Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms.

We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D 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 3D 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 critical role for the vascular endothelium in functional neurovascular coupling in the brain.

BACKGROUND: The functional modulation of blood flow in the brain is critical for brain health and is the basis of contrast in functional magnetic resonance imaging. There is evident coupling between increases in neuronal activity and increases in local blood flow; however, many aspects of this neurovascular coupling remain unexplained by current models. Based on the rapid dilation of distant pial arteries during cortical functional hyperemia, we hypothesized that endothelial signaling may play a key role in the long-range propagation of vasodilation during functional hyperemia in the brain. Although well characterized in the peripheral vasculature, endothelial involvement in functional neurovascular coupling has not been demonstrated. METHODS AND RESULTS: We combined in vivo exposed-cortex multispectral optical intrinsic signal imaging (MS-OISI) with a novel in vivo implementation of the light-dye technique to record the cortical hemodynamic response to somatosensory stimulus in rats before and after spatially selective endothelial disruption. We demonstrate that discrete interruption of endothelial signaling halts propagation of stimulus-evoked vasodilation in pial arteries, and that wide-field endothelial disruption in pial arteries significantly attenuates the hemodynamic response to stimulus, particularly the early, rapid increase and peak in hyperemia. CONCLUSIONS: Involvement of endothelial pathways in functional neurovascular coupling provides new explanations for the spatial and temporal features of the hemodynamic response to stimulus and could explain previous results that were interpreted as evidence for astrocyte-mediated control of functional hyperemia. Our results unify many aspects of blood flow regulation in the brain and body and prompt new investigation of direct links between systemic cardiovascular disease and neural deficits.

Calcium imaging of infrared-stimulated activity in rodent brain.

Infrared neural stimulation (INS) is a promising neurostimulation technique that can activate neural tissue with high spatial precision and without the need for exogenous agents. However, little is understood about how infrared light interacts with neural tissue on a cellular level, particularly within the living brain. In this study, we use calcium sensitive dye imaging on macroscopic and microscopic scales to explore the spatiotemporal effects of INS on cortical calcium dynamics. The INS-evoked calcium signal that was observed exhibited a fast and slow component suggesting activation of multiple cellular mechanisms. The slow component of the evoked signal exhibited wave-like properties suggesting network activation, and was verified to originate from astrocytes through pharmacology and 2-photon imaging. We also provide evidence that the fast calcium signal may have been evoked through modulation of glutamate transients. This study demonstrates that pulsed infrared light can induce intracellular calcium modulations in both astrocytes and neurons, providing new insights into the mechanisms of action of INS in the brain.

Direct, intraoperative observation of ~0.1 Hz hemodynamic oscillations in awake human cortex: implications for fMRI.

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.

A hands-on course teaching bioinstrumentation through the design and construction of a benchtop cardiac pacemaker.

We have developed a bioinstrumentation course that emphasizes practical application of engineering and biological concepts by having students focus on the development of a single biomedical device: a cardiac pacemaker. In creating their benchtop pacemaker, students learn about and design sensing circuitry, data acquisition and processing code, control system algorithms, and stimulation electronics. They also gain an understanding of cardiac anatomy and electrophysiology. The separate elements of the pacemaker created throughout the semester will be repeatedly tested, re-designed, and integrated with one another, culminating in an emulated pacemaker whose efficacy will be tested on North American bullfrogs. It is hypothesized that the hands-on learning in this course, coupled with the practical application of concepts in the context of a single biomedical device, will enhance students' skills in bioinstrumentation design.

Resolving the transition from negative to positive blood oxygen level-dependent responses in the developing brain.

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.

Dynamic contrast-enhanced optical imaging of in vivo organ function.

Conventional approaches to optical small animal molecular imaging suffer from poor resolution, limited sensitivity, and unreliable quantitation, often reducing their utility in practice. We previously demonstrated that the in vivo dynamics of an injected contrast agent could be exploited to provide high-contrast anatomical registration, owing to the temporal differences in each organ's response to the circulating fluorophore. This study extends this approach to explore whether dynamic contrast-enhanced optical imaging (DyCE) can allow noninvasive, in vivo assessment of organ function by quantifying the differing cellular uptake or wash-out dynamics of an agent in healthy and damaged organs. Specifically, we used DyCE to visualize and measure the organ-specific uptake dynamics of indocyanine green before and after induction of transient liver damage. DyCE imaging was performed longitudinally over nine days, and blood samples collected at each imaging session were analyzed for alanine aminotransferase (ALT), a liver enzyme assessed clinically as a measure of liver damage. We show that changes in DyCE-derived dynamics of liver and kidney dye uptake caused by liver damage correlate linearly with ALT concentrations, with an r2 value of 0.91. Our results demonstrate that DyCE can provide quantitative, in vivo, longitudinal measures of organ function with inexpensive and simple data acquisition.

In vivo optical imaging and dynamic contrast methods for biomedical research.

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).

High-speed vascular dynamics of the hemodynamic response.

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.

SPLASSH: Open source software for camera-based high-speed, multispectral in-vivo optical image acquisition.

Camera-based in-vivo optical imaging can provide detailed images of living tissue that reveal structure, function, and disease. High-speed, high resolution imaging can reveal dynamic events such as changes in blood flow and responses to stimulation. Despite these benefits, commercially available scientific cameras rarely include software that is suitable for in-vivo imaging applications, making this highly versatile form of optical imaging challenging and time-consuming to implement. To address this issue, we have developed a novel, open-source software package to control high-speed, multispectral optical imaging systems. The software integrates a number of modular functions through a custom graphical user interface (GUI) and provides extensive control over a wide range of inexpensive IEEE 1394 Firewire cameras. Multispectral illumination can be incorporated through the use of off-the-shelf light emitting diodes which the software synchronizes to image acquisition via a programmed microcontroller, allowing arbitrary high-speed illumination sequences. The complete software suite is available for free download. Here we describe the software's framework and provide details to guide users with development of this and similar software.

Ultra-fast multispectral optical imaging of cortical oxygenation, blood flow, and intracellular calcium dynamics.

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.

A system for high-resolution depth-resolved optical imaging of fluorescence and absorption contrast.

Laminar optical tomography (LOT) is a new three-dimensional in vivo functional optical imaging technique. Adopting a microscopy-based setup and diffuse optical tomography (DOT) imaging principles, LOT can perform both absorption- and fluorescence-contrast imaging with higher resolution (100-200 microm) than DOT and deeper penetration (2-3 mm) than laser scanning microscopy. These features, as well as a large field of view and acquisition speeds up to 100 frames per second, make LOT suitable for depth-resolved imaging of stratified tissues such as retina, skin, endothelial tissues and the cortex of the brain. In this paper, we provide a detailed description of a new LOT system design capable of imaging both absorption and fluorescence contrast, and present characterization of its performance using phantom studies.

Simultaneous multiwavelength laminar optical tomography.

Spatially resolved reflectance measurements can be used to characterize the depth-resolved optical properties of superficial tissues. However, until now, rapid acquisition of multiwavelength data has been hindered by multiplexing problems. We report on a novel multiwavelength laminar optical tomography system capable of acquiring data from multiple source-detector separations at three wavelengths simultaneously. Such data can allow in vivo depth-resolved spectroscopic imaging of absorbers, such as oxy- and deoxyhemoglobin, or of multiple fluorophores, that is unaffected by motion artifacts at frame rates exceeding 100 Hz. The system design and phantom validation studies are presented.

Hyperspectral in vivo two-photon microscopy of intrinsic contrast.

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.

Technical considerations in longitudinal multispectral small animal molecular imaging.

In a previous study, we investigated physical methods to reduce whole-body, diet-related autofluorescence interference in several mouse strains through changes in animal diet. Measurements of mice with an in vivo multispectral imaging system over a 21-day period allowed for the quantification of concentration changes in multiple in vivo fluorophores. To be an effective instrument, a multispectral imaging system requires a priori spectral knowledge, the form and importance of which is not necessarily intuitive, particularly when noninvasive in vivo longitudinal imaging studies are performed. Using an optimized spectral library from a previous autofluorescence-reduction study as a model, we investigated two additional spectral definition techniques to illustrate the results of poor spectral definition in a longitudinal fluorescence imaging study. Here we systematically evaluate these results and show how poor spectral definition can lead to physiologically irrelevant results. This study concludes that the proper selection of robust spectra corresponding to each specific fluorescent molecular label of interest is of integral importance to enable effective use of multispectral imaging techniques in longitudinal fluorescence studies.

Depth-resolved optical imaging and microscopy of vascular compartment dynamics during somatosensory stimulation.

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.

Depth-resolved optical imaging of transmural electrical propagation in perfused heart.

We present a study of the 3-dimensional (3D) propagation of electrical waves in the heart wall using Laminar Optical Tomography (LOT). Optical imaging contrast is provided by a voltage sensitive dye whose fluorescence reports changes in membrane potential. We examined the transmural propagation dynamics of electrical waves in the right ventricle of Langendorf perfused rat hearts, initiated either by endo-cardial or epi-cardial pacing. 3D images were acquired at an effective frame rate of 667Hz. We compare our experimental results to a mathematical model of electrical transmural propagation. We demonstrate that LOT can clearly resolve the direction of propagation of electrical waves within the cardiac wall, and that the dynamics observed agree well with the model of electrical propagation in rat ventricular tissue.