Monitoring Visual Cortical Activities During Progressive Retinal Degeneration Using Functional Bioluminescence Imaging.

Mouse models of inherited retinal degenerative diseases such as retinitis pigmentosa are characterized by degeneration of photoreceptors, which hinders the generation of signal to be transmitted to the visual cortex. By monitoring Ca2+-bioluminescence neural activity, we quantified changes in visual cortical activities in response to visual stimuli in RD10 mice during progression of retinal degeneration, which correlated with progressive deteriorations of electro-retinography signal from the eyes. The number of active neurons in the visual cortex, the intensity of Ca2+-bioluminescence response, and neural activation parameter showed progressive deterioration during aging. Further, we correlated the thinning of retina as measured by Optical Coherence Tomography with the decrease in visual cortical activities as retinal degeneration progressed. The present study establishes Ca2+-bioluminescence monitoring as a longitudinal imaging modality to characterize activities in visual cortex of retinal degenerative disease models and therapeutic interventions.

Biodistribution of adeno-associated virus type 2 carrying multi-characteristic opsin in dogs following intravitreal injection.

Gene therapy of retinal diseases using recombinant adeno-associated virus (rAAV) vector-based delivery has shown clinical success, and clinical trials based on rAAV-based optogenetic therapies are currently in progress. Recently, we have developed multi-characteristic opsin (MCO), which has been shown to effectively re-photosensitize photoreceptor-degenerated retina in mice leading to vision restoration at ambient light environment. Here, we report the biodistribution of the rAAV2 carried MCO (vMCO-I) in live samples and post-mortem organs following intraocular delivery in wild-type dogs. Immunohistochemistry showed that the intravitreal injection of vMCO-I resulted in gene transduction in the inner nuclear layer (INL) but did not induce detectable inflammatory or immune reaction in the dog retina. Vector DNA analysis of live body wastes and body fluids such as saliva and nasal secretions using quantitative polymerase chain reaction (qPCR) showed no correlative increase of vector copy in nasal secretions or saliva, minimal increase of vector copy in urine in the low-dose group 13 weeks after injection and in the faeces of the high-dose group at 3-13 weeks after injection suggesting clearance of the virus vector via urine and faeces. Further analysis of vector DNA extracted from faeces using PCR showed no transgene after 3 weeks post-injection. Intravitreal injection of vMCO-I resulted in few sporadic off-target presences of the vector in the mesenteric lymph node, liver, spleen and testis. This study showed that intravitreal rAAV2-based delivery of MCO-I for retinal gene therapy is safe.

Fe Core-Carbon Shell Nanoparticles as Advanced MRI Contrast Enhancer.

The aim of this study is to fabricate a hybrid composite of iron (Fe) core-carbon (C) shell nanoparticles with enhanced magnetic properties for contrast enhancement in magnetic resonance imaging (MRI). These new classes of magnetic core-shell nanoparticles are synthesized using a one-step top-down approach through the electric plasma discharge generated in the cavitation field in organic solvents by an ultrasonic horn. Transmission electron microscopy (TEM) observations revealed the core-shell nanoparticles with 10-85 nm in diameter with excellent dispersibility in water without any agglomeration. TEM showed the structural confirmation of Fe nanoparticles with body centered cubic (bcc) crystal structure. Magnetic multi-functional hybrid composites of Fe core-C shell nanoparticles were then evaluated as negative MRI contrast agents, displaying remarkably high transverse relaxivity (r2) of 70 mM-1·S-1 at 7 T. This simple one-step synthesis procedure is highly versatile and produces desired nanoparticles with high efficacy as MRI contrast agents and potential utility in other biomedical applications.

Quantitative analysis of nanoscale intranuclear structural alterations in hippocampal cells in chronic alcoholism via transmission electron microscopy imaging.

Chronic alcoholism is known to alter the morphology of the hippocampus, an important region of cognitive function in the brain. Therefore, to understand the effect of chronic alcoholism on hippocampal neural cells, we employed a mouse model of chronic alcoholism and quantified intranuclear nanoscale structural alterations in these cells. Transmission electron microscopy (TEM) images of hippocampal neurons were obtained, and the degree of structural alteration in terms of mass density fluctuation was determined using the light-localization properties of optical media generated from TEM imaging. The results, which were obtained at length scales ranging from ~30 to 200 nm, show that 10-12 week-old mice fed a Lieber-DeCarli liquid (alcoholic) diet had a higher degree of structural alteration than control mice fed a normal diet without alcohol. The degree of structural alteration became significantly distinguishable at a sample length of ~100 nm, which is the typical length scale of the building blocks of cells, such as DNA, RNA, proteins and lipids. Interestingly, different degrees of structural alteration at such length scales suggest possible structural rearrangement of chromatin inside the nuclei in chronic alcoholism.

Optical Techniques in Optogenetics.

Optogenetics is an innovative technique for optical control of cells. This field has exploded over the past decade or so and has given rise to great advances in neuroscience. A variety of applications both from the basic and applied research have emerged, turning the early ideas into a powerful paradigm for cell biology, neuroscience and medical research. This review aims at highlighting the basic concepts that are essential for a comprehensive understanding of optogenetics and some important biological/biomedical applications. Further, emphasis is placed on advancement in optogenetics-associated light-based methods for controlling gene expression, spatially-controlled optogenetic stimulation and detection of cellular activities.

Broad spectral excitation of opsin for enhanced stimulation of cells.

Optical stimulation of cells expressing light-sensitive proteins (opsins) has allowed targeted activation with cellular specificity. However, since narrow-band light has been used for excitation of these optogenetic probes, only active stimulation strategies are being attempted for clinical applications such as restoration of vision. Here, we report use of broad spectral excitation (white light) for optogenetic stimulation of opsin-sensitized cells. We found that ReaChR is optimally excited with white light offering significantly higher photocurrents compared to spectrally filtered narrow-band light stimulation. Our findings open up the possibility of passive stimulation strategy by use of natural sunlight for retinal stimulation, which could have benefits for ambient light stimulated vision restoration.

Pain inhibition by optogenetic activation of specific anterior cingulate cortical neurons.

Cumulative evidence from both humans and animals suggests that the anterior cingulate cortex (ACC) is important for pain-related perception, and thus a likely target for pain relief therapy. However, use of existing electrode based ACC stimulation has not significantly reduced pain, at least in part due to the lack of specificity and likely co-activation of both excitatory and inhibitory neurons. Herein, we report a dramatic reduction of pain behavior in transgenic mice by optogenetic stimulation of the inhibitory neural circuitry of the ACC expressing channelrhodopsin-2. Electrophysiological measurements confirmed that stimulation of ACC inhibitory neurons is associated with decreased neural activity in the ACC. Further, a distinct optogenetic stimulation intensity and frequency-dependent inhibition of spiking activity in the ACC was observed. Moreover, we confirmed specific electrophysiological responses from different neuronal units in the thalamus, in response to particular types of painful stimuli (i,e., formalin injection, pinch), which we found to be modulated by optogenetic control of the ACC inhibitory neurons. These results underscore the inhibition of the ACC as a clinical alternative in inhibiting chronic pain, and leads to a better understanding of the pain processing circuitry of the cingulate cortex.

Non-scanning fiber-optic near-infrared beam led to two-photon optogenetic stimulation in-vivo.

Stimulation of specific neurons expressing opsins in a targeted region to manipulate brain function has proved to be a powerful tool in neuroscience. However, the use of visible light for optogenetic stimulation is invasive due to low penetration depth and tissue damage owing to larger absorption and scattering. Here, we report, for the first time, in-depth non-scanning fiber-optic two-photon optogenetic stimulation (FO-TPOS) of neurons in-vivo in transgenic mouse models. In order to optimize the deep-brain stimulation strategy, we characterized two-photon activation efficacy at different near-infrared laser parameters. The significantly-enhanced in-depth stimulation efficiency of FO-TPOS as compared to conventional single-photon beam was demonstrated both by experiments and Monte Carlo simulation. The non-scanning FO-TPOS technology will lead to better understanding of the in-vivo neural circuitry because this technology permits more precise and less invasive anatomical delivery of stimulation.

Crystalline magnetic carbon nanoparticle assisted photothermal delivery into cells using CW near-infrared laser beam.

Efficient and targeted delivery of impermeable exogenous material such as small molecules, proteins, and plasmids into cells in culture as well as in vivo is of great importance for drug, vaccine and gene delivery for different therapeutic strategies. Though advent of optoporation by ultrafast laser microbeam has allowed spatial targeting in cells, the requirement of high peak power to create holes on the cell membrane is not practical and also challenging in vivo. Here, we report development and use of uniquely non-reactive crystalline magnetic carbon nanoparticles (CMCNPs) for photothermal delivery (PTD) of impermeable dyes and plasmids encoding light-sensitive proteins into cells using low power continuous wave near-infrared (NIR) laser beam. Further, we utilized the magnetic nature of these CMCNPs to localize them in desired region by external magnetic field, thus minimizing the required number of nanoparticles. We discovered that irradiation of the CMCNPs near the desired cell(s) with NIR laser beam leads to temperature rise that not only stretch the cell-membrane to ease delivery, it also creates fluid flow to allow mobilization of exogenous substances to the delivery. Due to significant absorption properties of the CMCNPs in the NIR therapeutic window, PTD under in vivo condition is highly possible.

Highly effective photonic cue for repulsive axonal guidance.

In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods). These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ∼90 µm.

Fiber-optic spanner.

Methods of controllable, noncontact rotation of optically trapped microscopic objects have garnered significant attention for tomographic imaging and microfluidic actuation. Here, we report development of a fiber-optic spanner and demonstrate controlled rotation of smooth muscle cells. The rotation is realized by introducing a transverse offset between two counterpropagating beams emanating from single-mode optical fibers. The rotation speed and surrounding microfluidic flow could be controlled by varying balanced laser beam powers. Further, we demonstrate simultaneous translation and rotation of the fiber-optically trapped cell by varying the laser power of one fiber-optic arm.

Magnetic-field-assisted photothermal therapy of cancer cells using Fe-doped carbon nanoparticles.

Photothermal therapy with assistance of nanoparticles offers a solution for the destruction of cancer cells without significant collateral damage to otherwise healthy cells. However, minimizing the required number of injected nanoparticles is a major challenge. Here, we introduce the use of magnetic carbon nanoparticles (MCNPs), localizing them in a desired region by applying an external magnetic-field, and irradiating the targeted cancer cells with a near-infrared laser beam. The MCNPs were prepared in benzene, using an electric plasma discharge, generated in the cavitation field of an ultrasonic horn. The CNPs were made ferromagnetic by use of Fe-electrodes to dope the CNPs, as confirmed by magnetometry. Transmission electron microscopy measurements showed the size distribution of these MCNPs to be in the range of 5 to 10 nm. For photothermal irradiation, a tunable continuous wave Ti: Sapphire laser beam was weakly focused on to the cell monolayer under an inverted fluorescence microscope. The response of different cell types to photothermal irradiation was investigated. Cell death in the presence of both MCNPs and laser beam was confirmed by morphological changes and propidium iodide fluorescence inclusion assay. The results of our study suggest that MCNP based photothermal therapy is a promising approach to remotely guide photothermal therapy.

Targeted microinjection into cells and retina using optoporation.

The laser microbeam has enabled highly precise noncontact delivery of exogenous materials into targeted cells without compromising cell viability, which has been a highly challenging task for traditional methods. Here, we report targeted delivery of impermeable substances into mammalian cells and goldfish retinal explants subsequent to ultrafast laser microbeam assisted injection. Introduction of impermeable dye into the cell through localized pore formation was confirmed by distinct fluorescence at the site of pore formation on the membrane and its spatiotemporal diffusion pattern through the nucleus. Indirect optoporation by bubble formation, external to cell, led to a similar spatial diffusion pattern but with a larger time constant for injection. Using optimized laser intensity, exposure, and a spatial irradiation pattern, desired spatial transfection patterns in goldfish retina explants were achieved as confirmed by the expression of injected plasmids encoded for light-activable channel rhodopsin-2 ion-channel, tagged with fluorescent protein. Laser assisted delivery of exogenous material into a specific area of three-dimensional neuronal tissue, such as the retina, will help to understand the functioning of neuronal circuitry of normal and degenerated retina.

Detachment and reorientation of cells using near-infrared laser microbeam.

Reorientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and reorientation of adherent cells. The detached cell was transported along the axial direction by scattering force and trapped at a higher plane inside the media using the same laser beam by a gravito-optical trap. The trapped cell could then be repositioned by movement of the sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was found to depend on the laser beam power. Viability of the detached and manipulated cell was found not to be compromised as confirmed by propidium iodide fluorescence exclusion assay. The reoriented cell was allowed to reattach to the substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for noncontact modification of cellular orientations that will enable study of intercellular interactions and design of engineered tissue.

Trapping and two-photon fluorescence excitation of microscopic objects using ultrafast single-fiber optical tweezers.

Analysis of trapped microscopic objects using fluorescence and Raman spectroscopy is gaining considerable interest. We report on the development of single fiber ultrafast optical tweezers and its use in simultaneous two-photon fluorescence (TPF) excitation of trapped fluorescent microscopic objects. Using this method, trapping depth of a few centimeters was achieved inside a colloidal sample with TPF from the trapped particle being visible to the naked eye. Owing to the propagation distance of the Bessel-like beam emerging from the axicon-fiber tip, a relatively longer streak of fluorescence was observed along the microsphere length. The cone angle of the axicon was engineered so as to provide better trapping stability and high axial confinement of TPF. Trapping of the floating objects led to stable fluorescence emission intensity over a long period of time, suitable for spectroscopic measurements. Furthermore, the stability of the fiber optic trapping was confirmed by holding and maneuvering the fiber by hand so as to move the trapped fluorescent particle in three dimensions. Apart from miniaturization capability into lab-on-a-chip microfluidic devices, the proposed noninvasive microaxicon tipped optical fiber can be used in multifunctional mode for in-depth trapping, rotation, sorting, and ablation, as well as for two-photon fluorescence excitation of a motile sample.

Spatially sculpted laser scissors for study of DNA damage and repair.

We present a simple and efficient method for controlled linear induction of DNA damage in live cells. By passing a pulsed laser beam through a cylindrical lens prior to expansion, an elongated elliptical beam profile is created with the ability to expose controlled linear patterns while keeping the beam and the sample stationary. The length and orientation of the beam at the sample plane were reliably controlled by an adjustable aperture and rotation of the cylindrical lens, respectively. Localized immunostaining by the DNA double strand break (DSB) markers phosphorylated H2AX (gamma H2AX) and Nbs1 in the nuclei of HeLa cells exposed to the "line scissors" was shown via confocal imaging. The line scissors method proved more efficient than the scanning mirror and scanning stage methods at induction of DNA DSB damage with the added benefit of having a greater potential for high throughput applications.

Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells.

Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed.

Manipulation of mammalian cells using a single-fiber optical microbeam.

The short working distance of microscope objectives has severely restricted the application of optical micromanipulation techniques at larger depths. We show the first use of fiber-optic tweezers toward controlled guidance of neuronal growth cones and stretching of neurons. Further, by mode locking, the fiber-optic tweezers beam was converted to fiber-optic scissors, enabling dissection of neuronal processes and thus allowing study of the subsequent response of neurons to localized injury. At high average powers, lysis of a three-dimensionally trapped cell was accomplished.

In-depth activation of channelrhodopsin 2-sensitized excitable cells with high spatial resolution using two-photon excitation with a near-infrared laser microbeam.

We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.

Controlled induction, enhancement, and guidance of neuronal growth cones by use of line optical tweezers.

We report an optical tweezers based approach for efficient and controlled manipulation of neuronal growth cones. The approach exploits asymmetric transverse gradient force created in a line optical tweezers to transport actin monomers in the desired growth direction. With this approach induction of artificial growth cones from the neuronal cell body and enhancement of the growth rate of the natural growth cones have been achieved. The use of this approach to bring two growth cones into close proximity for establishing a neuronal connection is also discussed.