Hybrid Multisite Silicon Neural Probe with Integrated Flexible Connector for Interchangeable Packaging

Multisite neural probes are a fundamental tool to study brain function. Hybrid silicon/polymer neural probes combine rigid silicon and flexible polymer parts into one single device and allow, for example, the precise integration of complex probe geometries, such as multishank designs, with flexible biocompatible cabling. Despite these advantages and benefiting from highly reproducible fabrication methods on both silicon and polymer substrates, they have not been widely available. This paper presents the development, fabrication, characterization, and in vivo electrophysiological assessment of a hybrid multisite multishank silicon probe with a monolithically integrated polyimide flexible interconnect cable. The fabrication process was optimized at wafer level, and several neural probes with 64 gold electrode sites equally distributed along 8 shanks with an integrated 8 µm thick highly flexible polyimide interconnect cable were produced. The monolithic integration of the polyimide cable in the same fabrication process removed the necessity of the postfabrication bonding of the cable to the probe. This is the highest electrode site density and thinnest flexible cable ever reported for a hybrid silicon/polymer probe. Additionally, to avoid the time-consuming bonding of the probe to definitive packaging, the flexible cable was designed to terminate in a connector pad that can mate with commercial zero-insertion force (ZIF) connectors for electronics interfacing. This allows great experimental flexibility because interchangeable packaging can be used according to experimental demands. High-density distributed in vivo electrophysiological recordings were obtained from the hybrid neural probes with low intrinsic noise and high signal-to-noise ratio (SNR).

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Hybrid multisite silicon neural probe with integrated flexible connector for interchangeable packaging

10.1101/2021.02.02.429112 ◽

2021 ◽

Author(s):

Ashley Novais ◽

Carlos Calaza ◽

José Fernandes ◽

Helder Fonseca ◽

João Gaspar ◽

...

Keyword(s):

Signal To Noise Ratio ◽

Intrinsic Noise ◽

High Signal ◽

Wafer Level ◽

Neural Probes ◽

Insertion Force ◽

Monolithically Integrated ◽

Electrophysiological Recordings ◽

Hybrid Silicon

AbstractMultisite neural probes are a fundamental tool to study brain function. Hybrid silicon/polymer neural probes, in particular, allow the integration of complex probe geometries, such as multi-shank designs, with flexible biocompatible cabling. Despite these advantages and benefiting from the highly reproducible fabrication methods on both silicon and polymer substrates, they have not been widely available. This paper presents the development, fabrication, characterization, and in vivo electrophysiological assessment of a hybrid multisite multi-shank silicon probe with a monolithically integrated polyimide flexible interconnect cable. The fabrication process was optimized on wafer level and several neural probes with 64 gold electrode sites equally distributed along 8 shanks with an integrated 8 μm-thick highly flexible polyimide interconnect cable were produce. To avoid the time-consuming bonding of the probe to definitive packaging, the flexible cable was designed to terminate in a connector pad that can mate with commercial zero-insertion force (ZIF) connectors for electronics interfacing. This allows great experimental flexibility since interchangeable packaging can be used according to experimental demands. High-density distributed in vivo electrophysiological recordings were obtained from the hybrid neural probes with low intrinsic noise and high signal-to-noise ratio (SNR).

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Double-Layer Flexible Neural Probe With Closely Spaced Electrodes for High-Density in vivo Brain Recordings

Frontiers in Neuroscience ◽

10.3389/fnins.2021.663174 ◽

2021 ◽

Vol 15 ◽

Author(s):

Sara Pimenta ◽

José A. Rodrigues ◽

Francisca Machado ◽

João F. Ribeiro ◽

Marino J. Maciel ◽

...

Keyword(s):

Double Layer ◽

Signal To Noise Ratio ◽

Low Cost ◽

High Signal ◽

Composition Analysis ◽

Assessment Tests ◽

Flexible Polymer ◽

Mouse Cortex ◽

Neural Probe

Flexible polymer neural probes are an attractive emerging approach for invasive brain recordings, given that they can minimize the risks of brain damage or glial scaring. However, densely packed electrode sites, which can facilitate neuronal data analysis, are not widely available in flexible probes. Here, we present a new flexible polyimide neural probe, based on standard and low-cost lithography processes, which has 32 closely spaced 10 μm diameter gold electrode sites at two different depths from the probe surface arranged in a matrix, with inter-site distances of only 5 μm. The double-layer design and fabrication approach implemented also provides additional stiffening just sufficient to prevent probe buckling during brain insertion. This approach avoids typical laborious augmentation strategies used to increase flexible probes’ mechanical rigidity while allowing a small brain insertion footprint. Chemical composition analysis and metrology of structural, mechanical, and electrical properties demonstrated the viability of this fabrication approach. Finally, in vivo functional assessment tests in the mouse cortex were performed as well as histological assessment of the insertion footprint, validating the biological applicability of this flexible neural probe for acquiring high quality neuronal recordings with high signal to noise ratio (SNR) and reduced acute trauma.

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Dextran as a Resorbable Coating Material for Flexible Neural Probes

Micromachines ◽

10.3390/mi10010061 ◽

2019 ◽

Vol 10 (1) ◽

pp. 61 ◽

Cited By ~ 5

Author(s):

Dries Kil ◽

Marta Bovet Carmona ◽

Frederik Ceyssens ◽

Marjolijn Deprez ◽

Luigi Brancato ◽

...

Keyword(s):

Immunological Response ◽

Scar Tissue ◽

Coating Material ◽

Dissolution Time ◽

Electrode Arrays ◽

Neural Probes ◽

Neuron Density ◽

Flexible Polymer ◽

In Vivo Testing

In the quest for chronically reliable and bio-tolerable brain interfaces there has been a steady evolution towards the use of highly flexible, polymer-based electrode arrays. The reduced mechanical mismatch between implant and brain tissue has shown to reduce the evoked immune response, which in turn has a positive effect on signal stability and noise. Unfortunately, the low stiffness of the implants also has practical repercussions, making surgical insertion extremely difficult. In this work we explore the use of dextran as a coating material that temporarily stiffens the implant, preventing buckling during insertion. The mechanical properties of dextran coated neural probes are characterized, as well as the different parameters which influence the dissolution rate. Tuning parameters, such as coating thickness and molecular weight of the used dextran, allows customization of the stiffness and dissolution time to precisely match the user’s needs. Finally, the immunological response to the coated electrodes was analyzed by performing a histological examination after four months of in vivo testing. The results indicated that a very limited amount of glial scar tissue was formed. Neurons have also infiltrated the area that was initially occupied by the dissolving dextran coating. There was no noticeable drop in neuron density around the site of implantation, confirming the suitability of the coating as a temporary aid during implantation of highly flexible polymer-based neural probes.

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Tantalum Oxide Nanoparticles as Versatile Contrast Agents for X-ray Computed Tomography

10.26434/chemrxiv.11845572.v1 ◽

2020 ◽

Author(s):

Shatadru Chakravarty ◽

Jeremy Hix ◽

Kaitlyn Wieweora ◽

Maximilian Volk ◽

Elizabeth Kenyon ◽

...

Keyword(s):

Computed Tomography ◽

Contrast Agents ◽

Mesoporous Silica Nanoparticles ◽

Sol Gel ◽

Tantalum Oxide ◽

High Signal ◽

Drug Delivery Vehicles ◽

X Ray Computed

Here we describe the synthesis, characterization and in vitro and in vivo performance of a series of tantalum oxide (TaOx) based nanoparticles (NPs) for computed tomography (CT). Five distinct versions of 9-12 nm diameter silane coated TaOx nanocrystals (NCs) were fabricated by a sol-gel method with varying degrees of hydrophilicity and with or without fluorescence, with the highest reported Ta content to date (78%). Highly hydrophilic NCs were left bare and were evaluated in vivo in mice for micro-CT of full body vasculature, where following intravenous injection, TaOx NCs demonstrate high CT contrast, circulation in blood for ~ 3 h, and eventual accumulation in RES organs; and following injection locally in the mammary gland, where the full ductal tree structure can be clearly delineated. Partially hydrophilic NCs were encapsulated within mesoporous silica nanoparticles (MSNPs; TaOx@MSNPs) and hydrophobic NCs were encapsulated within poly(lactic-co-glycolic acid) (PLGA; TaOx@PLGA) NPs, serving as potential CT-imagable drug delivery vehicles. Bolus intramuscular injections of TaOx@PLGA NPs and TaOx@MSNPs to mimic the accumulation of NPs at a tumor site produce high signal enhancement in mice. In vitro studies on bare NCs and formuated NPs demonstrate high cytocompatibility and low dissolution of TaOx. This work solidifies that TaOx-based NPs are versatile contrast agents for CT.

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Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

10.31219/osf.io/jf6pe ◽

2019 ◽

Author(s):

Sandeep Chakraborty

Keyword(s):

Cellular Response ◽

Sequencing Data ◽

Dna Breaks ◽

Precise Integration ◽

Guide Rna ◽

Double Strand Dna Breaks ◽

Human Therapy ◽

P53 Activation ◽

Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

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Aggregation-induced emission nanoparticles for in vivo three-photon fluorescence microscopic rat brain angiography

Journal of Innovative Optical Health Sciences ◽

10.1142/s1793545819500123 ◽

2019 ◽

Vol 12 (06) ◽

pp. 1950012 ◽

Cited By ~ 2

Author(s):

Hequn Zhang ◽

Weisi Xie ◽

Ming Chen ◽

Liang Zhu ◽

Zhe Feng ◽

...

Keyword(s):

Rat Brain ◽

Blood Vessels ◽

Laser Scanning ◽

Near Infrared ◽

Brain Disease ◽

Laser Scanning Microscopy ◽

High Signal ◽

Imaging Method ◽

Photon Fluorescence

Rodents are popular biological models for physiological and behavioral research in neuroscience and rats are better models than mice due to their higher genome similarity to human and more accessible surgical procedures. However, rat brain is larger than mice brain and it needs powerful imaging tools to implement better penetration against the scattering of the thicker brain tissue. Three-photon fluorescence microscopy (3PFM) combined with near-infrared (NIR) excitation has great potentials for brain circuits imaging because of its abilities of anti-scattering, deep-tissue imaging, and high signal-to-noise ratio (SNR). In this work, a type of AIE luminogen with red fluorescence was synthesized and encapsulated with Pluronic F-127 to make up form nanoparticles (NPs). Bright DCDPP-2TPA NPs were employed for in vivo three-photon fluorescent laser scanning microscopy of blood vessels in rats brain under 1550[Formula: see text]nm femtosecond laser excitation. A fine three-dimensional (3D) reconstruction up to the deepness of 600[Formula: see text][Formula: see text]m was achieved and the blood flow velocity of a selected vessel was measured in vivo as well. Our 3PFM deep brain imaging method simultaneously recorded the morphology and function of the brain blood vessels in vivo in the rat model. Using this angiography combined with the arsenal of rodent’s brain disease, models can accelerate the neuroscience research and clinical diagnosis of brain disease in the future.

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High-speed high-resolution laser diode-based photoacoustic microscopy for in vivo microvasculature imaging

Visual Computing for Industry Biomedicine and Art ◽

10.1186/s42492-020-00067-5 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Xiufeng Li ◽

Victor T C Tsang ◽

Lei Kang ◽

Yan Zhang ◽

Terence T W Wong

Keyword(s):

High Resolution ◽

High Speed ◽

High Performance ◽

Continuous Wave ◽

Signal To Noise Ratio ◽

Cost Effective ◽

High Signal ◽

Photoacoustic Microscopy ◽

Mouse Ear

AbstractLaser diodes (LDs) have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy (PAM). However, the spatial resolution and/or imaging speed of previously reported LD-based PAM systems have not been optimized simultaneously. In this paper, we developed a high-speed and high-resolution LD-based PAM system using a continuous wave LD, operating at a pulsed mode, with a repetition rate of 30 kHz, as an excitation source. A hybrid scanning mechanism that synchronizes a one-dimensional galvanometer mirror and a two-dimensional motorized stage is applied to achieve a fast imaging capability without signal averaging due to the high signal-to-noise ratio. By optimizing the optical system, a high lateral resolution of 4.8 μm has been achieved. In vivo microvasculature imaging of a mouse ear has been demonstrated to show the high performance of our LD-based PAM system.

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Methods for Voltage-Sensitive Dye Imaging of Rat Cortical Activity With High Signal-to-Noise Ratio

Journal of Neurophysiology ◽

10.1152/jn.01169.2006 ◽

2007 ◽

Vol 98 (1) ◽

pp. 502-512 ◽

Cited By ~ 81

Author(s):

Michael T. Lippert ◽

Kentaroh Takagaki ◽

Weifeng Xu ◽

Xiaoying Huang ◽

Jian-Young Wu

Keyword(s):

Dynamic Range ◽

Signal To Noise Ratio ◽

Field Potential ◽

High Sensitivity ◽

High Dynamic Range ◽

High Signal ◽

Blue Dye ◽

Voltage Sensitive Dye ◽

Imaging Device

We describe methods to achieve high sensitivity in voltage-sensitive dye (VSD) imaging from rat barrel and visual cortices in vivo with the use of a blue dye RH1691 and a high dynamic range imaging device (photodiode array). With an improved staining protocol and an off-line procedure to remove pulsation artifact, the sensitivity of VSD recording is comparable with that of local field potential recording from the same location. With this sensitivity, one can record from ∼500 individual detectors, each covering an area of cortical tissue 160 μm in diameter (total imaging field ∼4 mm in diameter) and a temporal resolution of 1,600 frames/s, without multiple-trial averaging. We can record 80–100 trials of intermittent 10-s trials from each imaging field before the VSD signal reduces to one half of its initial amplitude because of bleaching and wash-out. Taken together, the methods described in this report provide a useful tool for visualizing evoked and spontaneous waves from rodent cortex.

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Rationally Designing Simple Rod-Like Amphiphilic NIR-Emissive AIE Probes for Precise In-Vivo Detection of Aβ Fibrils/Plaques at A Super-Early Stage

10.26434/chemrxiv.13699222.v1 ◽

2021 ◽

Author(s):

Yipu Wang ◽

Dong Mei ◽

Xinyi Zhang ◽

Da-Hui Qu ◽

Ju Mei ◽

...

Keyword(s):

High Performance ◽

Ex Vivo ◽

Early Stage ◽

Signal To Noise Ratio ◽

High Signal ◽

In Vivo Detection ◽

Different Strains ◽

Aβ Fibrils

With increase of social aging, Alzheimer's disease (AD) has been one of the serious diseases threatening human health. The occurrence of Aβ fibrils or plaques is recognized as the hallmark of AD. Currently, optical imaging has stood out to be a promising technique for the imaging of Aβ fibrils/plaques and the diagnosis of AD. However, restricted by their poor blood-brain barrier (BBB) penetrability, short-wavelength excitation and emission, and aggregation-caused quenching (ACQ) effect, the clinically used gold-standard optical probes such as <a>thioflavin</a> T (ThT) and thioflavin S (ThS), are not effective enough in the early diagnosis of AD in vivo. Herein, we put forward an “all-in-one” design principle and demonstrate its feasibility in developing high-performance fluorescent probes which are specific to Aβ fibrils/plaques and promising for super-early in-vivo diagnosis of AD. As a proof of concept, a simple rod-like amphiphilic NIR fluorescent AIEgen, i.e., AIE-CNPy-AD, is developed by taking the specificity, BBB penetration ability, deep-tissue penetration capacity, high signal-to-noise ratio (SNR) into consideration. AIE-CNPy-AD is constituted by connecting the electron-donating and accepting moieties through single bonds and tagging with a propanesulfonate tail, giving rise to the NIR fluorescence, aggregation-induced emission (AIE) effect, amphiphilicity, and rod-like structure, which in turn result in high binding-affinity and excellent specificity to Aβ fibrils/plaques, satisfactory ability to penetrate BBB and deep tissues, ultrahigh SNR and sensitivity, and high-fidelity imaging capability. In-vitro, ex-vivo, and in-vivo <a>identifying of Aβ fibrils/plaques</a> in different strains of mice indicate that AIE-CNPy-AD holds the universality to the detection of Aβ fibrils/plaques. It is noteworthy that AIE-CNPy-AD is even able to trace the small and sparsely distributed Aβ fibrils/plaques in very young AD model mice such as 4-month-old APP/PS1 mice which are reported to be the youngest mice to have Aβ deposits in brains, suggesting its great potential in diagnosis and intervention of AD at a super-early stage.

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Chimeric RNA:DNA Donorguide Improves HDR in vitro and in vivo

10.1101/2021.05.28.446234 ◽

2021 ◽

Author(s):

Brandon W Simone ◽

Han B Lee ◽

Camden L Daby ◽

Santiago Restrepo-Castillo ◽

Hirotaka Ata ◽

...

Keyword(s):

Cell Types ◽

Repair Process ◽

Strand Break ◽

Genetic Alterations ◽

Precise Integration ◽

Genetic Changes ◽

Area Of Interest ◽

Temporal Localization

Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5' or 3' junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5' or 3' junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.

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