-
Cell Jan 2024Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at...
Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 μm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 μm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.
Topics: DNA; Enhancer Elements, Genetic; Gene Expression Regulation; Super Enhancers; Transcription, Genetic; SOXB1 Transcription Factors; Animals; Mice; Embryonic Stem Cells; Microscopy
PubMed: 38194964
DOI: 10.1016/j.cell.2023.12.005 -
Optics Express Oct 2023Spiral-phase-contrast imaging, which utilizes a spiral phase optical element, has proven to be effective in enhancing various aspects of imaging, such as edge contrast...
Spiral-phase-contrast imaging, which utilizes a spiral phase optical element, has proven to be effective in enhancing various aspects of imaging, such as edge contrast and shadow imaging. Typically, the implementation of spiral-phase-contrast imaging requires the formation of a Fourier plane through a 4f optical configuration in addition to an existing optical microscope. In this study, we present what we believe to be a novel single spiral-phase-objective, integrating a spiral phase plate, which can be easily and simply applied to a standard microscope, such as a conventional objective. Using a new hybrid design approach that combines ray-tracing and field-tracing simulations, we theoretically realized a well-defined and high-quality vortex beam through the spiral-phase-objective. The spiral-phase-objective was designed to have conditions that are practically manufacturable while providing predictable performance. To evaluate its capabilities, we utilized the designed spiral-phase-objective to investigate isotropic spiral phase contrast and anisotropic shadow imaging through field-tracing simulations, and explored the variation of edge contrast caused by changes in the thickness of the imaging object.
PubMed: 37859196
DOI: 10.1364/OE.499376 -
Microscopy (Oxford, England) Jun 2023Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely...
Microscopy has been essential to elucidate micro- and nano-scale processes in space and time and has provided insights into cell and organismic functions. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques.
Topics: Humans; Virus Diseases; Viruses; Microscopy, Atomic Force; Microscopy, Electron; Microscopy, Fluorescence; Cryoelectron Microscopy
PubMed: 37079744
DOI: 10.1093/jmicro/dfad024 -
Nature Communications Oct 2023In electron microscopy, detailed insights into nanoscale optical properties of materials are gained by spontaneous inelastic scattering leading to electron-energy loss...
In electron microscopy, detailed insights into nanoscale optical properties of materials are gained by spontaneous inelastic scattering leading to electron-energy loss and cathodoluminescence. Stimulated scattering in the presence of external sample excitation allows for mode- and polarization-selective photon-induced near-field electron microscopy (PINEM). This process imprints a spatial phase profile inherited from the optical fields onto the wave function of the probing electrons. Here, we introduce Lorentz-PINEM for the full-field, non-invasive imaging of complex optical near fields at high spatial resolution. We use energy-filtered defocus phase-contrast imaging and iterative phase retrieval to reconstruct the phase distribution of interfering surface-bound modes on a plasmonic nanotip. Our approach is universally applicable to retrieve the spatially varying phase of nanoscale fields and topological modes.
PubMed: 37848420
DOI: 10.1038/s41467-023-42054-3 -
Current Opinion in Biotechnology Feb 2024Despite remarkable progresses in quantitative phase imaging (QPI) microscopes, their wide acceptance is limited due to the lack of specificity compared with the... (Review)
Review
Despite remarkable progresses in quantitative phase imaging (QPI) microscopes, their wide acceptance is limited due to the lack of specificity compared with the well-established fluorescence microscopy. In fact, the absence of fluorescent tag prevents to identify subcellular structures in single cells, making challenging the interpretation of label-free 2D and 3D phase-contrast data. Great effort has been made by many groups worldwide to address and overcome such limitation. Different computational methods have been proposed and many more are currently under investigation to achieve label-free microscopic imaging at single-cell level to recognize and quantify different subcellular compartments. This route promises to bridge the gap between QPI and FM for real-world applications.
Topics: Quantitative Phase Imaging; Microscopy; Microscopy, Phase-Contrast
PubMed: 38142647
DOI: 10.1016/j.copbio.2023.103054 -
Optics Express Mar 2024Quantitative phase contrast microscopy (QPCM) can realize high-quality imaging of sub-organelles inside live cells without fluorescence labeling, yet it requires at...
Quantitative phase contrast microscopy (QPCM) can realize high-quality imaging of sub-organelles inside live cells without fluorescence labeling, yet it requires at least three phase-shifted intensity images. Herein, we combine a novel convolutional neural network with QPCM to quantitatively obtain the phase distribution of a sample by only using two phase-shifted intensity images. Furthermore, we upgraded the QPCM setup by using a phase-type spatial light modulator (SLM) to record two phase-shifted intensity images in one shot, allowing for real-time quantitative phase imaging of moving samples or dynamic processes. The proposed technique was demonstrated by imaging the fine structures and fast dynamic behaviors of sub-organelles inside live COS7 cells and 3T3 cells, including mitochondria and lipid droplets, with a lateral spatial resolution of 245 nm and an imaging speed of 250 frames per second (FPS). We imagine that the proposed technique can provide an effective way for the high spatiotemporal resolution, high contrast, and label-free dynamic imaging of living cells.
Topics: Animals; Mice; Quantitative Phase Imaging; Deep Learning; Mitochondria; Lipid Droplets
PubMed: 38571068
DOI: 10.1364/OE.520784 -
Journal of Hazardous Materials Aug 2023The PCM (phase contrast microscopy) method for asbestos counting needs special sample treatments, hence it is time consuming and rather expensive. As an alternative, we...
The PCM (phase contrast microscopy) method for asbestos counting needs special sample treatments, hence it is time consuming and rather expensive. As an alternative, we implemented a deep learning procedure on images directly acquired from the untreated airborne samples using standard Mixed Cellulose Ester (MCE) filters. Several samples with a mix of chrysotile and crocidolite with different concentration loads have been prepared. Using a 20x objective lens coupled with a backlight illumination system a number of 140 images were collected from these samples, which along with additional 13 highly fibre loaded artificial images constituted the database. About 7500 fibres were manually recognised and annotated following the National Institute for Occupational Safety and Health (NIOSH) fibre counting Method 7400 as input for the training and validation of the model. The best trained model provides a total precision of 0.84 with F1-Score of 0.77 at a confidence of 0.64. A further post-detection refinement to ignore detected fibres < 5 µm in length improves the final precision. This method can be considered as a reliable and competent alternative to conventional PCM.
Topics: United States; Deep Learning; Asbestos; Asbestos, Serpentine; Microscopy, Phase-Contrast; Asbestos, Crocidolite; Occupational Exposure
PubMed: 37178531
DOI: 10.1016/j.jhazmat.2023.131590 -
Accounts of Chemical Research Oct 2023Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine─the ribosome. This process is termed translation because... (Review)
Review
Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machine─the ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry.Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule-NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis.Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs.In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine () RNA and peptide solid-phase synthesis on 3'-aminoacylamino-adenosine resins, () native chemical ligations and Staudinger ligations, () tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and () enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging.Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis.
Topics: Cryoelectron Microscopy; RNA, Transfer; RNA; Amino Acids; Protein Biosynthesis; Peptides; Anti-Bacterial Agents; RNA, Messenger; Biology
PubMed: 37728742
DOI: 10.1021/acs.accounts.3c00412 -
Journal of Biomedical Optics Jun 2024Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two...
SIGNIFICANCE
Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast.
AIM
We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts.
APPROACH
BiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast.
RESULTS
BiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors.
CONCLUSIONS
BiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.
Topics: Animals; Microscopy; Tomography, Optical Coherence; Microscopy, Phase-Contrast
PubMed: 38584966
DOI: 10.1117/1.JBO.29.S2.S22704 -
Optics Express Jul 2023We demonstrate a propagation-based phase-contrast imaging method for full-field X-ray microscopy based on advanced Kirkpatrick-Baez (AKB) mirrors to achieve...
We demonstrate a propagation-based phase-contrast imaging method for full-field X-ray microscopy based on advanced Kirkpatrick-Baez (AKB) mirrors to achieve high-contrast observations of weak phase objects and correct field curvature aberrations. Through a demonstration performed at SPring-8, the phase contrast of weak phase objects such as polystyrene spheres and chemically fixed cells was successfully observed with high sensitivity (∼0.03 rad). Furthermore, the field of view of the AKB mirrors was expanded to the full area of the obtained images (25 × 30 µm) by correcting the field curvature aberration using reconstructed complex wavefields.
PubMed: 37710481
DOI: 10.1364/OE.493789