Recent advances in optical technologies such as multi-photon microscopy and optogenetics have revolutionized our ability to record and manipulate neuronal activity. Combining optical techniques with electrical recordings is of critical importance to connect the large body of neuroscience knowledge obtained from animal models to human studies mainly relying on electrophysiological recordings of brain-scale activity. However, integration of optical modalities with electrical recordings is challenging due to generation of light-induced artifacts. Here we report a transparent graphene microelectrode technology that eliminates light-induced artifacts to enable crosstalk-free integration of 2-photon microscopy, optogenetic stimulation, and cortical recordings in the same in vivo experiment. We achieve fabrication of crack- and residue-free graphene electrode surfaces yielding high optical transmittance for 2-photon imaging down to ~ 1 mm below the cortical surface. Transparent graphene microelectrode technology offers a practical pathway to investigate neuronal activity over multiple spatial scales extending from single neurons to large neuronal populations.
A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized because of experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data confirm that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar meshwork. These results present a hierarchical nanoscale picture of the plasma membrane.
The power spectrum of quantum dot (QD) fluorescence exhibits β f 1 noise, related to the intermittency of these nanosystems. As in other systems exhibiting f 1 noise, this power spectrum is not integrable at low frequencies, which appears to imply infinite total power. We report measurements of individual QDs that address this long-standing paradox. We find that the level of β f 1 noise decays with the observation time. The change of the spectrum with time places a bound on the total power. These observations are in stark contrast with most measurements of noise in macroscopic systems which do not exhibit any evidence for non-stationarity. We show that the traditional description of the power spectrum with a single exponent β is incomplete and three additional critical exponents characterize the dependence on experimental time.
Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.
We have systematically characterized the degradation of imaging quality with depth in deep brain multi-photon microscopy, utilizing our recently developed numerical model that computes wave propagation in scattering media. The signal-to-background ratio (SBR) and the resolution determined by the width of the point spread function are obtained as functions of depth. We compare the imaging quality of two-photon (2PM), three-photon (3PM), and non-degenerate two-photon microscopy (ND-2PM) for mouse brain imaging. We show that the imaging depth of 2PM and ND-2PM are fundamentally limited by the SBR, while the SBR remains approximately invariant with imaging depth for 3PM. Instead, the imaging depth of 3PM is limited by the degradation of the resolution, if there is sufficient laser power to maintain the signal level at large depth. The roles of the concentration of dye molecules, the numerical aperture of the input light, the anisotropy factor
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, noise level, input laser power, and the effect of temporal broadening are also discussed.
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