Browse Category: Imaging > Molecular

[Search within category]

(SD2023-232) Multi-Dimensional Widefield Infrared-encoding Spontaneous Emission Microscopy

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. 

Hyperspectral Microscopy Using A Phase Mask And Spectral Filter Array

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.

Novel Cell Penetrating Peptide for Drug Delivery

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.  

Spectral Fluctuation Raman Spectroscopy (SFRS)

The function of living tissue relies not only on its structure, but crucially on its dynamics at an array of timescales. Structural imaging of biological molecules at very high resolution has become routine in recent years, but these static snapshots provide little insight into the structural changes crucial for biological function. It is well known that changes in the geometry of macromolecules induce fluctuations in the Raman spectrum, but measurements of these fluctuations inherently suffer from poor signal strengths, meaning that dynamics at many timescales are obscured by the time-averaging necessary to obtain sufficient sensitivity.To address these problems, researchers at UC Berkeley have developed a method for probing the Raman spectrum, and hence dynamics of biological molecules at very high sensitivity and across timescales inaccessible to extant techniques. This technique, in fact, can in principle obtain arbitrarily fine spectral and temporal resolution, opening the door to, for example, probe everything from the dynamics of side chain rotations (picoseconds) to protein folding and domain motion (milliseconds).

(SD2023-060) Penalized Reference Matching algorithm with Stimulated Raman Scattering (PRM-SRS) microscopy: Multi-Molecular Detection of Hyperspectral images

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.

(SD2023-059) Super Resolution Microscopy with A-POD Deconvolution

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.

Nuclear Delivery and Transcriptional Repression with a Cell-penetrant MeCP2

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 System for Robust RNA Knockdown and Imaging in Eukaryotes

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.

Templated Synthesis Of Metal Nanorods

Brief description not available

(SD2021-181) Photo-activated Control of CRISPR-Cas9 Gene Editing

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.

2-D Polymer-Based Device for Serial X-Ray Crystallography

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

Facile, Excitation-Based Spectral Microscopy For Fast Multicolor Imaging And Quantitative Biosensing

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. 

Improved guide RNA and Protein Design for CasX-based Gene Editing Platform

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.

Remote Excitation Probe for Tip-Enhanced Raman Spectroscopy

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).  

High External-Efficiency Nanofocusing for Lens-Free Near-Field Optical Microscopy

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.  

New Bright Green Fluorescent Proteins

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. 

Novel Non-Immunogenic Positron Emission Tomography Gene Reporter

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.

Non-Immunogenic Positron Emission Tomography Gene Reporter Systems

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.

Microscale Device and Method for Purification of Radiopharmaceuticals

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.

Array Atomic Force Microscopy Enabling Simultaneous Multi-point and Multi-modal Nanoscale Analyses

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.

Development of Novel Fluorescent Puromycin Derivatives

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.

Scanning Terahertz Nanoscopy Probe

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

Cas12-mediated DNA Detection Reporter Molecules

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. 

Puromycin Activity-Based Sensing Probes For Molecular Imaging And Histochemistry

A novel class of puromycin activity-based sensing probes containing analyte-specific responsive triggers have been synthesized and utilized for molecular imaging and histochemistry. After specific reaction between the trigger on the probe and target analyte, free puromycin molecules will be released and incorporated into nascent peptides. These incorporated puromycin can be detected after immunostaining, thus offering a highly sensitive method for detection of target analytes due to no leakage problem (as found in some reported fluorescent probes) and high signal-to-noise level from immunostaining. The syntheses of the probes are highly versatile, and representative examples for detection of reactive oxygen species (ROS), reactive sulfur species (RSS), reactive carbonyl species (RCS), ROS scavengers, and redox active metal ions have been demonstrated. One exemplary probe is Peroxymycin-1, which contains H2O2-responsive aryl boronate conjugated to puromycin through carbamate linkage. Peroxymycin-1 shows robust performance on molecular imaging of H2O2 in cell culture and histochemical analysis of H2O2 level in tissue samples harvested from small animals. It has been further employed for detection of elevated H2O2 level in liver tissues from a murine model of non-alcoholic fatty liver disease (NAFLD), suggesting its potential for studying disease pathology associated with H2O2 as well as disease diagnosis and monitoring of treatment progress.

  • Go to Page: