Camera-based optical imaging of the exposed brain allows cortical hemodynamic responses to stimulation to be examined. Typical multispectral imaging systems utilize a camera and illumination at several wavelengths, allowing discrimination between changes in oxy-and deoxyhemoglobin concentration. However, most multispectral imaging systems utilize white light sources and mechanical filter wheels to multiplex illumination wavelengths, which are slow and difficult to synchronize at high frame rates. We present a new LED-based system capable of high-resolution multispectral imaging at frame rates exceeding 220 Hz. This improved performance enables simultaneous visualization of hemoglobin oxygenation dynamics within single vessels, changes in vessel diameters, blood flow dynamics from the motion of erythrocytes, and dynamically changing fluorescence.

While a range of cellular mechanisms have been proposed to underlie control of neurovascular coupling, a comprehensive, reconciliatory model has yet to be determined. To fit with such a model, it is essential that candidate mechanisms exhibit reaction times, spatial ranges and speeds of propagation that are consistent with the vascular manifestations of the ‘hemodynamic response’. Understanding these vascular dynamics is therefore a critical step towards developing a robust model of neurovascular coupling. In this study, we utilize highspeed optical imaging of exposed rodent somatosensory cortex to explore and characterize the spatiotemporal dynamics of surface vessels during functional hyperemia. Our high-speed, high resolution optical imaging approach allows us to study the hemodynamic response independently in individual vessels, and in discrete regions of the parenchyma with enough resolution to precisely characterize subtle spatial and temporal features of the response. Specifically, we explore when and where the first hemodynamic changes occur in response to stimuli, the direction and speed at which these changes propagate in arterioles and regions of the parenchyma, and the relative timing at which each of these compartments returns to its original baseline state. From these results, we are able to conclude that the hemodynamic response is initiated in the parenchyma and then spreads rapidly to surface arterioles. Following the initial onset we find evidence that the response spreads spatially outwards via the dilation of targeted arterioles. This propagation of vasodilation is independent of the direction of blood flow within each arteriole. We also find evidence of a decay phase that acts with a more uniform spatial dependence, rather than along targeted vessels, causing the periphery of the responding region to return to baseline first. We hypothesize that different underlying cellular mechanisms/signaling pathways are responsible for the response initiation and the response decay. Our results advance a fundamental understanding of the hemodynamic response, as well as our ability to evaluate potential cellular mechanisms for their involvement in neurovascular coupling.

Vasodilatory prostaglandins play a key role in neurovascular coupling (NVC),

In vivo two-photon imaging of intrinsic contrast can provide valuable information about structural tissue elements such as collagen and elastin and fluorescent metabolites such as nicotinamide adenine dinucleotide. Yet low signal and overlapping emission spectra can make it difficult to identify and delineate these species in vivo. We present a novel approach that combines excitation scanning with spectrally resolved emission two-photon microscopy, allowing distinct structures to be delineated based on their characteristic spectral fingerprints. The amounts of intrinsic fluorophores present in each voxel can also be evaluated. We demonstrate our method using in vivo imaging of nude mouse skin.Two-photon microscopy allows high-resolution imaging of in vivo intact tissues. Intrinsic contrast from nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), collagen, elastin, and keratin [1][2][3] can allow measurement of metabolism and native structure without the need to introduce artificial compounds. However, analysis of intrinsic fluorophores (and some exogenous contrast agents) is complicated by their broad overlapping emission spectra [4,5]. Reliable imaging of intrinsic contrast on microscopic-length scales in tissues such as the heart, brain, skin, and tumors could provide important insights into cellular function and allow improved interpretation and validation of diagnostics based on in vivo bulk-tissue measurements [3]. In this Letter we demonstrate a hyperspectral twophoton data acquisition and spectral unmixing technique that can delineate and quantify the concentrations of multiple intrinsic fluorophores and sources of second-harmonic generation (SHG) in vivo. We show that limited spectral data can be used to segment distinct structures within 3D volumes, based on the specific excitation-emission fingerprints of each structure's constituents. We also demonstrate that hyperspectral microscopy can allow extraction of the relative concentrations of specific intrinsic fluorophores, effectively yielding in vivo, spatially resolved chemical analysis of fine submicrometer structure.All measurements were made using our home-built two-photon microscope system, which includes three photomultiplier detectors (350-505 nm, 505-560 nm, and 560-650 nm emission ranges, R3896 Hamamatsu), a Ti:sapphire laser (MaiTai XF, Spectra Physics), and an Olympus XLUMPlanFl 20×/0.95 W objective, mounted on a fine z-adjustment stage (M-112.1DG, PI). Our integrated control software written in MATLAB allows synchronous tuning of the laser wavelength during imaging [6].Three male nude mice (40-45 g) were anesthetized with isoflurane and positioned on a warming pad on the microscope's x-y stage. One ear was moistened with water and held flat between a microscope slide and a glass cover slip. Data sets were acquired in two ways: Wavelength NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript scans consisted of 400 × 400 pixel images captured in synchrony with the laser being tuned from 710...