Although temporally focused wide-field two-photon microscopy (TFM) can perform depth resolved wide field imaging, it cannot avoid the image degradation due to scattering of excitation and emission photons when imaging in a turbid medium. Further, its axial resolution is inferior to standard point-scanning two-photon microscopy. We implemented a structured light illumination for TFM and have shown that it can effectively reject the out-of-focus scattered emission photons improving image contrast. Further, the depth resolution of the improved system is dictated by the spatial frequency of the structure light with the potential of attaining depth resolution better than point-scanning two-photon microscopy.

We introduce a multi-distance, frequency-domain, near-infrared spectroscopy (NIRS) method to measure the optical coefficients of two-layered media and the thickness of the top layer from diffuse reflectance measurements. This method features a direct solution based on diffusion theory and an inversion procedure based on the Levenberg-Marquardt algorithm. We have validated our method through Monte Carlo simulations, experiments on tissue-like phantoms, and measurements on the forehead of three human subjects. The Monte Carlo simulations and phantom measurements have shown that, in ideal two-layered samples, our method accurately recovers the top layer thickness (L), the absorption coefficient (µa) and the reduced scattering coefficient (µ′s) of both layers with deviations that are typically less than 10% for all parameters. Our method is aimed at absolute measurements of hemoglobin concentration and saturation in cerebral and extracerebral tissue of adult human subjects, where the top layer (layer 1) represents extracerebral tissue (scalp, skull, dura mater, subarachnoid space, etc.) and the bottom layer (layer 2) represents cerebral tissue. Human subject measurements have shown a significantly greater total hemoglobin concentration in cerebral tissue (82±14 µM) with respect to extracerebral tissue (30±7 µM). By contrast, there was no significant difference between the hemoglobin saturation measured in cerebral tissue (56%±10%) and extracerebral tissue (62%±6%). To our knowledge, this is the first time that an inversion procedure in the frequency domain with six unknown parameters with no other prior knowledge is used for the retrieval of the optical coefficients and top layer thickness with high accuracy on two-layered media. Our absolute measurements of cerebral hemoglobin concentration and saturation are based on the discrimination of extracerebral and cerebral tissue layers, and they can enhance the impact of NIRS for cerebral hemodynamics and oxygenation assessment both in the research arena and clinical practice.

We present near-infrared spectroscopy measurement of absolute cerebral hemoglobin concentration and saturation in a large sample of 36 healthy elderly (mean age, 85 AE 6 years) and 19 young adults (mean age, 28 AE 4 years). Non-invasive measurements were obtained on the forehead using a commercially available multi-distance frequency-domain system and analyzed using a diffusion theory model for a semi-infinite, homogeneous medium with semi-infinite boundary conditions. Our study included repeat measurements, taken five months apart, on 16 elderly volunteers that demonstrate intra-subject reproducibility of the absolute measurements with cross-correlation coefficients of 0.9 for absorption coefficient ðμ a Þ, oxy-hemoglobin concentration ð½HbO 2 Þ, and total hemoglobin concentration ð½HbTÞ, 0.7 for deoxy-hemoglobin concentration ð½HbÞ, 0.8 for hemoglobin oxygen saturation ðStO 2 Þ, and 0.7 for reduced scattering coefficient ðμ 0 s Þ. We found significant differences between the two age groups. Compared to young subjects, elderly subjects had lower cerebral ½HbO 2 , ½Hb, ½HbT, and StO 2 by 10 AE 4 μM, 4 AE 3 μM, 14 AE 5 μM, and 6% AE 5%, respectively. Our results demonstrate the reliability and robustness of multi-distance near-infrared spectroscopy measurements based on a homogeneous model in the human forehead on a large sample of human subjects. Absolute, non-invasive optical measurements on the brain, such as those presented here, can significantly advance the development of NIRS technology as a tool for monitoring resting/basal cerebral perfusion, hemodynamics, oxygenation, and metabolism.

Rationale and Objectives Perturbations in cerebral blood volume (CBV), blood flow (CBF), and metabolic rate of oxygen (CMRO2) lead to associated changes in tissue concentrations of oxy- and deoxy-hemoglobin (ΔO and ΔD), which can be measured by near-infrared spectroscopy (NIRS). A novel hemodynamic model has been introduced to relate physiological perturbations and measured quantities. We seek to use this model to determine functional traces of cbv(t) and cbf(t) − cmro2(t) from time-varying NIRS data, and cerebrovascular physiological parameters from oscillatory NIRS data (lowercase letters denote the relative changes in CBV, CBF, and CMRO2 with respect to baseline). Such a practical implementation of a quantitative hemodynamic model is an important step toward the clinical translation of NIRS. Materials and Methods In the time domain, we have simulated O(t) and D(t) traces induced by cerebral activation. In the frequency domain, we have performed a new analysis of frequency-resolved measurements of cerebral hemodynamic oscillations during a paced breathing paradigm. Results We have demonstrated that cbv(t) and cbf(t) − cmro2(t) can be reliably obtained from O(t) and D(t) using the model, and that the functional NIRS signals are delayed with respect to cbf(t) − cmro2(t) as a result of the blood transit time in the microvasculature. In the frequency domain, we have identified physiological parameters (e.g., blood transit time, cutoff frequency of autoregulation) that can be measured by frequency-resolved measurements of hemodynamic oscillations. Conclusions The ability to perform noninvasive measurements of cerebrovascular parameters has far-reaching clinical implications. Functional brain studies rely on measurements of CBV, CBF, and CMRO2, whereas the diagnosis and assessment of neurovascular disorders, traumatic brain injury, and stroke would benefit from measurements of local cerebral hemodynamics and autoregulation.

We report an experimental validation and applications of the new hemodynamic model presented in the companion article (Fantini, 2013, this issue) both in the frequency domain and in the time domain. In the frequency domain, we have performed diffuse optical measurements for coherent hemodynamics spectroscopy (CHS) on the brain and calf muscle of human subjects, showing that the hemodynamic model predictions (both in terms of spectral shapes and absolute spectral values) are confirmed experimentally. We show how the quantitative analysis based on the new model allows for autoregulation measurements from brain data, and provides an analytical description of near-infrared spiroximetry from muscle data. In the time domain, we have used data from the literature to perform a comparison between brain activation signals measured with functional near-infrared spectroscopy (fNIRS) or with blood oxygenation level dependent (BOLD) fMRI, and the corresponding signals predicted by the new model. This comparison shows an excellent agreement between the model predictions and the reported fNIRS and BOLD fMRI signals. This new hemodynamic model provides a valuable tool for brain studies with hemodynamic-based techniques.

Abstract. We present a pilot clinical application of coherent hemodynamics spectroscopy (CHS), a technique to investigate cerebral hemodynamics at the microcirculatory level. CHS relies on frequency-resolved measurements of induced cerebral hemodynamic oscillations that are measured with near-infrared spectroscopy (NIRS) and analyzed with a hemodynamic model. We have used cyclic inflation (200 mmHg) and deflation of a pneumatic cuff placed around the subject's thigh at seven frequencies in the range of 0.03 to 0.17 Hz to generate CHS spectra and to obtain a set of physiological parameters that include the blood transit times in the cerebral microcirculation, the cutoff frequency for cerebral autoregulation, and blood volume ratios across the three different compartments. We have investigated five hemodialysis patients, during the hemodialysis procedure, and six healthy subjects. We have found that the blood transit time in the cerebral microcirculation is significantly longer in hemodialysis patients with respect to healthy subjects. No significant differences were observed between the two groups in terms of autoregulation efficiency and blood volume ratios. The demonstration of the applicability of CHS in a clinical setting and its sensitivity to the highly important cerebral microcirculation may open up new opportunities for NIRS applications in research and in medical diagnostics and monitoring.

Brain microvascular pathology is a common finding in Alzheimer's disease and other dementias. However, the extent to which microvascular abnormalities cause or contribute to cognitive impairment is unclear. Noninvasive near-infrared spectroscopy (NIRS) can address this question, but its use for clarifying the role of microvascular dysfunction in dementia has been limited due to theoretical and practical considerations. We developed a new noninvasive NIRS method to obtain quantitative, dynamic measurements of absolute brain hemoglobin concentration and oxygen saturation and used it to show significant cerebrovascular impairments in a rat model of diet-induced vascular cognitive impairment. We fed young rats folate-deficient (FD) and control diets and measured absolute brain hemoglobin and hemodynamic parameters at rest and during transient mild hypoxia and hypercapnia. With respect to control animals, FD rats featured significantly lower brain hemoglobin concentration (72±4 μmol/L versus 95±6 μmol/L) and oxygen saturation (54%±3% versus 65%±2%). By contrast, resting arterial oxygen saturation was the same for both groups (96%±4%), indicating that decrements in brain hemoglobin oxygenation were independent of blood oxygen carrying capacity. Vasomotor reactivity in response to hypercapnia was also impaired in FD rats. Our results implicate microvascular abnormality and diminished oxygen delivery as a mechanism of cognitive impairment.

We present a quantitative near-IR spectroscopy study of the absolute values of oxygen saturation of hemoglobin before and after surgically induced testicular torsion in adult rabbits. Unilateral testicular torsions (0, 540, or 720 deg) on experimental testes and contralateral sham surgery on control testes are performed in four adult rabbits. A specially designed optical probe for measurements at multiple source-detector distances and a commercial frequency-domain tissue spectrometer are used to measure absolute values of testicular hemoglobin saturation. Our results show: (1) a consistent baseline absolute tissue hemoglobin saturation value of 78+/-5%, (2) a comparable tissue hemoglobin saturation of 77+/-6% after sham surgery, and (3) a significantly lower tissue hemoglobin saturation of 36+/-2% after 540- and 720-deg testicular torsion surgery. Our findings demonstrate the feasibility of performing frequency-domain, multidistance near-IR spectroscopy for absolute testicular oximetry in the assessment of testicular torsion. We conclude that near-IR spectroscopy has potential to serve as a clinical diagnostic and monitoring tool for the assessment of absolute testicular hemoglobin desaturation caused by torsion, with the possibility of serving as a complement to conventional color and spectral Doppler ultrasonography.