SignificanceWidefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale (“mesoscopic”) imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts.AimWe aim to develop a system for mesoscopic imaging of two fluorescent and two reflectance channels.ApproachExcitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm light-emitting diodes positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector.ResultsWe demonstrate the performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake, head-fixed mice with a curved “crystal skull” window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRABACh3.0. Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes.ConclusionsOur widefield microscope design with a single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.
The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada.
This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions.
The editorial shares a collective vision for Kyiv becoming a regional resource for teaching, practicing, and advancing neurophotonics - with a recipe for developing a summer school program as a forward-looking way to start.
Although the beneficial effects of regular physical exercise on brain aging and neurodegenerative diseases are well recognized, a clear understanding of how exercise leads to such benefits remains elusive. In this work, we investigated the effects of normal aging on cortical microvascular oxygenation, perfusion, and morphology and the impact of four months of voluntary wheel running on cortical microvascular oxygenation in 20 months old mice. We used two-photon microscopy to assess age-related and exercise-induced changes in the distributions of capillary oxygen partial pressure (PO2) and red-blood-cell flux across cortical depth in awake mice. Our finding suggests the mitigating effect of exercise on the progression of age-related changes in capillary oxygenation in deeper cortical layers which may be related to health-enhancing benefits of exercise in elderly individuals.
Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics’ agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, in this article we review an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion article, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed and provide an outlook for the future directions.
Neurophotonics editor-in-chief Anna Devor discusses neuroethics topics related to rapidly advancing neurotechnology, in conversation with Institute of Neuroethics founder Karen Rommelfanger.
Focused ultrasound (FUS) is an emerging technology for non-invasive and controlled blood-brain barrier (BBB) opening for drug delivery successfully tested in clinical trials. To improve the safety of this method, we use optical imaging techniques to better understand the relationship between repeated FUS-BBB opening, neuroinflammation, and alteration of neurovascular coupling in an animal model. We perform 1- and 2-photon microscopy in awake mice to image neuronal activity hemodynamics. Before and following FUS treatment, optical imaging sessions access changes in neuronal activity and/or hemodynamics; FUS treatments are repeated several times to approximate a clinical schedule.
Neurophotonics Editor in Chief Anna Devor reflects on need to cherish and cultivate diversity within the global neuroscience and neurophotonics community by creating inclusive environments to welcome young students in sharing the joy of science.
The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase – the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. I has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we test the hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue obtained with 2-photon phosphorescence lifetime microscopy (2PLM). We revisit our previously developed method for extraction of cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and apply this method to estimate baseline CMRO2 in awake mice across cortical layers. Our results show that, in contrary to the common notion, baseline CMRO2 in layer IV is not higher than that in upper layers. We speculate that the cytochrome oxidase band in layer IV may reflect higher metabolic demands during transient surges in neuronal activity. These findings are important for interpretation of cortical-layer-resolved Blood Oxygen Level Dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) studies.
An ongoing effort in many neuroscience laboratories is to perform chronic optical imaging and multiplexed electrophysiological recordings in awake behaving mice. We have adapted the use of conformal thin parylene C in conjunction with conductive polymer PEDOT:PSS coating on the electrode sites to realize wearable microelectrode array devices with sufficient optical transparency to allow simultaneous multiphoton imaging and optogenetic manipulation. These devices replace the current cranial glass windows providing broadband measurements of the extracellular potential as well as a see-through optical access. We have developed a mechanical solution for installation, connectorization and protection of the device and circuit board aiming for lifetime of several months while worn by a mouse. Our design allows access for high numerical aperture microscope objectives and does not obstruct the mouse face and whiskers enabling meaningful behaviors.
Recent advances in pluripotent stem cell technology have enabled generation of neuronal cell lines and cerebral organoids from human induced pluripotent stem cells (hiPSCs). These organoids are self-assembled, 3D cellular structures that resemble early developmental stages of the human brain. However, the lack of the natural brain microenvironment in cultured organoids can influence the phenotype and maturation of the reprogrammed neurons. To mitigate this limitation, cerebral organoids are transplanted into the mouse brain where their activity can be monitored using 2-photon imaging through cranial “windows.” Here, we will replace these windows with optically transparent graphene electrode microgrids to enable multimodal longitudinal monitoring and interrogation of neuronal activity in the graft and the surrounding host neuronal circuits. We envision that this technology can helpful for modeling of disease, examination of candidate treatments, and replacement therapy.
We report optical coherence tomography (OCT) imaging of localized fast optical signals (FOS) arising from whisker stimulation in awake mice. The activated voxels were identified by fitting the OCT intensity signal time course with a response function over a time scale of a few hundred milliseconds after the whisker stimulation. The significantly activated voxels were shown to be localized to the expected brain region for whisker stimulation. The ability to detect functional stimulation-evoked, depth resolved FOS with intrinsic contrast from the cortex provides a new tool for neural activity studies.
Significance: The cerebral metabolic rate of oxygen (CMRO2) is an important indicator of brain function and pathology. Knowledge about its magnitude is also required for proper interpretation of the blood oxygenation level-dependent (BOLD) signal measured with functional MRI. Despite the need for estimating CMRO2, no gold standard exists. Traditionally, the estimation of CMRO2 has been pursued with somewhat indirect approaches combining several different types of measurements with mathematical modeling of the underlying physiological processes. The recent ability to measure the level of oxygen (pO2) in cortex with two-photon resolution in in vivo conditions has provided a more direct way for estimating CMRO2, but has so far only been used to estimate the average CMRO2 close to cortical penetrating arterioles in rats.
Aim: The aim of this study was to propose a method to provide spatial maps of CMRO2 based on two-photon pO2 measurements.
Approach: The method has two key steps. First, the pO2 maps are spatially smoothed to reduce the effects of noise in the measurements. Next, the Laplace operator (a double spatial derivative) in two spatial dimensions is applied on the smoothed pO2 maps to obtain spatially resolved CMRO2 estimates.
Result: The smoothing introduces a bias, and a balance must be found where the effects of the noise are sufficiently reduced without introducing too much bias. In this model-based study, we explored this balance using synthetic model-based data, that is, data where the spatial maps of CMRO2 were preset and thus known. The corresponding pO2 maps were found by solving the Poisson equation, which relates CMRO2 and pO2. MATLAB code for using the method is provided.
Conclusion: Through this model-based study, we propose a method for estimating CMRO2 with high spatial resolution based on measurements of pO2 in cerebral cortex.
In classical Two photon microscopy (TPM), fluorescence excitation happens via absorption of two photons with the same energy. However, the energies of the two photons do not need to be the same: the sum of their energies must be equal to the total energy required for the ground state to excited state transition. This feature allows for non-degenerate two-photon excitation (ND-TPE), where excitation occurs via simultaneous absorption of two photons of different energies derived from two laser beams. ND-TPE has been exploited in fluorescence microscopy to extend the range of excitation wavelengths , increase resolution, increase penetration depth, and mitigate excitation outside of the focal volume.We use non-degenerate two-photon excitation where the two excitation beams are displaced in space outside the focal volume to increase the signal-to-background ratio (SBR), overcoming the fundamental penetration depth limit of conventional two-photon microscopy.
In non-degenerate two-photon excitation (ND-TPE), electronic transition of fluorophores happens via absorption of two photons with different energies. This contrasts with conventional - or degenerate - two-photon excitation (D-TPE), where two photons with identical energies are absorbed. ND-TPE can improve performance of two-photon microscopy by extending the excitation wavelength range, reducing out-of-focus excitation, and increasing resolution and penetration depth. However, a systematic study of fluorophore performance under ND-TPE is missing, which is critical for the selection of optimal excitation wavelength combinations. It is a well-known fact that degenerate two-photon absorption spectra often deviate from theoretical predictions based on one-photon absorption spectra. Therefore, it is not clear whether non-degenerate two-photon absorption spectra are predictable from the corresponding degenerate spectra. Using our sensitive fluorescence-based spectroscopy technique, we measured non-degenerate two-photon absorption cross-sections (ND-TPACS) of several commonly used fluorophores and generated 2-dimensional ND-TPACS maps. We observed that the shape of the measured ND-TPACS spectra follows the spectra of the degenerate two-photon absorption cross-sections (D-TPACS). However, ND-TPACS are higher in magnitude, which is predicted by the “resonant enhancement” phenomenon. Therefore, we show that ND-TPACS spectra are predictable from the corresponding degenerate D-TPACS spectra under consideration of resonant enhancement. Predictability of ND-TPACS spectra is an important finding that helps choosing the optimal combination of wavelengths for ND-TPE of a given fluorophore without prior experimental measurement of ND-TPACS.
KEYWORDS: Functional magnetic resonance imaging, Contamination, Blood, Two photon excitation microscopy, Angiography, Monte Carlo methods, Magnetism, Tissues, Brain, Capillaries
The vascular space occupancy (VASO) fMRI method probes changes in cerebral blood volume (CBV) under various physiological states, including neuronal activation in humans. However, it requires a careful choice of sequence parameters because the blood oxygen-level dependent (BOLD) effect offsets the VASO signal. Assessing this BOLD contamination as a function of pulse sequence parameters would improve the quantification of CBV changes with VASO. However, this task requires knowledge of the cerebral vascular geometry of the MRI voxel. Towards this end, optical microscopy can provide high-resolution 3D images of vasculature. Here, we use detailed angiograms of rodent brain acquired with two-photon microscopy to model fMRI signals (VASO and BOLD) from first principles using Monte Carlo diffusion of water protons. We present quantitative plots of VASO together with intra- and extravascular BOLD fractional signal changes as a function of echo time (TE), for spin echo (SE) and gradient echo (GRE) pulse sequences, at low to ultra-high magnetic fields. Our results indicate that at 3T, the BOLD contamination of the VASO response is under 12% for GRE and 2% for SE up to TE=6 ms, but this contamination is significantly higher at 7T and above. We also found GRE BOLD intravascular contributions of 85% at 1.5T, 55% at 3T and 4% at 7T and SE intravascular contributions of 70% at 1.5T, 40% at 3T and 10% at 7T. These results may provide important information to optimize the pulse sequence timing in human VASO and BOLD fMRI, leading the way to a wider application of these fMRI techniques in healthy and diseased brain.
Previously introduced bundles of hundreds or thousands of microfibers have the potential to extend optical access to deep brain regions, sampling fluorescence activity throughout a three-dimensional volume. Each fiber has a small diameter (8 μm) and follows a path of least resistance, splaying during insertion. By superimposing the fiber sensitivity profile for each fiber, we model the interface properties for a simulated neural population. Our modeling results suggest that for small (<200) bundles of fibers, each fiber will collect fluorescence from a small number of nonoverlapping neurons near the fiber apertures. As the number of fibers increases, the bundle delivers more uniform excitation power to the region, moving to a regime where fibers collect fluorescence from more neurons and there is greater overlap between neighboring fibers. Under these conditions, it becomes feasible to apply source separation to extract individual neural contributions. In addition, we demonstrate a source separation technique particularly suited to the interface. Our modeling helps establish performance expectations for this interface and provides a framework for estimating neural contributions under a range of conditions.
In non-degenerate 2-photon excitation (ND-2PE) microscopy, a fluorophore simultaneously absorbs two photons of different energies. We performed a ND-2PE study of fluorescent proteins and synthetic dyes (eg.eGFP, FITC, and etc.) continuously varying energies and numbers of both photons to create two-dimensional map of fluorescence landscapes. By using the best photon energy combination from our two-dimensional map, we found an increase in detected fluorescent image brightness with ND-2PE as we imaged cortical neurons labeled with enhanced green fluorescent protein (eGFP). It should be noted that the photons corresponding to longer wavelength will penetrate deeper into the tissue at reduced scattering. Additionally, using non-overlapping spatial modes carrying the photons at different energies will significantly reduce out of focus fluorescence from the large number of low energy photons, and by a proper choice of the number of high energy photons the ND-2PE fluorescence can be obtained from deep tissue. Experimentally, we strategically displaced two laser beams until they reached the sample plane such that the unwanted background in the excitation beam path was suppressed. In contrast, these two pump beams were well overlapped at focus which still produced sufficient number fluorescence photons for detection. In our experiment, the temporal alignment was achieved with optical delay line in the optical path of IR beam. With this technique we demonstrated experimentally that ND-2PE with side-by-side beams provided a better signal to background ratio in the scattering phantom as compared with D-2PE. The excitation volume of ND-2PE with side-by-side beam was also investigated and determined to be comparable in size with that of the D-2PE.
Cortical capillary blood flow and oxygenation are highly heterogeneous. Mapping absolute capillary blood flow and oxygenation along capillary path is a key step towards understanding how oxygen is transported and delivered in a complex microvascular network to enable adequate tissue oxygenation. In this work, we applied two-photon microscopic imaging of intravascular oxygen partial pressure (PO2) to measure both oxygen concentration and red blood cell (RBC) flux in cortical arterioles, capillaries, and venules. Imaging was performed in awake, head-restrained C57BL/6 mice (n=15), through a chronic sealed cranial window centered over the E1 whisker barrel. We obtained a detailed mapping of the resting state cortical microvascular PO2 in all arterioles and venules, and both PO2 and RBC flux in most capillaries down to 600 μm depth from the cortical surface (n=6,544 capillaries across all mice). Capillary RBC speed and density were also extracted and all measurements were co-registered with the microvascular angiograms. We characterized the distributions of capillary PO2 and flow as a function of branching order and cortical depth. The results show strong positive correlation between oxygenation and flow in the capillary segments, with an increased correlation in downstream capillaries. We have also observed homogenization of both oxygenation and flow in deeper cortical layers, which may imply a mechanism to improve oxygen delivery without increasing global blood flow in the area with increased metabolism.
We investigate the utility of non-degenerate 2-photon excitation (ND-2PE) as a strategy for extending the 2-photon imaging depth. For the ND-2PE scheme, two pulsed, synchronized laser sources of different wavelength each provide a photon for the 2-photon absorption process. By independently tuning their wavelengths, we are able to tune the excitation to tissue transparency windows while maintaining resonant fluorescence excitation. These transparency windows reduce excitation power loss resulting from scattering. In addition, by having two sources we are able to displace the beams in space except at their common focus; thus, reducing background fluorescence excitation. Finally, we show that ND-2PE inherently results in increased 2-photon absorption cross sections, resulting in increased fluorescence intensity. By combining beam displacement, tissue transparency and increased absorption cross sections, we achieve increased imaging depths as compared to degenerate 2-photon excitation with commonly used fluorophores.
We characterized cortical microvascular PO2 and blood flow changes in response to whisker stimulation in awake mice. The measurements were performed by combining two-photon microscopy imaging of the cortical oxygenation and optical coherence tomography imaging of the cerebral blood flow. In order to perform fast spatio-temporally resolved measurements of PO2, we used a newly-developed oxygen-sensitive probe PtG-2P, which has significantly higher brightness than the established two-photon-enhanced oxygen sensor PtP-C343. We characterized the performance of the new probe in vivo and mapped the amplitudes and shapes (e.g. initial dip, overshoot, and post stimulus undershoot) of the PO2 changes as a function of the vessel type (e.g., arterioles, capillaries, and venules) and a distance from the activation center. The measurements in the awake mice are not affected by the confounding factors of anesthesia on the animal physiology, including the level of cerebral metabolism and the amplitude and speed of neuronal and vascular responses. Our results will help to understand changes in oxygenation and blood flow on the cortical microvascular scale, will lead to improved understanding of the cerebral physiology, pathophysiology and will improve quantitative interpretation of fMRI signals.
The cerebral metabolic rate of oxygen (CMRO2) is an essential parameter for evaluating brain function and pathophysiology. Measurements of CMRO2 with high spatio-temporal resolution are critically important for understanding how the brain copes with metabolic and blood perfusion changes associated with various clinical conditions, such as stroke, periinfarct depolarizations, and various microvasculopathies (e.g., Alzheimer’s disease, chronic hypertension). CMRO2 measurements are also important for understanding the physiological underpinnings of functional Magnetic Resonance Imaging signals. However, the currently available approaches for quantifying CMRO2 rely on complex multimodal imaging and mathematical modeling. Here, we introduce a novel method that allows estimation of CMRO2 based on a single measurement modality - two-photon phosphorescence lifetime microscopy (2PLM) imaging of the partial pressure of oxygen (PO2) in cortical tissue. CMRO2 is estimated by fitting the changes of tissue PO2 around cortical penetrating arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline CMRO2 in anesthetized rats, and modulated tissue PO2 levels by manipulating the depth of anesthesia. This method has a spatial resolution of approximately 200 μm and it may provide CMRO2 measurements in individual cortical layers or within confined cortical regions such as in ischemic penumbra and the foci of functional activation.
Non-degenerate 2-photon excitation of a fluorophore with two laser beams of different photon energies may offer independent degree of freedom in tuning of the photon flux (i.e., the power) for each beam. Wereport a practical demonstration that the emission intensity of a fluorophore excited in the non-degenerate regime in scattering medium is more efficient than the commonly used degenerate 2-photon excitation. In our experiments we use spatially and temporally aligned Ti:Sapphiremode-locked laser and optical parametric oscillator beams operating at near infrared (NIR) and short-wavelength infrared (SWIR) optical frequencies, respectively. The non-degenerate 2-photon excitation mechanism takes advantage of the infrared wavelengths used in 3-photon microscopy to achieve increased penetration depth, while preserving relatively high 2-photon excitation cross section, exceeding that achievable with the 3-photon excitation. Importantly, independent control of power for each beam implies that the flux requirement for the higher photon energy NIR beam, which experiences higher scattering in biological tissue, can be relaxed at the expense of increasing the flux of the lower photon energy SWIR beam which experiences lower scattering, thus promising deeper penetration with higher efficiency of excitation.Applications for in vivo brain imaging will be also discussed.
The cerebral metabolic rate of oxygen (CMRO2) is an essential parameter for evaluating brain function and pathophysiology. However, the currently available approaches for quantifying CMRO2 rely on complex multimodal imaging and mathematical modeling. Here, we introduce a method that allows estimation of CMRO2 based on a single measurement modality—two-photon imaging of the partial pressure of oxygen (PO2) in cortical tissue. We employed two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen-sensitive nanoprobe PtP-C343 to map the tissue PO2 distribution around cortical penetrating arterioles. CMRO2 is subsequently estimated by fitting the changes of tissue PO2 around arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline CMRO2 in anesthetized rats and modulated tissue PO2 levels by manipulating the depth of anesthesia. This method provides CMRO2 measurements localized within ∼200 μm and it may provide oxygen consumption measurements in individual cortical layers or within confined cortical regions, such as in ischemic penumbra and the foci of functional activation.
Quantification of nicotinamide adenine dinucleotide (NADH) changes during functional brain activation and pathological conditions provides critical insight into brain metabolism. Of the different imaging modalities, two-photon laser scanning microscopy (TPLSM) is becoming an important tool for cellular-resolution measurements of NADH changes associated with cellular metabolic changes. However, NADH fluorescence emission is strongly absorbed by hemoglobin. As a result, in vivo measurements are significantly affected by the hemodynamics associated with physiological and pathophysiological manipulations. We model NADH fluorescence excitation and emission in TPLSM imaging based on precise maps of cerebral microvasculature. The effects of hemoglobin optical absorption and optical scattering from red blood cells, changes in blood volume and hemoglobin oxygen saturation, vessel size, and location with respect to imaging location are explored. A simple technique for correcting the measured NADH fluorescence intensity changes is provided, with the utilization of a parallel measurement of a physiologically inert fluorophore. The model is applied to TPLSM measurements of NADH fluorescence intensity changes in rat somatosensory cortex during mild hypoxia and hyperoxia. The general approach of the correction algorithm can be extended to other TPLSM measurements, where changes in the optical properties of the tissue confound physiological measurements, such as the detection of calcium dynamics.
We present a new method for imaging of oxygen in biological samples with micron-scale resolution and threedimensional
capability. Our technique combines phosphorescence quenching with multiphoton laser scanning
microscopy. It is termed two-photon phosphorescence lifetime microscopy (2PLM). 2PLM is made possible due to
specially designed dendritic probes, in which phosphorescence of metalloporphyrins upon two-photon excitation is
enhanced by intramolecular Förster-type energy transfer from two-photon antennae chromophores, located in the same
probe molecule. Oxygen sensitivity of the probes is regulated by dendritic encapsulation of the core metalloporphyrin,
while peripheral PEG groups on the dendrimer isolate the probes from contacts with biological macromolecules (e.g.
proteins, nucleic acids etc) in the environment. Instrumentation and resolution of the method are discussed along with
approaches to intracellular delivery of the probes. 2PLM was validated in pilot imaging experiments and used to obtain
depth-resolved high-resolution oxygen maps in vivo in the brain.
Absorption or fluorescence-based two-dimensional (2-D) optical imaging is widely employed in functional brain imaging. The image is a weighted sum of the real signal from the tissue at different depths. This weighting function is defined as "depth sensitivity." Characterizing depth sensitivity and spatial resolution is important to better interpret the functional imaging data. However, due to light scattering and absorption in biological tissues, our knowledge of these is incomplete. We use Monte Carlo simulations to carry out a systematic study of spatial resolution and depth sensitivity for 2-D optical imaging methods with configurations typically encountered in functional brain imaging. We found the following: (i) the spatial resolution is <200 μm for NA ≤0.2 or focal plane depth ≤300 μm. (ii) More than 97% of the signal comes from the top 500 μm of the tissue. (iii) For activated columns with lateral size larger than spatial resolution, changing numerical aperature (NA) and focal plane depth does not affect depth sensitivity. (iv) For either smaller columns or large columns covered by surface vessels, increasing NA and/or focal plane depth may improve depth sensitivity at deeper layers. Our results provide valuable guidance for the optimization of optical imaging systems and data interpretation.
The relationship between measurements of cerebral blood oxygenation and neuronal activity is highly complex and depends on both neurovascular and neurometabolic biological coupling. While measurements of blood oxygenation changes via optical and MRI techniques have been developed to map functional brain activity, there is evidence that the specific characteristics of these signals are sensitive to the underlying vascular physiology and structure of the brain. Since baseline blood flow and oxygen saturation may vary between sessions and across subjects, functional blood oxygenation changes may be a less reliable indicator of brain activity in comparison to blood flow and metabolic changes. In this work, we use a biomechanical model to examine the relationships between neural, vascular, metabolic, and hemodynamic responses to parametric whisker stimulation under both normal and hypercapnic conditions in a rat model. We find that the relationship between neural activity and oxy- and deoxyhemoglobin changes is sensitive to hypercapnia-induced changes in baseline cerebral blood flow. In contrast, the underlying relationships between evoked neural activity, blood flow, and model-estimated oxygen metabolism changes are unchanged by the hypercapnic challenge. We conclude that evoked changes in blood flow and cerebral oxygen metabolism are more closely associated with underlying evoked neuronal responses.
Laminar Optical Tomography (LOT) is a new medical imaging modality for high-resolution, depth-resolved, functional imaging of superficial tissue such as rodent cortex, skin and the retina. LOT uses visible laser light to image to depths of >2mm (far deeper than microscopy) and is highly sensitive to absorption and fluorescence contrast, enabling spectroscopic functional information such as hemoglobin oxygenation to be imaged with 100-200 micron resolution.
LOT has been used to image the hemodynamic response to stimulus in the somatosensory cortex of rats. The resulting three-dimensional (3D) images through the depth of the cortex can be used to delineate the arterial, capillary and venous responses, revealing new information about the intricacies of the oxygenation and blood flow dynamics related to neuronal activation. Additional applications of LOT are being explored, including the integration of 3D Voltage Sensitive Dye fluorescence imaging.
LOT imaging uses a system similar to a confocal microscope, quickly scanning a focused beam of light over the surface of the tissue (~8Hz frame rate). Light is detected from both the focus of the scanning beam, and also at increasing distances from the beam's focus. This scattered light has penetrated more deeply into the tissue, and allows features at different depths to be distinguished. An algorithm that includes photon migration modeling of light scattering converts the raw data into 3D images. The motivation for functional optical imaging will be outlined, the basic principles of LOT imaging will be described, and the latest in-vivo results will be presented.
Simultaneous optical coherence tomography (OCT) and video microscopy were performed on the rat somatosensory
cortex through a thinned skull during forepaw stimulation. Fractional change measurements in OCT images reveal a
functional signal timecourse similar to well understood hemodynamic signal timecourses measured with video
microscopy. The precise etiology of the observed OCT functional signal is still under investigation, but these results
suggest that OCT can provide high-resolution cross-sectional images of functional neuro-vascular activation.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.