Hyperspectral imaging (HSI) is an emerging imaging modality for medical applications, especially in disease diagnosis and image-guided surgery. HSI acquires a three-dimensional dataset called hypercube, with two spatial dimensions and one spectral dimension. Spatially resolved spectral imaging obtained by HSI provides diagnostic information about the tissue physiology, morphology, and composition. Researchers from UC San Diego developed a new method using a pair of femtosecond mid-infrared and visible excitation pulses to distinguish chromophores, including molecules and quantum dots, that possess nearly identical emission spectra using multiplexed conditions in a three-dimensional space. 

 The dynamic patterning of 3D optical point clouds has emerged as a key enabling technology in volumetric processing across a number of applications. In the context of biological microscopy, 3D point cloud patterning is employed for non-invasive all-optical interfacing with cell ensembles. In augmented and virtual reality (AR/VR), near-eye display systems can incorporate virtual 3D point cloud-based objects into real-world scenes, and in the realm of material processing, point cloud patterning can be mobilized for 3D nanofabrication via multiphoton or ultraviolet lithography. Volumetric point cloud patterning with spatial light modulators (SLMs) is therefore widely employed across these and other fields. However, existing hologram computation methods, such as iterative, look-up table-based and deep learning approaches, remain exceedingly slow and/or burdensome. Many require hardware-intensive resources and sacrifices to volume quality.To address this problem, UC Berkeley researchers have developed a new, non-iterative point cloud holography algorithm that employs fast deterministic calculations. Compared against existing iterative approaches, the algorithm’s relative speed advantage increases with SLM format, reaching >100,000´ for formats as low as 512x512, and optimally mobilizes time multiplexing to increase targeting throughput. 

Hyperspectral imaging, the practice of capturing detailed spectral (color) information from the output of an optical instrument such as a microscope or telescope, is useful in biological and astronomical research and in manufacturing. In addition to being bulky and expensive, existing hyperspectral imagers typically require scanning across a specimen, limiting temporal resolution and preventing dynamic objects from being effectively imaged. Snapshot methods which eliminate scanning are limited by a tradeoff between spatial and spectral resolution.In order to address these problems, researchers at UC Berkeley have developed a hyperspectral imager which can be attached to the output of any benchtop microscope. The imager is compact (about 6-inches), and can achieve a higher spatial resolution than traditional snapshot imagers. Additionally, this imager needs only one exposure to collect measurements for an arbitrary number of spectral filters, giving it unprecedented spectral resolution.

Professor Min Xue and his lab at the University of California, Riverside have developed a novel hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge that may be used for drug delivery purposes. This peptide is non-toxic and has been shown to transport a wide array of small-molecule cargos into a diverse panel of cells. It enables oral administration and absorption through the intestinal lining, and crosses the BBB in vivo. UCR EPP6 is advantageous over existing technologies since it is nontoxic, efficiently enables oral absorption and transport across the BBB.  Fig 1: A) Structure of the UCR EPP. B) Confocal images showing that EPP6 was able to transport different cargo molecules into the cells. C) Orally administered EPP6 is absorbed by the intestines, entering the blood circulation and reaching the brain.  

Lipids play crucial roles in many biological processes under physiological and pathological conditions. Mapping spatial distribution and examining metabolic dynamics of different lipids in cells and tissues in situ are critical for understanding aging and diseases. Commonly used imaging methods, including mass spectrometry-based technologies or labeled imaging techniques, tend to disrupt the native environment of cells/tissues and have limited spatial or spectral resolution, while traditional optical imaging techniques still lack the capacity to distinguish chemical differences between lipid subtypes.

Unlike traditionally-mapped Raman imaging, stimulated Raman scattering (SRS) imaging achieved the capability of imaging metabolic dynamics and a greatly improved signal-noise-ratio. However, its spatial resolution is still limited by the numerical aperture or scattering cross-section.

Methyl-CpG-binding-protein 2 (MeCP2) is a nuclear protein expressed in all cell types, especially neurons. Mutations in the MECP2 gene cause Rett syndrome (RTT), an incurable neurological disorder that disproportionately affects young girls. Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice, suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity.The inventors have discovered that ZF-tMeCP2, a conjugate of MeCP2(aa13-71, 313-484) and the cell-permeant mini-protein ZF5.3, binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters. Efficient nuclear delivery of ZF-tMeCP2 relies on a unique endosomal escape portal provided by HOPS-dependent endosomal fusion.In a comparative evaluation, the inventors observed the Tat conjugate of MeCP2 (Tat-tMeCP2) (1) degrades within the nucleus, (2) is not selective for methylated promoters, and (3) traffics in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of protein-derived therapeutics.

Type III CRISPR-Cas systems recognize and degrade RNA molecules using an RNA-guided mechanism that occurs widely in microbes for adaptive immunity against viruses. The inventors have demonstrated that this multi-protein system can be leveraged for programmable RNA knockdown of both nuclear and cytoplasmic transcripts in mammalian cells. Using single-vector delivery of the S. thermophilus Csm complex, RNA knockdown was achieved with high efficiency (90-99%) and minimal off-targets, outperforming existing technologies of shRNA- and Cas13-mediated knockdown. Furthermore, unlike Cas13, Csm is devoid of trans-cleavage activity and thus does not induce non-specific transcriptome-wide degradation and cytotoxicity. Catalytically inactivated Csm can also be used for programmable RNA-binding, which the inventors exploit for live-cell RNA imaging. This work demonstrates the feasibility and efficacy of multi-subunit CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.

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Researchers from UC San Diego introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability.

Researchers at the University of California, Davis have developed a single-use chip for the identification of protein crystals using X-ray based instruments.

Researchers at UCI have developed a novel method to combine common CCD (charge-coupled device) cameras with mid-infrared (MIR) technology in order to create an affordable and accessible spectroscopic camera for biochemical imaging.

The number of color channels that can be concurrently probed in fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput.    UC Berkeley researchers have discovered methods and systems for simultaneously imaging up to 6 subcellular targets, labeled by common fluorophores of substantial spectral overlap, in live cells at low (~1%) crosstalks and high temporal resolutions (down to ~10 ms), using a single, fixed fluorescence emission detection band. 

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The inventors have developed two new CasX gene-editing platforms (DpbCasXv2 and PlmCasXv2) through rationale structural engineering of the CasX protein and gRNA, which yield improved in vitro and in vivo behaviors. These platforms dramatically increase DNA cleavage activity and can be used as the basis for further improving CasX tools.The RNA-guided CRISPR-associated (Cas) protein CasX has been reported as a fundamentally distinct, RNA-guided platform compared to Cas9 and Cpf1. Structural studies revealed structural differences within the nucleotide-binding loops of CasX, with a compact protein size less than 1,000 amino acids, and guide RNA (gRNA) scaffold stem. These structural differences affect the active ternary complex assembly, leading to different in vivo and in vitro behaviors of these two enzymes.

Profs. Ruoxue Yan, Ming Liu, and their colleagues from the University of California, Riverside have developed a remote-excitation TERS (RE-TERS) probe that prevents uncoupled light from compromising the quality of TERS images. The probe utilizes silver nanoparticles as nanoantennas to mediate the coupling of light to surface plasmon polaritons (SPPs) in a sharp-tip silver nanowire to excite Raman signals remotely. The probe is easy to produce and overcomes the greatest obstacle that stands between scientists and TERS images with high spatial resolution at the nanoscale.  Fig 1. RE-TERS mapping of a chemical vapor deposition-grown molybdenum disulfide (MoS2) monolayer flake. (a) atomic force microscopy image of the MoS2 flake on an ultrasmooth gold substrate with the line-scan shown in (b). The markers indicate the edge of the MoS2 flake (green) and two wrinkles (light blue and orange).  

Profs. Ruoxue Yan, Ming Liu, and their colleagues from the University of California, Riverside have developed a two-step sequential broadband nanofocusing technique with an external nanofocusing efficiency of ~50% over nearly all the visible range on a fibre-coupled nanowire scanning probe. By integrating this with a basic portable scanning tunneling microscope, the technology captured images with spatial resolution as low as one nanometer at high resolution. The high performance and vast versatility offered by this fibre-based nanofocusing technique allows for the easy incorporation of nano-optical microscopy into various existing measurement platforms.  Fig. 1: High-resolution NSOM mapping. a, scanning tunnelling microscope topographic image of single wall carbon nanotubes on a gold film. Top inset: cross-sectional profile along the dashed line. Bottom inset: the possible configurations of the bundle.  

Fluorescent proteins (FP) have been widely used as research tools in both academia and pharma for many years.  Naturally occurring FP have been mutated to either be brighter, be monomers, and/or for easier folding and expression in cells.  The most common FP to date has been the green fluorescent protein (GFP) of the jelly fish Aequorea victoria which can be expressed in cells and fused with proteins of interest, and has proven to be an excellent tool to study protein localization, expression, signaling, etc. in real time via microscopy and other techniques. 

UCLA researchers in the Department of Pharmacology and Department of Microbiology, Immunology, & Molecular Genetics have developed a novel positron emission tomography reporter gene to preferentially trap radiolabeled deoxycytidine analogs.

UCLA researchers in the Department of Pharmacology and Department of Microbiology, Immunology, & Molecular Genetics have developed a novel dual gene positron emission tomography reporter system for the enhanced labeling of cells in vitro and in vivo.

UCLA researchers from the Departments of Molecular & Medical Pharmacology and Bioengineering have developed a novel method for the purification of radiopharmaceuticals for the on-demand production of positron emission tomography (PET) tracers.

Nanoscale multipoint structure-function analysis is essential for deciphering the complexity of multiscale physical and biological systems. Atomic force microscopy (AFM) allows nanoscale structure-function imaging in various operating environments and can be integrated seamlessly with disparate probe-based sensing and manipulation technologies. However, conventional AFMs only permit sequential single-point analysis. Widespread adoption of array AFMs for simultaneous multi-point study is still challenging due to the intrinsic limitations of existing technological approaches.

Puromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. Its mode of action is to inhibit protein synthesis by disrupting peptide transfer on ribosomes, leading to premature chain termination during protein translation. Puromycin blocks protein synthesis in both eukaryotes and prokaryotes and is routinely used as a research tool in cell culture. The native Puromycin is also used assays such as mRNA display. As such, derivatives have been synthesized in which the amino acid of the 3' end of adenosine based antibiotics is altered to change the compound's antibiotic activity. Other compounds have been synthesized with differing amino acids and functionalities to examine the effect it has on bacterial viability. The majority do not show useful absorption or emission profiles. What is needed is a method to track the compounds in biological systems.

UCLA researchers in the Department of Electrical Engineering have developed a Scanning Terahertz Nanoscopy (STN) system with significantly improved detection sensitivity and spatial resolution.

Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein (an effector protein, e.g., a type V Cas effector protein such as Cpf1) bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that continues to revolutionize the field of genome manipulation.    Cas12 is an RNA-guided protein that binds and cuts any matching DNA sequence. Binding of the Cas12-CRISPR RNA (crRNA) complex to a matching single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule activates the protein to non-specifically degrade any ssDNA in trans. Cas12a-dependent target binding can be coupled to a reporter molecule to provide a direct readout for DNA detection within a sample.  UC Berkeley researchers have developed compositions, systems, and kits having labeled single stranded reporter DNA molecules that provide a sensitive readout for detection of a target DNA.