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.

No abstract

We explore monitoring the death process of individual red blood cells (RBC) quantitatively by using thermal lens (TL) response. TL response is a noninvasive excitation/probe technique that reflects photothermal parameters (e.g., absorption, thermal diffusivity, size, etc.). Since these parameters of cells change significantly during certain biological processes, real-time TL response was performed to monitor RBC death process when incubated with ionomycin. Theoretical model developed was applied to curve-fit the TL response for extracting thermal diffusivity and size of cells. Thermal diffusivity of dying RBC is found increased by 1.7 times in comparison with healthy cell.

Thyroid nodule (TN), a discrete palpable swelling of the thyroid gland, is prevalent among 8% of the adult population. The important concern with these nodules is the differentiation between benign and malignant ones. Since conventional diagnostic techniques (e.g., ultrasound) are not accurate in diagnosis, clinical surgeons adopt to excision biopsy, a golden standard, which requires surgery and removal of the gland. We propose to apply Photoacoustic Spectral Response (PASR), a non-invasive and elasticity based diagnostic technique, onto TN for malignancy diagnosis. In this study, time domain photoacoustic signals were acquired through custom built experimental setup inside the surgical theatre and spectral information were obtained through signal processing. PA spectra could distinguish Follicular Variant of Papillary Thyroid Carcinoma (a thyroid cancer) from the other tissues with a high accuracy which an important challenge in conventional diagnostic techniques. Further, other tissue variants were also successfully differentiated through signal decomposition in a quantitative manner.

There is a strong need for non-invasive detection of normal tissue from diseased one and a better understanding of the factors involved in the infection’s growth. Continuous monitoring of tissue samples at different time points is highly desirable. We demonstrate using the photoacoustic spectral response technique (PASR) for in situ analysis in a developing chicken embryo as a model (CAM) for anti-angiogenesis and vascular development. The photoacoustic technique is an emerging modality that is based on the acoustic detection of optical absorption of biological samples. The detected PA signals and their spectral response were used as a signature to determine the vasculature development pathology. Continuous monitoring of vascular growth and an anti-drug (Cisplatin) effect on vasculature has been done. PASR was investigated for the 10th day, 11th day, and 12th day control and inoculated egg samples. It shows that the dominant frequency of the PA spectral response for 10th day control and inoculated eggs lies between 0.45–0.52 MHz, whereas for 11th day and 12th day control eggs lie at 0.61 ± 0.152 MHz and 0.67 ± 0.001 MHz, respectively. The inoculated 11th and 12th day eggs lie at 0.35 ± 0.156 MHz and 0.16 ± 0.004 MHz, respectively. PASR could monitor the change in growth within a span of one day, which was not possible through the conventional imaging approach. This would open up a potential diagnostic technique for continuous monitoring of CAM assays.

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.

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.

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.