Two-photon microscopy of cortical NADH fluorescence intensity changes: correcting contamination from the hemodynamic response

Baraghis, Edward; Devor, Anna; Fang, Qianqian; Srinivasan, Vivek J.; Wu, Wei‐Cheng; Lesage, Frédéric; Ayata, Cenk; Kasischke, Karl A.; Boas, David A.; Sakadžić, Sava

doi:10.1117/1.3633339

Cited by 22 publications

(26 citation statements)

References 48 publications

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“…Investigators have emphasized for many years that lifetime-based measurements of NADH autofluorescence offer multiple advantages over more common intensity-based measurements, including resolution of bound and unbound molecular conformations and insensitivity to both NADH concentration [9] and changes in tissue optical properties [47]. Coupling FLIM with 2P microscopy makes the technique especially useful for highresolution characterization of highly vascularized, scattering tissues such as the brain in vivo; however, attempts to rigorously interpret and validate the significance of FLIM-based NADH observations have not been reported until recently [48].…”

Section: Discussionmentioning

confidence: 99%

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo

Yaseen

Sutin

et al. 2017

Biomed. Opt. Express

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Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics. 2(2), 145-150 (1966). 33. L. K. Klaidman, A. C. Leung, and J. D. Adams, Jr., "High-performance liquid chromatography analysis of oxidized and reduced pyridine dinucleotides in specific brain regions," Anal.

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Section: Discussionmentioning

confidence: 99%

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo

Yaseen

Sutin

et al. 2017

Biomed. Opt. Express

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“…As described previously [34] and illustrated in Fig. 1a (inset), cerebral cortices were exposed via a sealed cranial window in the skulls of anesthetized and ventilated Sprague Dawley rats (male, 195–265 g, Charles River).…”

Section: 4 In Vivo Animal Studiesmentioning

confidence: 99%

In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH

Yaseen¹,

Sakadžić²,

Wu³

et al. 2013

Biomed. Opt. Express

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111

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Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism.

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“…However, isolating the NADH signal from confounding hemodynamic signals is challenging in vivo. 24 Therefore we segmented the vessels and removed them from the region of interest analysis.…”

Section: Nadh Imagingmentioning

confidence: 99%

Patterned Optogenetic Modulation of Neurovascular and Metabolic Signals

Richner

Baumgartner

Brodnick

et al. 2014

J Cereb Blood Flow Metab

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The hemodynamic and metabolic response of the cortex depends spatially and temporally on the activity of multiple cell types. Optogenetics enables specific cell types to be modulated with high temporal precision and is therefore an emerging method for studying neurovascular and neurometabolic coupling. Going beyond temporal investigations, we developed a microprojection system to apply spatial photostimulus patterns in vivo. We monitored vascular and metabolic fluorescence signals after photostimulation in Thy1-channelrhodopsin-2 mice. Cerebral arteries increased in diameter rapidly after photostimulation, while nearby veins showed a slower smaller response. The amplitude of the arterial response was depended on the area of cortex stimulated. The fluorescence signal emitted at 450/100 nm and excited with ultraviolet is indicative of reduced nicotinamide adenine dinucleotide, an endogenous fluorescent enzyme involved in glycolysis and the citric acid cycle. This fluorescence signal decreased quickly and transiently after optogenetic stimulation, suggesting that glucose metabolism is tightly locked to optogenetic stimulation. To verify optogenetic stimulation of the cortex, we used a transparent substrate microelectrode array to map cortical potentials resulting from optogenetic stimulation. Spatial optogenetic stimulation is a new tool for studying neurovascular and neurometabolic coupling.

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Two-photon microscopy of cortical NADH fluorescence intensity changes: correcting contamination from the hemodynamic response

Cited by 22 publications

References 48 publications

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo

In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH

Patterned Optogenetic Modulation of Neurovascular and Metabolic Signals

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