An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive 'liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

A multiwavelength backward-mode planar photoacoustic scanner for 3D imaging of soft tissues to depths of several millimeters with a spatial resolution in the tens to hundreds of micrometers range is described. The system comprises a tunable optical parametric oscillator laser system that provides nanosecond laser pulses between 600 and 1200 nm for generating the photoacoustic signals and an optical ultrasound mapping system based upon a Fabry-Perot polymer film sensor for detecting them. The system enables photoacoustic signals to be mapped in 2D over a 50 mm diameter aperture in steps of 10 microm with an optically defined element size of 64 microm. Two sensors were used, one with a 22 microm thick polymer film spacer and the other with a 38 mum thick spacer providing -3 dB acoustic bandwidths of 39 and 22 MHz, respectively. The measured noise equivalent pressure of the 38 microm sensor was 0.21 kPa over a 20 MHz measurement bandwidth. The instrument line-spread function (LSF) was measured as a function of position and the minimum lateral and vertical LSFs found to be 38 and 15 microm, respectively. To demonstrate the ability of the system to provide high-resolution 3D images, a range of absorbing objects were imaged. Among these was a blood vessel phantom that comprised a network of blood filled tubes of diameters ranging from 62 to 300 microm immersed in an optically scattering liquid. In addition, to demonstrate the applicability of the system to spectroscopic imaging, a phantom comprising tubes filled with dyes of different spectral characteristics was imaged at a range of wavelengths. It is considered that this type of instrument may provide a practicable alternative to piezoelectric-based photoacoustic systems for high-resolution structural and functional imaging of the skin microvasculature and other superficial structures.

Photoacoustic imaging allows absorption-based high-resolution spectroscopic in vivo imaging at a depth beyond that of optical microscopy. Until recently, photoacoustic imaging has largely been restricted to visualizing the vasculature through endogenous haemoglobin contrast, with most non-vascularized tissues remaining invisible unless exogenous contrast agents are administered. Genetically encodable photoacoustic contrast is attractive as it allows selective labelling of cells, permitting studies of, for example, specific genetic expression, cell growth or more complex biological behaviours in vivo. In this study we report a novel photoacoustic imaging scanner and a tyrosinase-based reporter system that causes human cell lines to synthesize the absorbing pigment eumelanin, thus providing strong photoacoustic contrast. Detailed threedimensional images of xenografts formed of tyrosinase-expressing cells implanted in mice are obtained in vivo to depths approaching 10 mm with a spatial resolution below 100 μm. This scheme is a powerful tool for studying cellular and genetic processes in deep mammalian tissues.O ptical techniques such as fluorescence or bioluminescence imaging are widely used to visualize biological tissues in vivo 1-4 . However, strong optical scattering fundamentally limits the penetration depth or spatial resolution. Microscopy and other techniques that utilize ballistic photons 4 can provide cellular resolution, but only to sub-millimetre penetration depths, while diffuse optical methods such as fluorescence optical tomography 1 can provide greater penetration depths (on the scale of centimetres) but with only limited spatial resolution (on the scale of millimetres). Photoacoustic imaging (PAI) offers the prospect of overcoming these limitations 5-8 . Here, ultrasound waves generated by the absorption of laser light by tissue chromophores are used to produce images of biological tissues based on optical absorption. Because acoustic waves are scattered much less than photons in soft tissues, PAI avoids the depth and spatial resolution limitations of purely optical imaging techniques: depths of a few centimetres with scalable spatial resolution ranging from tens to hundreds of micrometres (depending on depth) are readily achievable.Although strong absorption by haemoglobin enables the acquisition of exquisite three-dimensional photoacoustic (PA) images of the vasculature 9-14 , most cells and tissues are relatively weakly absorbing at visible and near-infrared wavelengths and are thus indistinguishable in the absence of exogenous contrast. The latter can be provided by nanoparticle-or dye-based targeted contrast agents 15-17 , but these can present challenges in achieving effective specific targeting and clearance. The use of reporter genes to provide genetically encoded exogenous PA contrast would avoid these limitations and has the further advantage of providing opportunities to study more complex biological behaviours such as cell growth dynamics and intracellular processes such as gene expressi...

The ability to identify a specific cancer using minimally invasive biopsy holds great promise for improving the diagnosis, treatment selection, and prediction of prognosis in cancer. Using whole-genome methylation data from The Cancer Genome Atlas (TCGA) and machine learning methods, we evaluated the utility of DNA methylation for differentiating tumor tissue and normal tissue for four common cancers (breast, colon, liver, and lung). We identified cancer markers in a training cohort of 1,619 tumor samples and 173 matched adjacent normal tissue samples. We replicated our findings in a separate TCGA cohort of 791 tumor samples and 93 matched adjacent normal tissue samples, as well as an independent Chinese cohort of 394 tumor samples and 324 matched adjacent normal tissue samples. The DNA methylation analysis could predict cancer versus normal tissue with more than 95% accuracy in these three cohorts, demonstrating accuracy comparable to typical diagnostic methods. This analysis also correctly identified 29 of 30 colorectal cancer metastases to the liver and 32 of 34 colorectal cancer metastases to the lung. We also found that methylation patterns can predict prognosis and survival. We correlated differential methylation of CpG sites predictive of cancer with expression of associated genes known to be important in cancer biology, showing decreased expression with increased methylation, as expected. We verified gene expression profiles in a mouse model of hepatocellular carcinoma. Taken together, these findings demonstrate the utility of methylation biomarkers for the molecular characterization of cancer, with implications for diagnosis and prognosis.

The reconstruction of photoacoustic images typically neglects the effect of acoustic absorption on the measured time domain signals. Here, a method to compensate for acoustic absorption in photoacoustic tomography is described. The approach is based on time-reversal image reconstruction and an absorbing equation of state which separately accounts for acoustic absorption and dispersion following a frequency power law. Absorption compensation in the inverse problem is achieved by reversing the absorption proportionality coefficient in sign but leaving the equivalent dispersion parameter unchanged. The reconstruction is regularized by filtering the absorption and dispersion terms in the spatial frequency domain using a Tukey window. This maintains the correct frequency dependence of these parameters within the filter pass band. The method is valid in one, two and three dimensions, and for arbitrary power law absorption parameters. The approach is verified through several numerical experiments. The reconstruction of a carbon fibre phantom and the vasculature in the abdomen of a mouse are also presented. When absorption compensation is included, a general improvement in the image magnitude and resolution is seen, particularly for deeper features.

14 15Exquisitely sensitive broadband detectors are needed to expand the capabilities of biomedical ultrasound, 30The sensitive detection of broadband ultrasound waves in the hundreds of kHz to tens of beam as required to achieve small element size for low directional sensitivity. 107The strong optical confinement afforded by the planoconcave microresonator design creates the opportunity 108 to maximise sensitivity in two ways. The first is by increasing the mirror reflectivity, trapping light for longer 109 and increasing the number of significant round trips in the cavity, leading to a higher Q-factor and thus a higher showing the 50% cut-off for the modelled response of a disk-shaped purely spatially averaging sensor of diameter 2mm. c, 161Directional response of 100μm sensor at selected frequencies as compared to the modelled response of a disk-shaped 162 spatially averaging receiver of diameter 2mm. For all data: w " = 12.5μm. 164Along with the NEP measurements in figure 1, the frequency response data in figure 2

The use of a novel all-optical photoacoustic scanner for imaging the development of tumor vasculature and its response to a therapeutic vascular disrupting agent is described. The scanner employs a Fabry-Perot polymer film ultrasound sensor for mapping the photoacoustic waves and an image reconstruction algorithm based upon attenuation-compensated acoustic time reversal. The system was used to noninvasively image human colorectal tumor xenografts implanted subcutaneously in mice. Label-free three-dimensional in vivo images of whole tumors to depths of almost 10 mm with sub-100-micron spatial resolution were acquired in a longitudinal manner. This enabled the development of tumor-related vascular features, such as vessel tortuosity, feeding vessel recruitment, and necrosis to be visualized over time. The system was also used to study the temporal evolution of the response of the tumor vasculature following the administration of a therapeutic vascular disrupting agent (OXi4503). This revealed the well-known destruction and recovery phases associated with this agent. These studies illustrate the broader potential of this technology as an imaging tool for the preclinical and clinical study of tumors and other pathologies characterized by changes in the vasculature.

Polydimethylsiloxane (PDMS) is widely used in biomedical science and can form composites that have broad applicability. One promising application where PDMS composites offer several advantages is optical ultrasound generation via the photoacoustic effect. Here, methods to create these PDMS composites are reviewed and classified. It is highlighted how the composites can be applied to a range of substrates, from micrometer‐scale, temperature‐sensitive optical fibers to centimeter‐scale curved and planar surfaces. The resulting composites have enabled all‐optical ultrasound imaging of biological tissues both ex vivo and in vivo, with high spatial resolution and with clinically relevant contrast. In addition, the first 3D all‐optical pulse‐echo ultrasound imaging of ex vivo human tissue, using a PDMS‐multiwalled carbon nanotube composite and a fiber‐optic ultrasound receiver, is presented. Gold nanoparticle‐PDMS and crystal violet‐PDMS composites with prominent absorption at one wavelength range for pulse‐echo ultrasound imaging and transmission at a second wavelength range for photoacoustic imaging are also presented. Using these devices, images of diseased human vascular tissue with both structural and molecular contrast are obtained. With a broader perspective, literature on recent advances in PDMS microfabrication from different fields is highlighted, and methods for incorporating them into new generations of optical ultrasound generators are suggested.