The human brain receives twenty percent of cardiac output. Resultantly, the blood supply meets approximately 2.5 times the average metabolic demand of the brain. The main payload of this blood supply is molecular oxygen bound to erythrocytes and dissociated into plasma, where both short- and long-term deprivation can have significant physiological impact. During a stroke, the supply of blood carrying the oxygen is severely reduced or completely diminished, resulting in decreased neuronal function and cell death from ischemia despite the seemingly global oversupply versus metabolic demand. Therefore, understanding the bottlenecks in supply is vital for characterizing the cerebrovascular plumbing, which can benefit from both quantitative and high resolution mapping.
Unraveling the cerebrovascular architecture is an active area of physiological research, currently only understood from the perspective of large regional supply routes dominated by specific arterial branches of the carotid arteries. In brief, supply routes stemming from the meninges feed the cerebral cortex, where branches of the anterior, medial, and posterior cerebral arteries supply large segments of the functional gray matter. More locally, arterioles are critical to supplying the cortical microvascular network, which begin as pial arterioles on the surface and then dive into the cortical layers. These vessels are responsible for oxygenating the gray matter and predominantly return to draining venules on the cortical surface.
We utilize multiphoton fluorescence and phosphorescence lifetime microscopy to render the microvascular architecture and dissolved oxygen concentrations, respectively, in vessels supplying the cortical layers of the mouse brain in vivo. The nonlinear imaging technique provides exceptionally localized excitation within the tissue volume, enabling three-dimensional imaging by scanning and stepping the illumination in space. By using fluorescent contrast agents and proteins both the vascular and tissue environments can be labeled precisely in vivo. Beyond providing contrast, specialized phosphorescent probes can be utilized to optically sense the local dissolved oxygen tension (pO2) within the nonlinear excitation volume. Given the precise excitation technique, sensitive detection elements and electronics are utilized to register the emission signals with high SNR and temporal resolution.
We present a review of imaging deep-tissue structures with multiphoton microscopy. We examine the effects of light scattering and absorption due to the optical properties of biological sample and identify 1300 nm and 1700 nm as ideal excitation wavelengths. We summarize the availability of fluorophores for multiphoton microscopy as well as ultrafast laser sources to excite available fluorophores. Lastly, we discuss the applications of multiphoton microscopy for neuroscience.
Two-color multiphoton microscopy through wavelength mixing of synchronized lasers has been shown to increase the spectral window of excitable fluorophores without the need for wavelength tuning. However, most currently available dual output laser sources rely on the costly and complicated optical parametric generation approach. In this report, we detail a relatively simple and low cost diamond Raman laser pumped by a ytterbium fiber amplifier emitting at 1055 nm, which generates a first Stokes emission centered at 1240 nm with a pulse width of 100 fs. The two excitation wavelengths of 1055 and 1240 nm, along with the effective two-color excitation wavelength of 1140 nm, provide an almost complete coverage of fluorophores excitable within the range of 1000–1300 nm. When compared with 1055 nm excitation, two-color excitation at 1140 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (for example, tdKatushka2). We demonstrate multicolor imaging of tdKatushka2 and Hoechst 33342 via simultaneous two-color two-photon, and two-color three-photon microscopy in engineered 3D multicellular spheroids. We further discuss potential benefits and applications for two-color three-photon excitation. In addition, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation.
We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. By combining the high repetition rate (511 kHz) and high pulse energy (400 nJ) of our amplifier laser system, we demonstrate imaging of vasculature labeled with Texas Red and Indocyanine Green, and neurons expressing tdTomato and yellow fluorescent protein. We measure the blood flow speed of a single capillary at a depth of 1.2 mm, and image vasculature to a depth of 1.53 mm with fine axial steps (5 μm) and reasonable acquisition times. The high image quality enabled analysis of vascular morphology at depths to 1.45 mm.
Here we demonstrate that a mode-locked ytterbium fiber laser for two-photon fluorescence microscopy can be built for $13,000. The laser emits at a wavelength of 1060 nm with a usable average power of 1 W at a repetition rate of 40 MHz and a compressed pulse width of 81 fs at the sample. The laser is used to obtain deep in vivo two-color images of layer-V pyramidal neurons expressing YFP and vasculature labelled with Texas Red at depths up to 900 µm. The sub-1 µm features of dendritic spines can be resolved at a 200 µm depth.
Monitoring the progression of the vascular structure and cerebral blood flow (CBF) after brain injury is vital to understand the neurovascular recovery process. Multiexposure speckle imaging (MESI) provides a quantitatively accurate technique for chronically measuring the postocclusion CBF perfusion of the infarct and peri-infarct regions in rodent stroke models, while multiphoton microscopy offers direct visualization of the microvascular structure. In this paper, we present imaging outcomes extending 35 days after photo-thrombotic occlusion, tracking the progression of the vasculature throughout this period. We compare MESI flow estimates within the unresolvable parenchyma with subsurface microvascular volume fractions taken with two-photon microscopy in the same regions to assess how the vascular density influences the surface-integrated MESI flow values. The MESI flow measurements and volume fractions are shown to have high correlations (r=0.90) within areas of recovering vasculature in the peri-infarct region. We also observe vascular reorientation occurring within the microvascular structure throughout the 35-day postocclusion period. With the combination of a chronic mouse model and relatively noninvasive optical imaging techniques, we present an imaging protocol for monitoring long-term vascular progression after photo-thrombotic occlusion with the potential to test the efficacy of rehabilitation and pharmacological therapies.
Occlusions in single cortical microvessels lead to a reduction in oxygen supply, but this decrement has not been able to be quantified in three dimensions at the level of individual vessels using a single instrument. We demonstrate a combined optical system using two-photon phosphorescence lifetime and fluorescence microscopy (2PLM) to characterize the partial pressure of oxygen (pO2) in single descending cortical arterioles in the mouse brain before and after generating a targeted photothrombotic occlusion. Integrated real-time Laser Speckle Contrast Imaging (LSCI) provides wide-field perfusion maps that are used to monitor and guide the occlusion process while 2PLM maps changes in intravascular oxygen tension. We present the technique’s utility in highlighting the effects of vascular networking on the residual intravascular oxygen tensions measured after occlusion in three dimensions.
BACKGROUND AND OBJECTIVES: The macrophage is an important early cellular marker related to risk of future rupture of atherosclerotic plaques. Two-channel two-photon luminescence (TPL) microscopy combined with optical coherence tomography (OCT) was used to detect, and further characterize the distribution of aorta-based macrophages using plasmonic gold nanorose as an imaging contrast agent. STUDY DESIGN AND MATERIALS AND METHODS: Nanorose uptake by macrophages was identified by TPL microscopy in macrophage cell culture. Ex vivo aorta segments (8 x 8 x 2 mm^3) rich in macrophages from a rabbit model of aorta inflammation were imaged by TPL microscopy in combination with OCT. Aorta histological sections (5 µm in thickness) were also imaged by TPL microscopy. RESULTS: Merged two-channel TPL images showed the lateral and depth distribution of nanorose-loaded macrophages (confirmed by RAM-11 stain) and other aorta components (e.g., elastin fiber and lipid droplet), suggesting that nanorose-loaded macrophages are diffusively distributed and mostly detected superficially within 20 µm from the luminal surface of the aorta. Moreover, OCT images depicted detailed surface structure of the diseased aorta. CONCLUSIONS: Results suggest that TPL microscopy combined with OCT can simultaneously reveal macrophage distribution with respect to aorta surface structure, which has the potential to detect vulnerable plaques and monitor plaque-based macrophages overtime during cardiovascular interventions.
BACKGROUND AND OBJECTIVES: Gold nanoparticles (GNPs) such as gold nanoshells (GNSs) and gold nanorods (GNRs) have been explored in a number of in vitro and in vivo studies as imaging contrast and cancer therapy agents due to their highly desirable spectral and molecular properties. While the organ-level biodistribution of these particles has been reported previously, little is known about the cellular level or intra-organ biodistribution. The objective of this study was to demonstrate the use of intrinsic two-photon-induced photoluminescence (TPIP) to study the cellular level biodistribution of GNPs. STUDY DESIGN/MATERIALS AND METHODS: Tumor xenografts were created in 27 male nude mice (Swiss nu/nu) using HCT 116 cells (CCL-247; American Type Culture Collection (ATCC), Manassas, VA, human colorectal cancer cell line). GNSs and GNRs were systemically injected 24 hours prior to tumor harvesting. A skin flap with the tumor was excised and sectioned as 8 µm thick tissues for imaging GNPs under a custom-built multiphoton microscope. For multiplexed imaging, nuclei, cytoplasm, and blood vessels were demonstrated by hematoxylin and eosin (H&E) staining, YOYO-1 iodide staining, and CD31-immunofluorescence staining. RESULTS: Distribution features of GNPs at the tumor site were determined from TPIP images. GNSs and GNRs had a heterogeneous distribution with higher accumulation at the tumor cortex than tumor core. GNPs were also observed in unique patterns surrounding the perivascular region. While most GNSs were confined at the distance of approximately 400 µm inside the tumor edge, GNRs were shown up to 1.5 mm penetration inside the edge. CONCLUSIONS: We have demonstrated the use of TPIP imaging in a multiplexed fashion to image both GNPs and nuclei, cytoplasm, or vasculature simultaneously. We also confirmed that TPIP imaging enabled visualization of GNP distribution patterns within the tumor and other critical organs. These results suggest that direct luminescence-based imaging of metal nanoparticles holds a valuable and promising position in understanding the accumulation kinetics of GNPs. In addition, these techniques will be increasingly important as the use of these particles progress to human clinical trials where standard histopathology techniques are used to analyze their effects.
We demonstrate a method to improve the measurement sensitivity of two-photon frequency-domain lifetime measurements in poor signal to background conditions. This technique uses sinusoidal modulation of the two-photon excitation source and detection of the second harmonic of the modulation frequency that appears in the emission. Additionally, we present the mathematical model which describes how the observed phase shift and amplitude demodulation factor of two-photon phosphorescence emission are related to the phosphorescence lifetime and modulation frequency. We demonstrate the validity of the model by showing the existence of new frequency terms in the phosphorescence emission generated from the quadratic nature of two-photon absorption and by showing that the phase shift and demodulation match theory for all frequency components.
An instrument is demonstrated that is capable of three-dimensional (3D) vasculature imaging and pO2 quantification with high spatial resolution. The instrument combines two-photon (2P) microscopy with phosphorescence quenching to measure pO2. The instrument was demonstrated by performing depth-resolved microvascular pO2 measurements of rat cortical vessels down to 120μm below the surface. 2P excitation of porphyrin was confirmed, and measured pO2 values were consistent with previously published data for normoxic and hyperoxic conditions. The ability to perform 3D pO2 measurements using optical techniques will allow researchers to overcome existing limitations imposed by polarographic electrodes, magnetic resonance techniques, and surface-only pO2 measurement techniques.
Gold nanoshells (dielectric silica core/gold shell) are a novel class of hybrid metal nanoparticles whose unique optical properties have spawned new applications including more sensitive molecular assays and cancer therapy. We report a new photo-physical property of nanoshells (NS) whereby these particles glow brightly when excited by near-infrared light. We characterized the luminescence brightness of NS, comparing to that of gold nanorods (NR) and fluorescent beads (FB). We find that NS are as bright as NR and 140 times brighter than FB. To demonstrate the potential application of this bright two-photon-induced photoluminescence (TPIP) signal for biological imaging, we imaged the 3D distribution of gold nanoshells targeted to murine tumors.
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.
A numerical model was developed to simulate the effects of tissue optical properties, objective numerical aperture (N.A.), and instrument performance on two-photon-excited fluorescence imaging of turbid samples. Model data are compared with measurements of fluorescent microspheres in a tissuelike scattering phantom. Our results show that the measured two-photon-excited signal decays exponentially with increasing focal depth. The overall decay constant is a function of absorption and scattering parameters at both excitation and emission wavelengths. The generation of two-photon fluorescence is shown to be independent of the scattering anisotropy, g, except for g > 0.95. The N.A. for which the maximum signal is collected varies with depth, although this effect is not seen until the focal plane is greater than two scattering mean free paths into the sample. Overall, measurements and model results indicate that resolution in two-photon microscopy is dependent solely on the ability to deliver sufficient ballistic photon density to the focal volume. As a result we show that lateral resolution in two-photon microscopy is largely unaffected by tissue optical properties in the range typically encountered in soft tissues, although the maximum imaging depth is strongly dependent on absorption and scattering coefficients, scattering anisotropy, and objective N.A..