Probing near-infrared photorelaxation pathways in eumelanins and pheomelanins.

Ultraviolet-visible spectroscopy readily discerns the two types of melanin pigments (eumelanin and pheomelanin), although fundamental details regarding the optical properties and pigment heterogeneity are more difficult to disentangle via analysis of the broad featureless absorption spectrum alone. We employed nonlinear transient absorption spectroscopy to study different melanin pigments at near-infrared wavelengths. Excited-state absorption, ground-state depletion, and stimulated emission signal contributions were distinguished for natural and synthetic eumelanins and pheomelanins. A starker contrast among the pigments is observed in the nonlinear excitation regime because they all exhibit distinct transient absorptive amplitudes, phase shifts, and nonexponential population dynamics spanning the femtosecond-nanosecond range. In this manner, different pigments within the pheomelanin subclass were distinguished in synthetic and human hair samples. These results highlight the potential of nonlinear spectroscopies to deliver an in situ analysis of natural melanins in tissue that are otherwise difficult to extract and purify.

Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.

Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe.

Photoacoustic tomography (PAT) of genetically encoded probes allows for imaging of targeted biological processes deep in tissues with high spatial resolution; however, high background signals from blood can limit the achievable detection sensitivity. Here we describe a reversibly switchable nonfluorescent bacterial phytochrome for use in multiscale photoacoustic imaging, BphP1, with the most red-shifted absorption among genetically encoded probes. BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution. We monitored tumor growth and metastasis with ∼ 100-μm resolution at depths approaching 10 mm using photoacoustic computed tomography, and we imaged individual cancer cells with a suboptical-diffraction resolution of ∼ 140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at several scales.

Development of hemispheric specialization for lexical pitch-accent in Japanese infants.

Infants' speech perception abilities change through the first year of life, from broad sensitivity to a wide range of speech contrasts to becoming more finely attuned to their native language. What remains unclear, however, is how this perceptual change relates to brain responses to native language contrasts in terms of the functional specialization of the left and right hemispheres. Here, to elucidate the developmental changes in functional lateralization accompanying this perceptual change, we conducted two experiments on Japanese infants using Japanese lexical pitch-accent, which changes word meanings with the pitch pattern within words. In the first behavioral experiment, using visual habituation, we confirmed that infants at both 4 and 10 months have sensitivities to the lexical pitch-accent pattern change embedded in disyllabic words. In the second experiment, near-infrared spectroscopy was used to measure cortical hemodynamic responses in the left and right hemispheres to the same lexical pitch-accent pattern changes and their pure tone counterparts. We found that brain responses to the pitch change within words differed between 4- and 10-month-old infants in terms of functional lateralization: Left hemisphere dominance for the perception of the pitch change embedded in words was seen only in the 10-month-olds. These results suggest that the perceptual change in Japanese lexical pitch-accent may be related to a shift in functional lateralization from bilateral to left hemisphere dominance.