High Sensitivity Singlet Oxygen Luminescence Sensor Using Computational Spectroscopy and Solid-State Detector.

This paper presents a technique for high sensitivity measurement of singlet oxygen luminescence generated during photodynamic therapy (PDT) and ultraviolet (UV) irradiation on skin. The high measurement sensitivity is achieved by using a computational spectroscopy (CS) approach that provides improved photon detection efficiency compared to spectral filtering methodology. A solid-state InGaAs photodiode is used as the CS detector, which significantly reduces system cost and improves robustness compared to photomultiplier tubes. The spectral resolution enables high-accuracy determination and subtraction of photosensitizer fluorescence baseline without the need for time-gating. This allows for high sensitivity detection of singlet oxygen luminescence emission generated by continuous wave light sources, such as solar simulator sources and those commonly used in PDT clinics. The value of the technology is demonstrated during in vivo and ex vivo experiments that show the correlation of measured singlet oxygen with PDT treatment efficacy and the illumination intensity on the skin. These results demonstrate the potential use of the technology as a dosimeter to guide PDT treatment and as an analytical tool supporting the development of improved sunscreen products for skin cancer prevention.

Assessment of breast cancer surgical margins with multimodal optical microscopy: A feasibility clinical study.

Providing surgical margin information during breast cancer surgery is crucial for the success of the procedure. The margin is defined as the distance from the tumor to the cut surface of the resection specimen. The consensus among surgeons and radiation oncologists is that there should be no tumor left within 1 to maximum 2 mm from the surface of the surgical specimen. If a positive margin remains, there is substantial risk for tumor recurrence, which may also result in potentially reduced cosmesis and eventual need for mastectomy. In this paper we report a novel multimodal optical imaging instrument based on combined high-resolution confocal microscopy-optical coherence tomography imaging for assessing the presence of potential positive margins on surgical specimens. Since rapid specimen analysis is critical during surgery, this instrument also includes a fluorescence imaging channel to enable rapid identification of the areas of the specimen that have potential positive margins. This is possible by specimen incubation with a cancer specific agent prior to imaging. In this study we used a quenched contrast agent, which is activated by cancer specific enzymes, such as urokinase plasminogen activators (uPA). Using this agent or a similar one, one may limit the use of high-resolution optical imaging to only fluorescence-highlighted areas for visualizing tissue morphology at the sub-cellular scale and confirming or ruling out cancer presence. Preliminary evaluation of this technology was performed on 20 surgical specimens and testing of the optical imaging findings was performed against histopathology. The combination of the three imaging modes allowed for high correlation between optical image analysis and histological ground-truth. The initial results are encouraging, showing instrument capability to assess margins on clinical specimens with a positive predictive value of 1.0 and a negative predictive value of 0.83.

Application of mild hypothermia successfully mitigates neural injury in a 3D in-vitro model of traumatic brain injury.

Therapeutic hypothermia (TH) is an attractive target for mild traumatic brain injury (mTBI) treatment, yet significant gaps in our mechanistic understanding of TH, especially at the cellular level, remain and need to be addressed for significant forward progress to be made. Using a recently-established 3D in-vitro neural hydrogel model for mTBI we investigated the efficacy of TH after compressive impact injury and established critical treatment parameters including target cooling temperature, and time windows for application and maintenance of TH. Across four temperatures evaluated (31.5, 33, 35, and 37°C), 33°C was found to be most neuroprotective after 24 and 48 hours post-injury. Assessment of TH administration onset time and duration showed that TH should be administered within 4 hours post-injury and be maintained for at least 6 hours for achieving maximum viability. Cellular imaging showed TH reduced the percentage of cells positive for caspases 3/7 and increased the expression of calpastatin, an endogenous neuroprotectant. These findings provide significant new insight into the biological parameter space that renders TH effective in mitigating the deleterious effects of cellular mTBI and provides a quantitative foundation for the future development of animal and preclinical treatment protocols.

Modular approach for resolving and mapping complex neural and other cellular structures and their associated deformation fields in three dimensions.

Understanding the biological implications of cellular mechanotransduction, especially in the context of pathogenesis, requires the accurate resolution of material deformation and strain fields surrounding the cells. This is particularly challenging for cells displaying branched, 3D architectures. Here, we provide a modular approach for 3D image segmentation and strain mapping of topologically complex structures. We describe how to use our approach, using neural cells and networks as an example. In addition to describing how to implement the computational analysis, we provide details of a cell culture protocol that can be used to generate neural networks for analysis and experimentation. This protocol allows for transformation of matrix-induced strains, and their full resolution across single cells or networks in three dimensions. The protocol also provides analyses to compute both the locally varying cytoskeletal strains and the average strain experienced by cells. An additional module allows spatial correlation of these strain maps with cytoskeletal features, including neurite disruptions such as neuronal blebs. Image processing and strain mapping take ≥3 h, with the exact time required being dependent on use case, software familiarity, and file size.

Strain and rate-dependent neuronal injury in a 3D in vitro compression model of traumatic brain injury.

In the United States over 1.7 million cases of traumatic brain injury are reported yearly, but predictive correlation of cellular injury to impact tissue strain is still lacking, particularly for neuronal injury resulting from compression. Given the prevalence of compressive deformations in most blunt head trauma, this information is critically important for the development of future mitigation and diagnosis strategies. Using a 3D in vitro neuronal compression model, we investigated the role of impact strain and strain rate on neuronal lifetime, viability, and pathomorphology. We find that strain magnitude and rate have profound, yet distinctively different effects on the injury pathology. While strain magnitude affects the time of neuronal death, strain rate influences the pathomorphology and extent of population injury. Cellular injury is not initiated through localized deformation of the cytoskeleton but rather driven by excess strain on the entire cell. Furthermore we find that, mechanoporation, one of the key pathological trigger mechanisms in stretch and shear neuronal injuries, was not observed under compression.