood Vasculature Monitoring Application in a Chick-Embryo Chorioallantoic Membrane (CAM) Model.

Photoacoustic (PA) spectral response technique has shown good promise in efficient preclinical tissue diagnosis by depicting mechano-biological properties due to high spatial resolution and penetration depth. The conventional PA-based system is a pump-probe technique that utilizes neodymium-doped yttrium aluminum garnet pulsed laser as a pump and an ultrasound sensor as a probe. For biomedical studies, high-speed PA signals need to be acquired, requiring higher bandwidth ultrasound sensors. While the bandwidth increases, they exhibit a very low signal-to-noise ratio that inhibits acquiring PA signals of biomedical samples. An interferometer-based probe has recently been investigated as a potential ultrasound probe for obtaining PA signals as an alternative. This optical PA detection technique offers high sensitivity by combining low acoustic impedance with high electromechanical coupling. However, there is a lack of exploration of the same for real-time biomedical studies. This work shows the development of a homodyne Mach-Zehnder interferometer-based PA spectral response (PASR) followed by a correlation study between the conventional ultrasound sensor and the interferometer-based sensor. Further, this study demonstrates the capability of continuous monitoring of vascular growth and the effect of an antidrug (Cisplatin) on the vasculature tested on a chick-embryo chorioallantoic membrane model. PASR was able to monitor growth changes within one day, which was not possible with conventional methods. This opens up potential possibilities for using this technique in biomedical applications.

Biomedical Application of Photoacoustics: A Plethora of Opportunities.

The photoacoustic (PA) technique is a non-invasive, non-ionizing hybrid technique that exploits laser irradiation for sample excitation and acquires an ultrasound signal generated due to thermoelastic expansion of the sample. Being a hybrid technique, PA possesses the inherent advantages of conventional optical (high resolution) and ultrasonic (high depth of penetration in biological tissue) techniques and eliminates some of the major limitations of these conventional techniques. Hence, PA has been employed for different biomedical applications. In this review, we first discuss the basic physics of PA. Then, we discuss different aspects of PA techniques, which includes PA imaging and also PA frequency spectral analysis. The theory of PA signal generation, detection and analysis is also detailed in this work. Later, we also discuss the major biomedical application area of PA technique.

Photoacoustic Spectral Sensing Technique for Diagnosis of Biological Tissue Coagulation: In-Vitro Study.

Thermal coagulation of abnormal tissues has evolved as a therapeutic technique for different diseases including cancer. Tissue heating beyond 55 °C causes coagulation that leads to cell death. Noninvasive diagnosis of thermally coagulated tissues is pragmatic for performing efficient therapy as well as reducing damage of surrounding healthy tissues. We propose a noninvasive, elasticity-based photoacoustic spectral sensing technique for differentiating normal and coagulated tissues. Photoacoustic diagnosis is performed for quantitative differentiation of normal and coagulated excised chicken liver and muscle tissues in vitro by characterizing a dominant frequency of photoacoustic frequency spectrum. Pronounced distinction in the spectral parameter (i.e., dominant frequency) was observed due to change in tissue elastic property. We confirmed nearly two-fold increase in dominant frequencies for the coagulated muscle and liver tissues as compared to the normal ones. A density increase caused by tissue coagulation is clearly reflected in the dominant frequency composition. Experimental results were consistent over five different sample sets, delineating the potential of proposed technique to diagnose biological tissue coagulation and thus monitor thermal coagulation therapy in clinical applications.

Development of a compact laser-diode based frequency domain photoacoustic sensing system: Application of human breast cancer diagnosis.

We present the development of a laser diode based photoacoustic spectral response (PASR) setup capable of diagnosing human breast cancer tissues through the use of mechanobiological properties of the tissue. A detailed description of the laser driver is provided, highlighting the important characteristics of the developed driver. Furthermore, the amplifier development is described. The developed laser diode based PASR system has been characterized using standard samples. Subsequently, the developed experiment has been applied onto diagnosis of human breast tumors. Energy has been used as a parameter to differentiate between normal and malignant tissues. The results were statistically consistent and then compared with standard histopathology for correlation.

Application of continuous-wave photoacoustic sensing to red blood cell morphology.

The feasibility of continuous wave laser-based photoacoustic (CWPA) response technique in detecting the morphological changes in cells during the biological studies, through the features extracted from CWPA signal (i.e., amplitude) is demonstrated here. Various hematological disorders (e.g., sickle cell anemia, thalesemia) produce distinct changes at the cellular level morphologically. In order to explore the photoacoustic response technique to detect these morphological changes, we have applied CWPA technique onto the blood samples. Results of our preliminary study show a distinct change in the signal amplitude of photoacoustic (PA) signal due to a change in the concentration of blood, which signifies the sensitivity of the technique towards red blood cell (RBC) count (related to hematological disease like anemia). Further hypotonic and hypertonic solutions were induced in blood to produce morphological changes in RBCs (i.e., swollen and shrink, respectively) as compared to the normal RBCs. Experiments were performed using continuous wave laser-based photoacoustic response technique to verify the morphological changes in these RBCs. A distinct change in the PA signal amplitude was found for the distinct nature of RBCs (swollen, shrink, and normal). Thus, this can serve as a diagnostic signature for different biological studies based on morphological changes at cellular level. The experiments were also performed using conventional pulsed laser photoacoustic response technique which uses nano-second pulsed laser and the results obtained from both PA techniques were validated to produce identical changes. This demonstrates the utility of continuous wave laser-based photoacoustic technique for different biological studies related to morphological cellular disorders.

Quantitative Differentiation of Pneumonia from Normal Lungs: Diagnostic Assessment Using Photoacoustic Spectral Response.

Pneumonia is an acute lung infection that takes life of many young children in developing countries. Early stage (red hepatization) detection of pneumonia would be pragmatic to control mortality rate. Detection of this disease at early stages demands the knowledge of pathology, making it difficult to screen noninvasively. We propose photoacoustic spectral response (PASR), a noninvasive elasticity-dependent technique for early stage pneumonia detection. We report the quantitative red hepatization detection of pneumonia through median frequency, spectral energy, and variance. Significant contrast in spectral parameters due to change in sample elasticity is found. The tissue-mimicking phantom study illustrates a 39% increase in median frequency for 1.5 times the change in density. On applying to formalin-fixed pneumonia-affected goat lungs, it provides a distinct change in spectral parameters between pneumonia affected areas and normal lungs. The obtained PASR results were found to be highly correlating to standard histopathology. The proposed technique therefore has potential to be a regular diagnostic tool for early pneumonia detection.

Quantitative photoacoustic characterization of blood clot in blood: A mechanobiological assessment through spectral information.

Formation of blood clots, called thrombus, can happen due to hyper-coagulation of blood. Thrombi, while moving through blood vessels can impede blood flow, an important criterion for many critical diseases like deep vein thrombosis and heart attacks. Understanding mechanical properties of clot formation is vital for assessment of severity of thrombosis and proper treatment. However, biomechanics of thrombus is less known to clinicians and not very well investigated. Photoacoustic (PA) spectral response, a non-invasive technique, is proposed to investigate the mechanism of formation of blood clots through elasticity and also differentiate clots from blood. Distinct shift (increase in frequency) of the PA response dominant frequency during clot formation is reported. In addition, quantitative differentiation of blood clots from blood has been achieved through parameters like dominant frequency and spectral energy of PA spectral response. Nearly twofold increases in dominant frequency in blood clots compared to blood were found in the PA spectral response. Significant changes in energy also help in quantitatively differentiating clots from blood, in the blood. Our results reveal that increase in density during clot formation is reflected in the PA spectral response, a significant step towards understanding the mechanobiology of thrombus formation. Hence, the proposed tool, in addition to detecting thrombus formation, could reveal mechanical properties of the sample through quantitative photoacoustic spectral parameters.

Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope.

Photothermal microscopy (PTM), a noninvasive pump-probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications.

Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion.

Red blood cells (RBCs) have been found to undergo "programmed cell death," or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.

Integration of laser trapping for continuous and selective monitoring of photothermal response of a single microparticle.

Photothermal response (PTR) is an established pump and probe technique for real-time sensing of biological assays. Continuous and selective PTR monitoring is difficult owing to the Brownian motion changing the relative position of the target with respect to the beams. Integration of laser trapping with PTR is proposed as a solution. The proposed method is verified on red polystyrene microparticles. PTR is continuously monitored for 30 min. Results show that the mean relaxation time variation of the acquired signals is less than 5%. The proposed method is then applied to human red blood cells for continuous and selective PTR.