The preservation of certain labile cancer biomarkers with formaldehyde-based fixatives can be considerably affected by preanalytical factors such as quality of fixation. Currently, there are no technologies capable of quantifying a fixative’s concentration or the formation of cross-links in tissue specimens. This work examined the ability to detect formalin diffusion into a histological specimen in real time. As formaldehyde passively diffused into tissue, an ultrasound time-of-flight (TOF) shift of several nanoseconds was generated due to the distinct sound velocities of formalin and exchangeable fluid within the tissue. This signal was resolved with a developed digital acoustic interferometry algorithm, which compared the phase differential between signals and computed the absolute TOF with subnanosecond precision. The TOF was measured repeatedly across the tissue sample for several hours until diffusive equilibrium was realized. The change in TOF from 6-mm thick ex vivo human tonsil fit a single-exponential decay (Radj2≥0.98) with rate constants that varied drastically spatially between 2 and 10 h (σ=2.9 h) due to substantial heterogeneity. This technology may prove essential to personalized cancer diagnostics by documenting and tracking biospecimen preanalytical fixation, guaranteeing their suitability for diagnostic assays, and speeding the workflow in clinical histopathology laboratories.
Recently, it has been demonstrated that the preservation of cancer biomarkers, such as phosphorylated protein epitopes, in formalin-fixed paraffin-embedded tissue is highly dependent on the localized concentration of the crosslinking agent. This study details a real-time diffusion monitoring system based on the acoustic time-of-flight (TOF) between pairs of 4 MHz focused transducers. Diffusion affects TOF because of the distinct acoustic velocities of formalin and interstitial fluid. Tissue is placed between the transducers and vertically translated to obtain TOF values at multiple locations with a spatial resolution of approximately 1 mm. Imaging is repeated for several hours until osmotic equilibrium is reached. A post-processing technique, analogous to digital acoustic interferometry, enables detection of subnanosecond TOF differences. Reference subtraction is used to compensate for environmental effects. Diffusion measurements with TOF monitoring ex vivo human tonsil tissue are well-correlated with a single exponential curve (R2>0.98) with a magnitude of up to 50 ns, depending on the tissue size (2-6 mm). The average exponential decay constant of 2 and 6 mm diameter samples are 20 and 315 minutes, respectively, although times varied significantly throughout the tissue (σmax=174 min). This technique can precisely monitor diffusion progression and could be used to mitigate effects from tissue heterogeneity and intersample variability, enabling improved preservation of cancer biomarkers distinctly sensitive to degradation during preanalytical tissue processing.
Understanding the tumor microenvironment is critical to characterizing how cancers operate and predicting their response to treatment. We describe a novel, high-resolution coregistered photoacoustic (PA) and pulse echo (PE) ultrasound system used to image the tumor microenvironment. Compared to traditional optical systems, the platform provides complementary contrast and important depth information. Three mice are implanted with a dorsal skin flap window chamber and injected with PC-3 prostate tumor cells transfected with green fluorescent protein. The ensuing tumor invasion is mapped during three weeks or more using simultaneous PA and PE imaging at 25 MHz, combined with optical and fluorescent techniques. Pulse echo imaging provides details of tumor structure and the surrounding environment with 100-μm3 resolution. Tumor size increases dramatically with an average volumetric growth rate of 5.35 mm3/day, correlating well with 2-D fluorescent imaging (R = 0.97, p < 0.01). Photoacoustic imaging is able to track the underlying vascular network and identify hemorrhaging, while PA spectroscopy helps classify blood vessels according to their optical absorption spectrum, suggesting variation in blood oxygen saturation. Photoacoustic and PE imaging are safe, translational modalities that provide enhanced depth resolution and complementary contrast to track the tumor microenvironment, evaluate new cancer therapies, and develop molecular contrast agents in vivo.
Understanding the tumor microenvironment is critical to characterizing how cancers operate and predicting how they
will eventually respond to treatment. The mouse window chamber model is an excellent tool for cancer research,
because it enables high resolution tumor imaging and cross-validation using multiple modalities. We describe a novel
multimodality imaging system that incorporates three dimensional (3D) photoacoustics with pulse echo ultrasound for
imaging the tumor microenvironment and tracking tissue growth in mice. Three mice were implanted with a dorsal skin
flap window chamber. PC-3 prostate tumor cells, expressing green fluorescent protein (GFP), were injected into the skin.
The ensuing tumor invasion was mapped using photoacoustic and pulse echo imaging, as well as optical and fluorescent
imaging for comparison and cross validation. The photoacoustic imaging and spectroscopy system, consisting of a
tunable (680-1000nm) pulsed laser and 25 MHz ultrasound transducer, revealed near infrared absorbing regions,
primarily blood vessels. Pulse echo images, obtained simultaneously, provided details of the tumor microstructure and
growth with 100-μm3 resolution. The tumor size in all three mice increased between three and five fold during 3+ weeks
of imaging. Results were consistent with the optical and fluorescent images. Photoacoustic imaging revealed detailed
maps of the tumor vasculature, whereas photoacoustic spectroscopy identified regions of oxygenated and deoxygenated
blood vessels. The 3D photoacoustic and pulse echo imaging system provided complementary information to track the
tumor microenvironment, evaluate new cancer therapies, and develop molecular imaging agents in vivo. Finally, these
safe and noninvasive techniques are potentially applicable for human cancer imaging.
Multimedia applications require besides basic communication services the particular support of guaranteed end-to-end services. In addition, these services need to be of a multipoint nature, where group communications take place in a guaranteed fashion as well. Therefore, a Quality-of-Service- driven specification of application requirements for multipoint scenarios and the guaranteed provision has been developed. At the same time, multipoint dynamics in terms of joining or leaving participants have been integrated, still guaranteeing end-to-end performance. In contrast to other communication frameworks, Quality-of-Service mechanisms and multipoint communication support are not considered as orthogonal, but an integration of these aspects leads to an efficient and highly flexible approach. Resource reservation, flexible protocol stacks, and heterogeneous network support are provided to guarantee multipoint multimedia application communication services in an end-to-end fashion.
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