Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy.

The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo.

Noninvasive Antemortem Detection of Retinal Prions by a Fluorescent Tracer.

BACKGROUND: Neurodegenerative diseases are widespread yet challenging to diagnose and stage antemortem. As an extension of the central nervous system, the eye harbors retina ganglion cells vulnerable to degeneration, and visual symptoms are often an early manifestation of neurodegenerative disease. OBJECTIVE: Here we test whether prion protein aggregates could be detected in the eyes of live mice using an amyloid-binding fluorescent probe and high-resolution retinal microscopy. METHODS: We performed retinal imaging on an experimental mouse model of prion-associated cerebral amyloid angiopathy in a longitudinal study. An amyloid-binding fluorophore was intravenously administered, and retinal imaging was performed at timepoints corresponding to early, mid-, and terminal prion disease. Retinal amyloid deposits were quantified and compared to the amyloid load in the brain. RESULTS: We report that by early prion disease (50% timepoint), discrete fluorescent foci appeared adjacent to the optic disc. By later timepoints, the fluorescent foci surrounded the optic disc and tracked along retinal vasculature. CONCLUSION: The progression of perivascular amyloid can be directly monitored in the eye by live imaging, illustrating the utility of this technology for diagnosing and monitoring the progression of cerebral amyloid angiopathy.

ARCAM-1 Facilitates Fluorescence Detection of Amyloid-Containing Deposits in the Retina.

Purpose: To investigate the use of an amyloid-targeting fluorescent probe, ARCAM-1, to identify amyloid-containing deposits in the retina of a transgenic mouse model of Alzheimer's disease (AD) and in human postmortem AD patients. Methods: Aged APP/PS1 transgenic AD and wild-type (WT) mice were given an intraperitoneal (IP) injection of ARCAM-1 and their retinas imaged in vivo using a fluorescence ophthalmoscope. Eyes were enucleated and dissected for ex vivo inspection of retinal amyloid deposits. Additionally, formalin-fixed eyes from human AD and control patients were dissected, and the retinas were stained using ARCAM-1 or with an anti-amyloid-ß antibody. Confocal microscopy was used to image amyloid-containing deposits stained with ARCAM-1 or with immunostaining. Results: Four out of eight APP/PS1 mice showed the presence of amyloid aggregates in the retina during antemortem imaging. Retinas from three human AD patients stained with ARCAM-1 showed an apparent increased density of fluorescently labeled amyloid-containing deposits compared to the retinas from two healthy, cognitively normal (CN) patients. Immunolabeling confirmed the presence of amyloid deposits in both the retinal neuronal layers and in retinal vasculature. Conclusions: ARCAM-1 facilitates antemortem detection of amyloid aggregates in the retina of a mouse model for AD, and postmortem detection of amyloid-containing deposits in human retinal tissues from AD patients. These results support the hypothesis of AD pathology manifesting in the eye and highlight a novel area for fluorophore development for the optical detection of retinal amyloid in AD patients. Translational Relevance: This paper represents an initial examination for potential translation of an amyloid-targeting fluorescent probe to a retinal imaging agent for aiding in the diagnosis of Alzheimer's disease.

Voltage Imaging with a NIR-Absorbing Phosphine Oxide Rhodamine Voltage Reporter.

The development of fluorescent dyes that emit and absorb light at wavelengths greater than 700 nm and that respond to biochemical and biophysical events in living systems remains an outstanding challenge for noninvasive optical imaging. Here, we report the design, synthesis, and application of near-infrared (NIR)-absorbing and -emitting optical voltmeter based on a sulfonated, phosphine-oxide (po) rhodamine for voltage imaging in intact retinas. We find that po-rhodamine based voltage reporters, or poRhoVRs, display NIR excitation and emission profiles at greater than 700 nm, show a range of voltage sensitivities (13 to 43% ΔF/F per 100 mV in HEK cells), and can be combined with existing optical sensors, like Ca2+-sensitive fluorescent proteins (GCaMP), and actuators, like light-activated opsins ChannelRhodopsin-2 (ChR2). Simultaneous voltage and Ca2+ imaging reveals differences in activity dynamics in rat hippocampal neurons, and pairing poRhoVR with blue-light based ChR2 affords all-optical electrophysiology. In ex vivo retinas isolated from a mouse model of retinal degeneration, poRhoVR, together with GCaMP-based Ca2+ imaging and traditional multielectrode array (MEA) recording, can provide a comprehensive physiological activity profile of neuronal activity, revealing differences in voltage and Ca2+ dynamics within hyperactive networks of the mouse retina. Taken together, these experiments establish that poRhoVR will open new horizons in optical interrogation of cellular and neuronal physiology in intact systems.

Identification of Patients with Preeclampsia by Measuring Fluorescence of an Amyloid-Binding Aryl Cyano Amide in Human Urine Samples.

Preeclampsia (PE) is a hypertensive disorder of pregnancy and one of the leading contributors to both maternal and perinatal morbidity and mortality. Reliable diagnostic parameters unique to the disorder that accurately define and diagnose PE are currently unavailable. Recent studies have revealed that PE is accompanied by the accumulation of amyloidogenic deposits in the placenta and the presence of congophilic amyloid-like protein aggregates in the urine. Here, we evaluate the capability of an amyloid-targeting aryl cyano amide (ARCAM-1) fluorophore to identify PE patients from analysis of urine samples. Our results reveal that this probe can distinguish patients with PE from gestationally healthy patients and patients suffering from non-PE hypertension, highlighting the potential for amyloid-targeting fluorophores to help identify PE patients during pregnancy.

Translational opportunities for amyloid-targeting fluorophores.

Advances in diagnostic medicine have led to an increased awareness and heightened concern for the high prevalence of amyloid-associated neurodegenerative diseases, especially in the elderly. These diseases have characteristic late stage symptoms that often make it possible to distinguish one disorder from another, though methods to diagnose neurodegeneration pre-symptomatically remain a critical challenge. At the molecular level, misfolded protein aggregates known as amyloids are ubiquitously found in many neurodegenerative diseases, and have been suggested to appear before clinical symptoms manifest. Amyloids have, thus, become a valuable potential diagnostic target for chemists, and recent work by many groups have shown that they can be selectively targeted by small molecule fluorescent probes. Here, we summarize some of the exciting work currently under investigation in the area of fluorescence-based amyloid detection and highlight recent efforts to expand the utility of amyloid-targeting fluorophores as clinical tools for disease diagnostics.

Solvation-Guided Design of Fluorescent Probes for Discrimination of Amyloids.

The deposition of insoluble protein aggregates in the brain is a hallmark of many neurodegenerative diseases. While their exact role in neurodegeneration remains unclear, the presence of these amyloid deposits often precedes clinical symptoms. As a result, recent progress in imaging methods that utilize amyloid-specific small molecule probes have become a promising avenue for antemortem disease diagnosis. Here, we present a series of amino-aryl cyanoacrylate (AACA) fluorophores that show a turn-on fluorescence signal upon binding to amyloids in solution and in tissue. Using a theoretical model for environmental sensitivity of fluorescence together with ab initio computational modeling of the effects of polar environment on electron density distribution and conformational dynamics, we designed, synthesized, and evaluated a set of fluorophores that (1) bind to aggregated forms of Alzheimer's-related ß-amyloid peptides with low micromolar to high nanomolar affinities and (2) have the capability to fluorescently discriminate different amyloids based on differences in amino acid composition within the binding pocket through exploitation of their solvatochromic properties. These studies showcase the rational design of a family of amyloid-binding imaging agents that could be integrated with new optical approaches for the clinical diagnosis of amyloidoses, where accurate identification of the specific neurodegenerative disease could aid in the selection of a proper course for treatment.

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum-Stable Membranes with Low Leakage.

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5-30-fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time-dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells.

Real-Time Monitoring of Alzheimer's-Related Amyloid Aggregation via Probe Enhancement-Fluorescence Correlation Spectroscopy.

This work describes the use of fluorescence correlation spectroscopy (FCS) and a novel amyloid-binding fluorescent probe, ARCAM 1, to monitor the aggregation of the Alzheimer's disease-associated amyloid ß-peptide (Aß). ARCAM 1 exhibits a large increase in fluorescence emission upon binding to Aß assemblies, making it an excellent candidate for probe enhancement FCS (PE-FCS). ARCAM 1 binding does not change Aß aggregation kinetics. It also exhibits greater dynamic range as a probe in reporting aggregate size by FCS in Aß, when compared to thioflavin T (ThT) or an Aß peptide modified with a fluorophore. Using fluorescent burst analysis (via PE-FCS) to follow aggregation of Aß, we detected soluble aggregates at significantly earlier time points compared to typical bulk fluorescence measurements. Autocorrelation analysis revealed the size of these early Aß assemblies. These results indicate that PE-FCS/ARCAM 1 based assays can detect and provide size characterization of small Aß aggregation intermediates during the assembly process, which could enable monitoring and study of such aggregates that transiently accumulate in biofluids of patients with Alzheimer's and other neurodegenerative diseases.