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We report a three-dimensional microscopy technique—swept, confocally-aligned planar excitation (SCAPE) microscopy—that allows volumetric imaging of living samples at ultrahigh speeds. Although confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image three-dimensional volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light sheet in a single-objective, en face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless three-dimensional imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae. A swept light-sheet microscopy scheme allows volumetric imaging of living samples at high speed.

The adult brain exhibits a local increase in cortical blood flow in response to external stimulus. However, broadly varying hemodynamic responses in the brains of newborn and young infants have been reported. Particular controversy exists over whether the \"true\" neonatal response to stimulation consists of a decrease or an increase in local deoxyhemoglobin, corresponding to a positive (adult-like) or negative blood oxygen level-dependent (BOLD) signal in functional magnetic resonance imaging (fMRI), respectively. A major difficulty with previous studies has been the variability in human subjects and measurement paradigms. Here, we present a systematic study in neonatal rats that charts the evolution of the cortical blood flow response during postnatal development using exposed-cortex multispectral optical imaging. We demonstrate that postnatal-day-12-13 rats (equivalent to human newborns) exhibit an \"inverted\" hemodynamic response (increasing deoxyhemoglobin, negative BOLD) with early signs of oxygen consumption followed by delayed, active constriction of pial arteries. We observed that the hemodynamic response then matures via development of an initial hyperemic (positive BOLD) phase that eventually masks oxygen consumption and balances vasoconstriction toward adulthood. We also observed that neonatal responses are particularly susceptible to stimulus-evoked systemic blood pressure increases, leading to cortical hyperemia that resembles adult positive BOLD responses. We propose that this confound may account for much of the variability in prior studies of neonatal cortical hemodynamics. Our results suggest that functional magnetic resonance imaging studies of infant and child development may be profoundly influenced by the maturing neurovascular and autoregulatory systems of the neonatal brain.

The cortical hemodynamic response to somatosensory stimulus is investigated at the level of individual vascular compartments using both depth-resolved optical imaging and in-vivo two-photon microscopy. We utilize a new imaging and spatiotemporal analysis approach that exploits the different characteristic dynamics of responding arteries, arterioles, capillaries and veins to isolate their three-dimensional spatial extent within the cortex. This spatial delineation is validated using vascular casts. Temporal delineation is supported by in-vivo two-photon microscopy of the temporal dynamics and vascular mechanisms of the arteriolar and venous responses. Using these techniques we have been able to characterize the roles of the different vascular compartments in generating and controlling the hemodynamic response to somatosensory stimulus. We find that changes in arteriolar total hemoglobin concentration agree well with arteriolar dilation dynamics, which in turn correspond closely with changes in venous blood flow. For 4-s stimuli, we see only small changes in venous hemoglobin concentration, and do not detect measurable dilation or ballooning in the veins. Instead, we see significant evidence of capillary hyperemia. We compare our findings to historical observations of the composite hemodynamic response from other modalities including functional magnetic resonance imaging. Implications of our results are discussed with respect to mathematical models of cortical hemodynamics, and to current theories on the mechanisms underlying neurovascular coupling. We also conclude that our spatiotemporal analysis approach is capable of isolating and localizing signals from the capillary bed local to neuronal activation, and holds promise for improving the specificity of other hemodynamic imaging modalities.

An almost sinusoidal, large amplitude ~0.1 Hz oscillation in cortical hemodynamics has been repeatedly observed in species ranging from mice to humans. However, the occurrence of 'slow sinusoidal hemodynamic oscillations' (SSHOs) in human functional magnetic resonance imaging (fMRI) studies is rarely noted or considered. As a result, little investigation into the cause of SSHOs has been undertaken, and their potential to confound fMRI analysis, as well as their possible value as a functional biomarker has been largely overlooked. Here, we report direct observation of large-amplitude, sinusoidal ~0.1 Hz hemodynamic oscillations in the cortex of an awake human undergoing surgical resection of a brain tumor. Intraoperative multispectral optical intrinsic signal imaging (MS-OISI) revealed that SSHOs were spatially localized to distinct regions of the cortex, exhibited wave-like propagation, and involved oscillations in the diameter of specific pial arterioles, indicating that the effect was not the result of systemic blood pressure oscillations. fMRI data collected from the same subject 4 days prior to surgery demonstrates that ~0.1 Hz oscillations in the BOLD signal can be detected around the same region. Intraoperative optical imaging data from a patient undergoing epilepsy surgery, in whom sinusoidal oscillations were not observed, is shown for comparison. This direct observation of the '0.1 Hz wave' in the awake human brain, using both intraoperative imaging and pre-operative fMRI, confirms that SSHOs occur in the human brain, and can be detected by fMRI. We discuss the possible physiological basis of this oscillation and its potential link to brain pathologies, highlighting its relevance to resting-state fMRI and its potential as a novel target for functional diagnosis and delineation of neurological disease.

This paper provides an overview of optical imaging methods commonly applied to basic research applications. Optical imaging is well suited for non-clinical use, since it can exploit an enormous range of endogenous and exogenous forms of contrast that provide information about the structure and function of tissues ranging from single cells to entire organisms. An additional benefit of optical imaging that is often under-exploited is its ability to acquire data at high speeds; a feature that enables it to not only observe static distributions of contrast, but to probe and characterize dynamic events related to physiology, disease progression and acute interventions in real time. The benefits and limitations of in vivo optical imaging for biomedical research applications are described, followed by a perspective on future applications of optical imaging for basic research centred on a recently introduced real-time imaging technique called dynamic contrast-enhanced small animal molecular imaging (DyCE).

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 high-speed 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 appears to initiate 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 the fundamental understanding of the hemodynamic response, as well as our ability to evaluate potential cellular mechanisms for their involvement in neurovascular coupling. ►High frame rate optical imaging resolves rapid dynamics of cortical vasculature. ►Functional response begins in the parenchyma, before dilation of pial arterioles. ►Response proceeds with retrograde propagation of vasodilation in pial arterioles. ►Direction of propagation in arterioles is independent of direction of blood flow. ►Return to baseline may have different underlying mechanism compared to onset.

An almost sinusoidal, large amplitude ~0.1Hz oscillation in cortical hemodynamics has been repeatedly observed in species ranging from mice to humans. However, the occurrence of ‘slow sinusoidal hemodynamic oscillations’ (SSHOs) in human functional magnetic resonance imaging (fMRI) studies is rarely noted or considered. As a result, little investigation into the cause of SSHOs has been undertaken, and their potential to confound fMRI analysis, as well as their possible value as a functional biomarker has been largely overlooked. Here, we report direct observation of large-amplitude, sinusoidal ~0.1Hz hemodynamic oscillations in the cortex of an awake human undergoing surgical resection of a brain tumor. Intraoperative multispectral optical intrinsic signal imaging (MS-OISI) revealed that SSHOs were spatially localized to distinct regions of the cortex, exhibited wave-like propagation, and involved oscillations in the diameter of specific pial arterioles, indicating that the effect was not the result of systemic blood pressure oscillations. fMRI data collected from the same subject 4days prior to surgery demonstrates that ~0.1Hz oscillations in the BOLD signal can be detected around the same region. Intraoperative optical imaging data from a patient undergoing epilepsy surgery, in whom sinusoidal oscillations were not observed, is shown for comparison. This direct observation of the ‘0.1Hz wave’ in the awake human brain, using both intraoperative imaging and pre-operative fMRI, confirms that SSHOs occur in the human brain, and can be detected by fMRI. We discuss the possible physiological basis of this oscillation and its potential link to brain pathologies, highlighting its relevance to resting-state fMRI and its potential as a novel target for functional diagnosis and delineation of neurological disease. •Direct observation of ~0.1Hz oscillation in awake human cortex.•Oscillation in frequency range of resting state function connectivity analysis.•Oscillation delineates vascular networks.•~0.1Hz oscillations are also observed in pre-operative fMRI BOLD signal.•Potential novel target for functional diagnosis of neurological disease.

Functional optical imaging encompasses the use of optical imaging techniques to study living biological systems in their native environments. Optical imaging techniques are well-suited for functional imaging because they are minimally-invasive, use non ionizing radiation, and derive contrast from a wide range of biological molecules. Modern transgenic labeling techniques, active and inactive exogenous agents, and intrinsic sources of contrast provide specific and dynamic markers of in-vivo processes at subcellular resolution. A central challenge in building functional optical imaging systems is to acquire data at high enough spatial and temporal resolutions to be able to resolve the in-vivo process(es) under study. This challenge is particularly highlighted within neuroscience where considerable effort in the field has focused on studying the structural and functional relationships within complete neurovascular units in the living brain. Many existing functional optical techniques are limited in meeting this challenge by their imaging geometries, light source(s), and/or hardware implementations. In this thesis we describe the design, construction, and application of novel 2D and 3D optical imaging systems to address this central challenge with a specific focus on functional neuroimaging applications. The 2D system is an ultra-fast, multispectral, wide-field imaging system capable of imaging 7.5 times faster than existing technologies. Its camera-first design allows for the fastest possible image acquisition rates because it is not limited by synchronization challenges that have hindered previous multispectral systems. We present the development of this system from a bench top instrument to a portable, low-cost, modular, open source, laptop based instrument. The constructed systems can acquire multispectral images at > 75 frames per second with image resolutions up to 512 x 512 pixels. This increased speed means that spectral analysis more accurately reflects the instantaneous state of tissues and allows for significantly improved tracking of moving objects. We describe 3 quantitative applications of these systems to in-vivo research and clinical studies of cortical imaging and calcium signaling in stem cells. The design and source code of the portable system was released to the greater scientific community to help make high-speed, multispectral imaging more accessible to a larger number of dynamic imaging applications, and to foster further development of the software package. The second system we developed is an entirely new, high-speed, 3D fluorescence microscopy platform called Laser-Scanning Intersecting Plane Tomography (L-SIPT). L-SIPT uses a novel combination of light-sheet illumination and off-axis detection to provide en-face 3D imaging of samples. L-SIPT allows samples to move freely in their native environments, enabling a range of experiments not possible with previous 3D optical imaging techniques. The constructed system is capable of acquiring 3D images at rates >20 volumes per second (VPS) with volume resolutions of 1400 x 50 x 150 pixels, over a 200 fold increase over conventional laser scanning microscopes. Spatial resolution is set by choice of telescope design. We developed custom opto-mechanical components, computer raytracing models to guide system design and to characterize the technique's fundamental resolution limits, and phantoms and biological samples to refine the system's performance capabilities. We describe initial applications development of the system to image freely moving, transgenic Drosophila melanogaster larvae, 3D calcium signaling and hemodynamics in transgenic and exogenously labeled rodent cortex in-vivo, and 3D calcium signaling in acute transgenic rodent cortical brain slices in-vitro.

The adult brain exhibits a local increase in cortical blood flow in response to external stimulus. However, broadly varying hemodynamic responses in the brains of newborn and young infants have been reported. Particular controversy exists over whether the “true” neonatal response to stimulation consists of a decrease or an increase in local deoxyhemoglobin, corresponding to a positive (adult-like) or negative blood oxygen level-dependent (BOLD) signal in functional magnetic resonance imaging (fMRI), respectively. A major difficulty with previous studies has been the variability in human subjects and measurement paradigms. Here, we present a systematic study in neonatal rats that charts the evolution of the cortical blood flow response during postnatal development using exposed-cortex multispectral optical imaging. We demonstrate that postnatal-day-12–13 rats (equivalent to human newborns) exhibit an “inverted” hemodynamic response (increasing deoxyhemoglobin, negative BOLD) with early signs of oxygen consumption followed by delayed, active constriction of pial arteries. We observed that the hemodynamic response then matures via development of an initial hyperemic (positive BOLD) phase that eventually masks oxygen consumption and balances vasoconstriction toward adulthood. We also observed that neonatal responses are particularly susceptible to stimulus-evoked systemic blood pressure increases, leading to cortical hyperemia that resembles adult positive BOLD responses. We propose that this confound may account for much of the variability in prior studies of neonatal cortical hemodynamics. Our results suggest that functional magnetic resonance imaging studies of infant and child development may be profoundly influenced by the maturing neurovascular and autoregulatory systems of the neonatal brain.