Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications

BACKGROUND: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. PRINCIPAL FINDINGS: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. CONCLUSION: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups

Principles and Fundamentals of Optical Imaging

In this chapter I will give a brief general introduction to optical imaging and then discuss in more detail some of the methods specifically used for imaging cortical dynamics today. Absorption and fluorescence microscopy can be used to form direct, diffraction-limited images but standard methods are often only applicable to superficial layers of cortical tissue. Two-photon microscopy takes an intermediate role since the illumination pathway is diffraction-limited but the detection pathway is not. Losses in the illumination path can be compensated using higher laser power. Since the detection pathway does not require image formation, the method can substantially increase the imaging depth. Understanding the role of scattering is important in this case since non-descanned detection can substantially enhance the imaging performance. Finally, I will discuss some of the most widely used imaging methods that all rely on diffuse scattering such as diffuse optical tomography, laser speckle imaging, and intrinsic optical imaging. These purely scattering-based methods offer a much higher imaging depth, although at a substantially reduced spatial resolution

Imaging optically scattering objects with ultrasound-modulated optical tomography

We show the feasibility of imaging objects having different optical scattering coefficients relative to the surrounding scattering medium using ultrasound-modulated optical tomography (UOT). While the spatial resolution depends on ultrasound parameters, the image contrast depends on the difference in scattering coefficient between the object and the surrounding medium. Experimental measurements obtained with a CCD-based speckle contrast detection scheme are in agreement with Monte Carlo simulations and analytical calculations. This study complements previous UOT experiments that demonstrated optical absorption contrast. (C) 2007 Optical Society of America.X113030sciescopu

Identification of n = 4, Δn = 0 transitions in the spectra of nickel-like cadmium ions from a capillary discharge plasma column

Includes bibliographical references (pages 418-419).Spectra of Nickel-like Cadmium (CdXXI) ions in the 12.7-18.4 nm wavelength region obtained with a high current capillary discharge have been analyzed. Fifty-three 3d9 4p-3d9 4d and 3d9 4d-3d9 4f CdXXI lines were identified with the assistance of calculations performed using the Slater-Condon method with generalized least-squares fits of the energy parameters. The average deviation between the measured and theoretical wavelengths is (λexp-λth) = 0:0065nm. The results demonstrate that fast capillary discharges can produce high quality spectra for the study of multiply charged ions with charges of up to at least Z = 20

Multimodal reconstruction of microvascular-flow distributions using combined two-photon microscopy and Doppler optical coherence tomography

Computing microvascular cerebral blood flow ([Formula: see text]) in real cortical angiograms is challenging. Here, we investigated whether the use of Doppler optical coherence tomography (DOCT) flow measurements in individual vessel segments can help in reconstructing [Formula: see text] across the entire vasculature of a truncated cortical angiogram. A [Formula: see text] computational framework integrating DOCT measurements is presented. Simulations performed on a synthetic angiogram showed that the addition of DOCT measurements, especially close to large inflowing or outflowing vessels, reduces the impact of pressure boundary conditions and estimated vessel resistances resulting in a more accurate reconstruction of [Formula: see text]. Our technique was then applied to reconstruct microvascular flow distributions in the mouse cortex down to [Formula: see text] by combining two-photon laser scanning microscopy angiography with DOCT

Validation and optimization of hypercapnic-calibrated fMRI from oxygen-sensitive two-photon microscopy

Hypercapnic-calibrated fMRI allows the estimation of the relative changes in the cerebral metabolic rate of oxygen (rCMRO(2)) from combined BOLD and arterial spin labelling measurements during a functional task, and promises to permit more quantitative analyses of brain activity patterns. The estimation relies on a macroscopic model of the BOLD effect that balances oxygen delivery and consumption to predict haemoglobin oxygenation and the BOLD signal. The accuracy of calibrated fMRI approaches has not been firmly established, which is limiting their broader adoption. We use our recently developed microscopic vascular anatomical network model in mice as a ground truth simulator to test the accuracy of macroscopic, lumped-parameter BOLD models. In particular, we investigate the original Davis model and a more recent heuristic simplification. We find that these macroscopic models are inaccurate using the originally defined parameters, but that the accuracy can be significantly improved by redefining the model parameters to take on new values. In particular, we find that the parameter α that relates cerebral blood-volume changes to cerebral blood-flow changes is significantly smaller than typically assumed and that the optimal value changes with magnetic field strength. The results are encouraging in that they support the use of simple BOLD models to quantify BOLD signals, but further work is needed to understand the physiological interpretation of the redefined model parameters. This article is part of the themed issue ‘Interpreting BOLD: a dialogue between cognitive and cellular neuroscience’

Magnetic resonance fingerprinting based on realistic vasculature in mice

Magnetic resonance fingerprinting (MRF) was recently proposed as a novel strategy for MR data acquisition and analysis. A variant of MRF called vascular MRF (vMRF) followed, that extracted maps of three parameters of physiological importance: cerebral oxygen saturation (SatO(2)), mean vessel radius and cerebral blood volume (CBV). However, this estimation was based on idealized 2-dimensional simulations of vascular networks using random cylinders and the empirical Bloch equations convolved with a diffusion kernel. Here we focus on studying the vascular MR fingerprint using real mouse angiograms and physiological values as the substrate for the MR simulations. The MR signal is calculated ab initio with a Monte Carlo approximation, by tracking the accumulated phase from a large number of protons diffusing within the angiogram. We first study the identifiability of parameters in simulations, showing that parameters are fully estimable at realistically high signal-to-noise ratios (SNR) when the same angiogram is used for dictionary generation and parameter estimation, but that large biases in the estimates persist when the angiograms are different. Despite these biases, simulations show that differences in parameters remain estimable. We then applied this methodology to data acquired using the GESFIDE sequence with SPIONs injected into 9 young wild type and 9 old atherosclerotic mice. Both the pre injection signal and the ratio of post-to-pre injection signals were modeled, using 5-dimensional dictionaries. The vMRF methodology extracted significant differences in SatO(2), mean vessel radius and CBV between the two groups, consistent across brain regions and dictionaries. Further validation work is essential before vMRF can gain wider application

Determinants of Optogenetic Cortical Spreading Depolarizations

Cortical spreading depolarization (SD) is the electrophysiological event underlying migraine aura, and a critical contributor to secondary damage after brain injury. Experimental models of SD have been used for decades in migraine and brain injury research; however, they are highly invasive and often cause primary tissue injury, diminishing their translational value. Here we present a non-invasive method to trigger SDs using light-induced depolarization in transgenic mice expressing channelrhodopsin-2 in neurons (Thy1-ChR2-YFP). Focal illumination (470 nm, 1-10 mW) through intact skull using an optical fiber evokes power-dependent steady extracellular potential shifts and local elevations of extracellular [K+] that culminate in an SD when power exceeds a threshold. Using the model, we show that homozygous mice are significantly more susceptible to SD (i.e., lower light thresholds) than heterozygous ChR2 mice. Moreover, we show SD susceptibility differs significantly among cortical divisions (motor, whisker barrel, sensory, visual, in decreasing order of susceptibility), which correlates with relative channelrhodopsin-2 expression. Furthermore, the NMDA receptor antagonist MK-801 blocks the transition to SD without diminishing extracellular potential shifts. Altogether, our data show that the optogenetic SD model is highly suitable for examining physiological or pharmacological modulation of SD in acute and longitudinal studies.Functional Genomics of Muscle, Nerve and Brain Disorder

In Vivo Imaging of Flavoprotein Fluorescence During Hypoxia Reveals the Importance of Direct Arterial Oxygen Supply to Cerebral Cortex Tissue.

Live imaging of mitochondrial function is crucial to understand the important role played by these organelles in a wide range of diseases. The mitochondrial redox potential is a particularly informative measure of mitochondrial function, and can be monitored using the endogenous green fluorescence of oxidized mitochondrial flavoproteins. Here, we have observed flavoprotein fluorescence in the exposed murine cerebral cortex in vivo using confocal imaging; the mitochondrial origin of the signal was confirmed using agents known to manipulate mitochondrial redox potential. The effects of cerebral oxygenation on flavoprotein fluorescence were determined by manipulating the inspired oxygen concentration. We report that flavoprotein fluorescence is sensitive to reductions in cortical oxygenation, such that reductions in inspired oxygen resulted in loss of flavoprotein fluorescence with the exception of a preserved 'halo' of signal in periarterial regions. The findings are consistent with reports that arteries play an important role in supplying oxygen directly to tissue in the cerebral cortex, maintaining mitochondrial function