Description: This topic is eligible for the DARPA Direct to Phase II Pilot Program. Please see section 7.0 of the DARPA instructions for additional information. To be eligible, offerors are required to provide information demonstrating the scientific and technical merit and feasibility of a Phase I project. DARPA will not evaluate the offeror's related Phase II proposal where it determines that the offeror has failed to demonstrate the scientific and technical merit and feasibility of the Phase I project. Offerors must choose between submitting a Phase I proposal OR a Direct to Phase II proposal, and may not submit both for the same topic. OBJECTIVE: Improve the ability to interrogate and ultimately modulate the neural influences of inflammation and immune function by enabling the design of new discovery tools that probe the bioelectrical states of human cells. DESCRIPTION: Today's warfighter is subject to a wide range of physical demands that include significant musculoskeletal stress and injury related to heavy equipment and gear, high physical exertion in tactical environments, and potential exposure to a variety of known and emerging pathogens and toxins. These external stressors typically elicit acute and/or chronic inflammatory responses as the body initiates the healing process. Advances at the interface of the nervous and immune systems are critical to understanding inflammation and ultimately optimizing human performance via the reduction of inflammation. While the nervous and immune systems were previously thought to serve and act distinctly, a picture of an integrated communication and protective system is beginning to emerge (1). Recent developments in neurophysiology and immunology have begun to make the connection between neural reflexes and inflammation/immunity (2). Better descriptions of the ways in which peripheral neurons interact with immune cells may enable a more comprehensive understanding of inflammation, leading to potential therapies or approaches to speed healing. Additionally, the development of techniques to track and functionally manipulate ion flows in cells has started to elucidate the possible roles of voltage gradients in the regulation of cell behaviors (3). Bioelectrical cell signaling studies are already providing insights in developmental and cancer biology (4, 5), and further technological advancements in the field would enable the characterization of the neural/immune interface. Microelectrodes are powerful tools for quantitative, real-time, single- and multi-cell electrophysiology that require direct physical contact, which can limit their utility for various types of measurement. Additionally, they are limited in terms of spatial resolution and the ability to localize bioelectric activity and structure at the same time. Furthermore, electrical methods are often designed to measure events that exceed action potential thresholds, whilst important bioelectric activity can occur at voltage levels below action potential thresholds. Ideal tools for bioelectric measurements would offer sub-cellular resolution, high contrast, high dynamic range, and the ability to probe large fields of view with many cells in real-time. Current optical methods have advantages, yet also suffer in sensitivity (signal to noise) and require temporal averaging, thereby reducing temporal resolution. Thus, efforts to measure important bioelectric codes that guide cell fate and influence immunity still suffer from trade-offs in spatial, temporal, and voltage resolution, as well as the ability to record many cells and structures simultaneously. This solicitation calls for the development of new voltage- and/or field-sensitive dyes designed to probe the bioelectrical states of cells in neural as well as non-neural systems. Commonly available voltage dyes focus on calcium signaling and provide insight into fast processes (millisecond timescale). Certain bioelectrical interrogations may benefit from different chemistries and timescales, as well as the ability to detect sub-threshold activation of neurons and other cells, thereby requiring a finer voltage (and/or electric field) resolution. Additional challenges with current state-of-the-art voltage dyes include high cost for the volume of dye typically required for in vivo studies, low contrast, and low signal-to-noise ratios. Recent examples of methods with promise to improve performance for voltage and field sensing include fluorescence resonance energy transfer (FRET) probes, voltage-sensitive proteins, and other nanoparticles such as quantum dots and NV-diamonds. Combined with new optical imaging approaches that offer unprecedented fields of view at sub-cellular resolution, successful advancements in probe technology will enable transition to academic, government, and commercial researchers to propel the field by offering, for example, lower cost, better stability, greater brightness, a broader range of absorption and emission wavelengths, higher voltage/field resolution, calibration simplicity, and increased specificity in targeting of cell regions. PHASE I: Design a new voltage-sensitive probe that addresses at least one of the following technical challenges for measuring bioelectrical states of cells in neural as well as non-neural systems: a) Improvement of voltage and/or electric field resolution to detect sub-threshold activation of neurons and other cells (e.g., approach the ability to report differences of several mV). b) Ability to probe at longer (seconds to minutes) or shorter (microseconds) timescales than currently available (milliseconds). c) Extension to multiple cell types and/or specific cell regions not currently accessible. Phase I deliverables will include a technical report detailing the experiments and results supporting the successful demonstration of a new voltage-sensitive probe that meets the selected technical challenge. Phase I deliverables will also include a Phase II transition plan for demonstrating sufficient reproducibility of the developed probe and potential research advancement to merit commercialization. The Phase II transition plan will include a description of the commercialization path, any barriers to market entry, and any identified early adoption partners. DIRECT TO PHASE II - Offerors interested in submitting a Direct to Phase II proposal in response to this topic must provide documentation to substantiate that the scientific and technical merit and feasibility described in the Phase I section of this topic has been met and describes the potential commercial applications. Documentation should include all relevant information including, but not limited to: technical reports, test data, prototype designs/models, and performance goals/results. Read and follow Section 7.0 of the DARPA Instructions. PHASE II: Seek to finalize and validate the voltage-sensitive probe developed in Phase I and demonstrate the utility beyond currently available probes to address the selected technical challenge. Additionally, establish and describe the market competitiveness of the new probe in the context of the combination of features that are desirable for a specific, defined application of the probe. This could include manufacture cost, probe stability, brightness, absorption and emission wavelength availability, sensitivity, and calibration simplicity. If successful, Phase II deliverables will include a detailed technical profile of the new voltage-sensitive probe that fully illustrates the advanced capabilities for the selected challenge, establishes the desirable parameters that validates the probe as a viable tool, and describes the commercialization path. PHASE III: The successful development of a new voltage-sensitive probe will enable a mechanistic understanding for modulating inflammation and immune function via neural interfaces. This capability is critical to understanding inflammation and its relation to human performance in order to address DoD challenges. The ultimate realization of the underlying technologies will advance healing and performance in the context of physical demands specific to the warfighter, including musculoskeletal stress and injury related to heavy equipment and gear, high physical exertion in tactical environments, and potential exposure to a variety of known and emerging pathogens and toxins. The successful development of a new voltage-sensitive probe will propel advances in the biotechnology and pharmaceutical sectors with the end goal of understanding the bioelectrical underpinnings of inflammation and immunity to promote health and healing. Additionally, the ability to modulate the neural influences of inflammation and immune function has significant implications for a wide range of therapeutic and diagnostic applications in addition to healing and human performance. New probes are in demand that would have high impact for other areas of healthcare, including tumor detection, stem cell research, and regenerative medicine.