This PDF file contains the front matter associated with SPIE Proceedings Volume 8804, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

In this article, we show experimental results of time-resolved optical spectroscopy performed with small distance between launching and detecting fibers. It was already demonstrated that depth discrimination is independent of source-detector separation and that measurements at small source detector distance provide better contrast and spatial resolution. The main disadvantage is represent by the huge increase in early photons (scarcely diffused by tissue) peak that can saturate the dynamic range of most detectors, hiding information carried by late photons. Thanks to a fast-gated Single- Photon Avalanche Diode (SPAD) module, we are able to reject the peak of early photons and to obtain high-dynamic range acquisitions. We exploit fast-gated SPAD module to perform for the first time functional near-infrared spectroscopy (fNIRS) at small source-detector distance for in vivo measurements and we demonstrate the possibility to detect non-invasively the dynamics of oxygenated and deoxygenated haemoglobin occurring in the motor cortex during a motor task. We also show the improvement in terms of signal amplitude and Signal-to-Noise Ratio (SNR) obtained exploiting fast-gated SPAD performances with respect to “non-gated” measurements.

We demonstrate the simultaneous imaging of cortical blood flow and haemoglobin oxygenation of the exposed cortex in rats using laser speckle contrast analysis (LASCA) and RGB reflectometry. Spatial and temporal resolved changes in blood flow and oxygenation are observed for single forepaw stimulation.

We recorded maps of cortical and systemic hemodynamic responses (oxy-hemoglobin, O₂Hb and deoxy-hemoglobin, HHb) during incremental neuromuscular electrical stimulation (NMES) of the right forearm in nine subjects by a 32- channel time domain fNIRS (TD-fNIRS) instrument. Statistical parametric maps (SPM) relative to the different current stimulations (under and over the maximal tolerated intensity-MTI) versus the 10%MTI were generated. Exploiting the temporal information contained in the TD-fNIRS signal it was possible to create different maps referring to the deeper (cortical activations) and the more superficial (systemic changes) head layers. The increasing of the stimulation current on the right forearm muscle produced a significantly larger bilateral sensorimotor and prefrontal cortical activations (i.e. increase in the O2Hb and decrease in HHb) than the systemic changes. Physiological parameters (heart rate, breathing rate and skin conductance) were also monitored.

Fluorescence light sheet microscopy has known a true renaissance in the last years. In fact, since optical sectioning is achieved in a wide-field detection scheme, this technique allows high resolution three-dimensional imaging with high frame rate. Light sheet microscopy is therefore an ideal candidate for reconstructing macroscopic specimens with micron resolution: coupled with clearing protocols based on refractive index matching it has been exploited to image entire mouse brains without physical sectioning. Use of clearing protocols poses several challenges to light sheet microscopy. First of all, residual light scattering inside the tissue expands the excitation light sheet, leading to the excitation of out-of-focus planes, and thus frustrating the very principle of light sheet illumination. To reject out-of-focus contributions we recently coupled light sheet illumination with confocal detection, achieving significant contrast enhancement in real time. Another issue which often arises when working with clearing agents is the refractive index mismatch between the clearing and the medium objective design medium. This introduces severe spherical aberration, which leads to broadening of the point spread function and to a strong reduction in its peak value: When imaging deep (several mm) inside macroscopic specimens, the signal can be reduced by more than an order of magnitude. We investigated the possibility of correcting such spherical aberration by introducing extra optical devices in the detection path.

We combined the advantage of an ultrafast random access microscope with novel labelling technologies to study the intra- and inter-cellular action potential propagation in neurons and cardiac myocytes with sub-millisecond time resolution. The random accesses microscopy was used in combination with a new fluorinated voltage sensitive dye with improved photostability to record membrane potential from multiple Purkinje cells with near simultaneous sampling. The RAMP system rapidly scanned between lines drawn in the membranes of neurons to perform multiplex measurements of the TPF signal. This recording was achieved by rapidly positioning the laser excitation with the AOD to sample a patch of membrane from each cell in <100 μs; for recording from five cells, multiplexing permits a temporal resolution of 400 μs sufficient to capture every spike. The system is capable to record spontaneous activity over 800 ms from five neighbouring cells simultaneously, showing that spiking is not temporally correlated. The system was also used to investigate the electrical properties of tubular system (TATS) in isolated rat ventricular myocytes.

In the adult nervous system, different neuronal classes show different regenerative behavior. Although previous studies demonstrated that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury, we have hitherto no details about the real dynamics of fiber regeneration. We set up a model of singularly axotomized climbing fibers (CF) to investigate their reparative properties in the adult central nervous system (CNS) in vivo. Here we describe the approach followed to characterize the reactive plasticity after injury.

The optical properties of the human head in the range from 600 nm to 1100 nm have been non-invasively in-vivo investigated by various research groups using different diffuse optics techniques and data analysis methods.

5-ALA-induced protoporphyrin IX (PpIX) fluorescence enables to guiding in intra-operative surgical glioma resection. However at present, it has yet to be shown that this method is able to identify infiltrative component of glioma. In extracted tumor tissues we measured a two-peaked emission in low grade gliomas and in the infiltrative component of glioblastomas due to multiple photochemical states of PpIX. This spectral complexity of the fluorescence emission spectrum brings new limits in the sensibility of current methods to measured PpIX concentration. We propose new measured parameters, by taking into consideration the spectral complexity, to overcome these limitations in sensitivity. These parameters clearly distinguish the solid component of glioblastomas from low grade gliomas and infiltrative component of glioblastomas.

To visualize hemodynamics in cerebral cortex of in vivo rat brain during cortical spreading depression, we investigate a spectral reflectance imaging technique based on the Wiener estimation for a digital RGB camera.

Functional near-infrared spectroscopy (fNIRS) is a non-invasive optical technique able to measure hemodynamic response in the brain cortex. Among the different approaches the fNIRS can be based on, the time resolved one allows a straightforward relationship between the photon detection time and its path within the medium, improving the discrimination of the information content relative to the different layers the tissues are composed of. Thus absorption and scattering properties of the probed tissue can be estimated, and from them the oxy- and deoxy-hemoglobin concentration. However, an open issue in the optical imaging studies is still the accuracy in separating the superficial hemodynamic changes from those happening in deeper regions of the head and more likely involving the cerebral cortex. In fact a crucial point is the precise estimate of the time dependent pathlength spent by photons within the perturbed medium. A novel method for the calculus of the absorption properties in time domain fNIRS, based on a refined computation of photon pathlength in multilayered media, is proposed. The method takes into account the non-ideality of the measurement system (its instrument response function) and the heterogeneous structure of the head. The better accuracy in computing the optical pathlength can improve the NIRS data analysis, especially for the deeper layer. Simulations and preliminary analysis on in vivo data have been performed to validate the method and are here presented.

Using Monte Carlo simulations we demonstrate that a realistic absorption inhomogeneity embedded in a diffusive medium can be effectively mimicked by a small black object of a proper volume (Equivalence Relation). Applying this concept we propose the construction of simple and well reproducible inhomogeneous phantoms.