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.

Pleiotropy in the wild: the dormancy gene DOG1 exerts cascading control on life cycles.

In the wild, organismal life cycles occur within seasonal cycles, so shifts in the timing of developmental transitions can alter the seasonal environment experienced subsequently. Effects of genes that control the timing of prior developmental events can therefore be magnified in the wild because they determine seasonal conditions experienced by subsequent life stages, which can influence subsequent phenotypic expression. We examined such environmentally induced pleiotropy of developmental-timing genes in a field experiment with Arabidopsis thaliana. When studied in the field under natural seasonal variation, an A. thaliana seed-dormancy gene, Delay Of Germination 1 (DOG1), was found to influence not only germination, but also flowering time, overall life history, and fitness. Flowering time of the previous generation, in turn, imposed maternal effects that altered germination, the effects of DOG1 alleles, and the direction of natural selection on these alleles. Thus under natural conditions, germination genes act as flowering genes and potentially vice versa. These results illustrate how seasonal environmental variation can alter pleiotropic effects of developmental-timing genes, such that effects of genes that regulate prior life stages ramify to influence subsequent life stages. In this case, one gene acting at the seed stage impacted the entire life cycle.

Availability of doctors at primary health centres of Andhra Pradesh, India.

BACKGROUND: Availability of doctors at primary health centres (PHCs) is a major concern. We measured the operational availability of doctors in PHCs and examined the effect of residential distance and private practice. METHODS: Thirty-six health centres, consisting of six randomly selected PHCs from six purposively chosen districts in Andhra Pradesh were studied. Cross-sectional data on residential distance, private practice and attendance pattern of 38 operationally available medical officers were analysed. RESULTS: About 80% of doctors residing within 20 km attended their PHC on all 6 days of a week, compared with only 33% for those staying >40 km away. Among those staying >20 km away from their PHC, the odds of absenteeism by privately practising doctors was 3-24 times more than for those not practising privately. CONCLUSION: Residential distance seems to affect the operational availability of doctors. Private practice by doctors living within 20 km from the PHC does not seem to affect their operational availability. However, private practice by doctors staying >20 km away from their PHC may affect their availability.

DOG1 expression is predicted by the seed-maturation environment and contributes to geographical variation in germination in Arabidopsis thaliana.

Seasonal germination timing of Arabidopsis thaliana strongly influences overall life history expression and is the target of intense natural selection. This seasonal germination timing depends strongly on the interaction between genetics and seasonal environments both before and after seed dispersal. DELAY OF GERMINATION 1 (DOG1) is the first gene that has been identified to be associated with natural variation in primary dormancy in A. thaliana. Here, we report interaccession variation in DOG1 expression and document that DOG1 expression is associated with seed-maturation temperature effects on germination; DOG1 expression increased when seeds were matured at low temperature, and this increased expression was associated with increased dormancy of those seeds. Variation in DOG1 expression suggests a geographical structure such that southern accessions, which are more dormant, tend to initiate DOG1 expression earlier during seed maturation and achieved higher expression levels at the end of silique development than did northern accessions. Although elimination of the synthesis of phytohormone abscisic acid (ABA) results in the elimination of maternal temperature effects on dormancy, DOG1 expression predicted dormancy better than expression of genes involved in ABA metabolism.

Interfacing live cells with nanocarbon substrates.

Nanocarbon materials, including single-walled carbon nanotubes (SWCNTs) and graphene, promise various novel biomedical applications (e.g., nanoelectronic biosensing). In this Letter, we study the ability of SWCNT networks and reduced graphene oxide (rGO) films in interfacing several types of cells, such as neuroendocrine PC12 cells, oligodendroglia cells, and osteoblasts. It was found that rGO is biocompatible with all these cell types, whereas the SWCNT network is inhibitory to the proliferation, viability, and neuritegenesis of PC12 cells, and the proliferation of osteoblasts. These observations could be attributed to the distinct nanotopographic features of these two kinds of nanocarbon substrates.

Major flowering time gene, flowering locus C, regulates seed germination in Arabidopsis thaliana.

Flowering locus C (FLC) is a major regulator of flowering responses to seasonal environmental factors. Here, we document that FLC also regulates another major life-history transition-seed germination, and that natural variation at the FLC locus and in FLC expression is associated with natural variation in temperature-dependent germination. FLC-mediated germination acts through additional genes in the flowering pathway (FT, SOC1, and AP1) before involving the abscisic acid catabolic pathway (via CYP707A2) and gibberellins biosynthetic pathway (via GA20ox1) in seeds. Also, FLC regulation of germination is largely maternally controlled, with FLC peaking and FT, SOC1, and AP1 levels declining at late stages of seed maturation. High FLC expression during seed maturation is associated with altered expression of hormonal genes (CYP707A2 and GA20ox1) in germinating seeds, indicating that gene expression before the physiological independence of seeds can influence gene expression well after any physical connection between maternal plants and seeds exists. The major role of FLC in temperature-dependent germination documented here reveals a much broader adaptive significance of natural variation in FLC. Therefore, pleiotropy between these major life stages likely influences patterns of natural selection on this important gene, making FLC a promising case for examining how pleiotropy influences adaptive evolution.

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.

Diversification of phytochrome contributions to germination as a function of seed-maturation environment.

Environmental conditions during seed maturation influence germination, but the genetic basis of maternal environmental effects on germination is virtually unknown. Using single and multiple mutants of phytochromes, it is shown here that different phytochromes contributed to germination differently, depending on seed-maturation conditions. Arabidopsis thaliana wild-type seeds that were matured under cool temperatures were intensely dormant compared with seeds matured at warmer temperature, and this dormancy was broken only after warm seed-stratification followed by cold seed-stratification. The warm-cold stratification broke dormancy in fresh seeds but not in dry after-ripened seeds. Functional PHYB and PHYD were necessary to break cool-induced dormancy, which indicates a previously unknown and ecologically important function for PHYD. Disruption of PHYA in combination with PHYD (but not PHYB) restored germination to near wild-type levels, indicating that PHYA contributes to the maintenance of cool-induced dormancy on a phyD background. Effects of seed-maturation temperature were much stronger than effects of seed-maturation photoperiod. PHYB contributed to germination somewhat more strongly in seeds matured under short days, whereas PHYD contributed to germination somewhat more strongly in seeds matured under long days. The variable contributions of different phytochromes to germination as a function of seed-maturation conditions reveal further functional diversification of the phytochromes during the process of germination. This study identifies among the first genes to be associated with maternal environmental effects on germination.

Phytochrome mediates germination responses to multiple seasonal cues.

We identified a new role of phytochrome in mediating germination responses to seasonal cues and thereby identified for the first time a gene involved in maternal environmental effects on germination. We examined the germination responses of a mutant, hy2-1, which is deficient in the phytochrome chromophore. The background genotype, Landsberg erecta (Ler), lacked dormancy in most treatments, while hy2-1 required cold stratification for germination in a manner that resembled a more dormant ecotype, Columbia (Col). Unlike Col, hy2-1 was not induced into dormancy by warm stratification. Therefore, the down-regulation of phytochrome-mediated germination pathways results in sensitivity to cold, but we found no evidence that reduced phytochrome activity enables the warm-induction of dormancy. Cool temperatures during seed maturation induced dormancy. The hy2-1 mutants did not overcome this dormancy, indicating that phytochrome-mediated pathways are required to break cold-induced dormancy. Ler did not respond to post-stratification temperature, but hy2-1 did respond, suggesting phytochrome pathways are involved in germination responses to temperature. In summary, phytochromes mediate dormancy and germination responses to seasonal cues experienced both during seed maturation and after dispersal. Phytochromes therefore appear to be involved in mediating seasonal germination timing, a trait of great ecological importance and one that is under strong natural selection.