Luminescent materials are widely used for imaging and sensing owing to their high sensitivity, rapid response and facile detection by many optical technologies1. Typically materials must be chemically tailored to achieve intense, photostable fluorescence, oxygen-sensitive phosphorescence or dual emission for ratiometric sensing, often by blending two dyes in a matrix. Dual-emissive materials combining all of these features in one easily tunable molecular platform are desirable, but when fluorescence and phosphorescence originate from the same dye, it can be challenging to vary relative fluorescence/phosphorescence intensities for practical sensing applications. Heavy-atom substitution 2 alone increases phosphorescence by a given, not variable amount. Here, we report a strategy for modulating fluorescence/phosphorescence for a single-component, dual-emissive, iodide-substituted difluoroboron dibenzoylmethane-poly(lactic acid) (BF 2 dbm(I)PLA) solid-state sensor material. This is accomplished through systematic variation of the PLA chain length in controlled solvent-free lactide polymerization 3 combined with heavy-atom substitution2. We demonstrate the versatility of this approach by showing that films made from low-molecular-weight BF 2 dbm(I)PLA with weak fluorescence and strong phosphorescence are promising as 'turn on' sensors for aerodynamics applications 4 , and that nanoparticles fabricated from a higher-molecularweight polymer with balanced fluorescence and phosphorescence intensities serve as ratiometric tumour hypoxia imaging agents.Ratiometric sensing addresses challenges associated with sensor concentration and obviates the need for specialized luminescence lifetime instrumentation. For example, Kopelman and co-workers developed a powerful nanosensor (PEEBLEs) for ratiometric oxygen sensing comprising a cyanine dye standard and an O 2 -sensitive Pt porphyrin phosphor in a sol-gel matrix and demonstrated its utility for cell biology 5 . Single-component dye-polymer conjugates with both fluorescence and phosphorescence offer advantages over threecomponent dye/standard/matrix mixtures. Dual-emissive materials possess an internal rather than external standard in ratiometric sensing schemes, and thus, 1:1 fluorophore/phosphor stoichiometry, resulting in greater sample homogeneity and minimal dye leaching. Readily processable biomaterials combined with photostable, optically tunable dyes that emit brightly even in aqueous environments are beneficial for imaging and sensing in biomedical contexts. Previously, we discovered that difluoroboron dibenzoylmethane-poly(lactic acid) (BF 2 dbmPLA; as in Fig. 1, with hydrogen in place of iodide) exhibits unusual, long-lived green room-temperature phosphorescence (RTP) in addition to intense blue fluorescence 6 . Given that thermal decay pathways are often accessible, it is rare to see RTP from organic compounds; typically organized media7 , 8 or heavy atoms9 are required for RTP. This feature adds enhanced capability to the excellent fluorescence properties o...

A flexible and fast Monte Carlo-based model of diffuse reflectance has been developed for the extraction of the absorption and scattering properties of turbid media, such as human tissues. This method is valid for a wide range of optical properties and is easily adaptable to existing probe geometries, provided a single phantom calibration measurement is made. A condensed Monte Carlo method was used to speed up the forward simulations. This model was validated by use of two sets of liquid-tissue phantoms containing Nigrosin or hemoglobin as absorbers and polystyrene spheres as scatterers. The phantoms had a wide range of absorption (0-20 cm(-1)) and reduced scattering coefficients (7-33 cm(-1)). Mie theory and a spectrophotometer were used to determine the absorption and reduced scattering coefficients of the phantoms. The diffuse reflectance spectra of the phantoms were measured over a wavelength range of 350-850 nm. It was found that optical properties could be extracted from the experimentally measured diffuse reflectance spectra with an average error of 3% or less for phantoms containing hemoglobin and 12% or less for phantoms containing Nigrosin.

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.tissue engineering | contractile force | self-repair | angiogenesis | window chamber

Exercise has been shown to improve postischemia perfusion of normal tissues; we investigated whether these effects extend to solid tumors. Estrogen receptor–negative (ER-, 4T1) and ER+ (E0771) tumor cells were implanted orthotopically into syngeneic mice (BALB/c, N = 11–12 per group) randomly assigned to exercise or sedentary control. Tumor growth, perfusion, hypoxia, and components of the angiogenic and apoptotic cascades were assessed by MRI, immunohistochemistry, western blotting, and quantitative polymerase chain reaction and analyzed with one-way and repeated measures analysis of variance and linear regression. All statistical tests were two-sided. Exercise statistically significantly reduced tumor growth and was associated with a 1.4-fold increase in apoptosis (sedentary vs exercise: 1544 cells/mm2, 95% CI = 1223 to 1865 vs 2168 cells/mm2, 95% CI = 1620 to 2717; P = .048), increased microvessel density (P = .004), vessel maturity (P = .006) and perfusion, and reduced intratumoral hypoxia (P = .012), compared with sedentary controls. We also tested whether exercise could improve chemotherapy (cyclophosphamide) efficacy. Exercise plus chemotherapy prolonged growth delay compared with chemotherapy alone (P < .001) in the orthotopic 4T1 model (n = 17 per group). Exercise is a potential novel adjuvant treatment of breast cancer.

Optical techniques for functional imaging in mice have a number of key advantages over other common imaging modalities such as magnetic resonance imaging, positron emission tomography or computed tomography, including high resolution, low cost and an extensive library of available contrast agents and reporter genes. A major challenge to such work is the limited penetration depth imposed by tissue turbidity. We describe a window chamber technique by which these limitations can be avoided. This facilitates the study of a wide range of processes, with potential endpoints including longitudinal gene expression, vascular remodeling and angiogenesis, and tumor growth and invasion. We further describe several quantitative imaging and analysis techniques for characterizing in vivo fluorescence properties and functional endpoints, including vascular morphology and oxygenation. The procedure takes ~2 h to complete, plus up to several weeks for tumor growth and treatment procedures.

The Monte Carlo-based inverse model of diffuse reflectance described in part I of this pair of companion papers was applied to the diffuse reflectance spectra of a set of 17 malignant and 24 normal-benign ex vivo human breast tissue samples. This model allows extraction of physically meaningful tissue parameters, which include the concentration of absorbers and the size and density of scatterers present in tissue. It was assumed that intrinsic absorption could be attributed to oxygenated and deoxygenated hemoglobin and beta-carotene, that scattering could be modeled by spheres of a uniform size distribution, and that the refractive indices of the spheres and the surrounding medium are known. The tissue diffuse reflectance spectra were evaluated over a wavelength range of 400-600 nm. The extracted parameters that showed the statistically most significant differences between malignant and nonmalignant breast tissues were hemoglobin saturation and the mean reduced scattering coefficient. Malignant tissues showed decreased hemoglobin saturation and an increased mean reduced scattering coefficient compared with nonmalignant tissues. A support vector machine classification algorithm was then used to classify a sample as malignant or nonmalignant based on these two extracted parameters and produced a cross-validated sensitivity and specificity of 82% and 92%, respectively.

Dual emissive luminescence properties of solid-state difluoroboron β-diketonate-poly(lactic acid) (BF2bdk-PLA) materials have been utilized as biological oxygen sensors. Dyes with red-shifted absorption and emission are important for multiplexing and in vivo imaging, thus hydroxyl-functionalized dinaphthoylmethane initiators and dye-PLA conjugates BF2dnm(X)PLA (X = H, Br, I) with extended conjugation were synthesized. The luminescent materials show red-shifted absorbance (~435 nm) and fluorescence tunability by molecular weight. Fluorescence colors range from yellow (~530 nm) in 10 – 12 kDa polymers to green (~490 nm) in 20 – 30 kDa polymers. Room-temperature phosphorescence (RTP) and thermally activated delayed fluorescence (TADF) are present under a nitrogen atmosphere. For the iodine-substituted derivative, BF2dnm(I)PLA, clearly distinguishable fluorescence (green) and phosphorescence (orange) peaks are present, making it ideal for ratiometric oxygen-sensing and imaging. Bromide and hydrogen analogues with weaker relative phosphorescence intensities and longer phosphorescence lifetimes can be used as highly sensitive, concentration independent, lifetime-based oxygen sensors or for gated emission detection. BF2dnm(I)PLA nanoparticles were taken up by T41 mouse mammary cells and successfully demonstrated differences in vitro ratiometric measurement of oxygen.

The features extracted from the Monte Carlo based analysis were hemoglobin saturation, total hemoglobin concentration, beta-carotene concentration and the mean (wavelength averaged) reduced scattering coefficient. Beta-carotene concentration was positively correlated and the mean reduced scattering coefficient was negatively correlated with percent adipose tissue content in normal breast tissues. In addition, there was a statistically significant decrease in the beta-carotene concentration and hemoglobin saturation, and a statistically significant increase in the mean reduced scattering coefficient in malignant tissues compared to non-malignant tissues. The features extracted from the partial least squares analysis were a set of principal components. A subset of principal components showed that the diffuse reflectance spectra of malignant breast tissues displayed an increased intensity over wavelength range of 440-510 nm and a decreased intensity over wavelength range of 510-600 nm, relative to that of non-malignant breast tissues. The diagnostic performance of the classification algorithms based on both feature extraction techniques yielded similar sensitivities and specificities of approximately 80% for discriminating between malignant and non-malignant breast tissues. While both methods yielded similar classification accuracies, the model based approach provided insight into the physiological and structural features that discriminate between malignant and non-malignant breast tissues.