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Targeted NIR Fluorescent Mechanically Interlocked Molecules-Peptide Bioconjugate for Live Cancer Cells Submitochondrial Stimulated Emission Depletion Super-Resolution Microscopy.
Bioconjug Chem
January 2025
Department of Chemistry, Organic Chemistry Section, Jadavpur University, Kolkata 700032, India.
Herein, a water-soluble, ultrabright, near-infrared (NIR) fluorescent, mechanically interlocked molecules (MIMs)-peptide bioconjugate is designed with dual targeting capabilities. Cancer cell surface overexpressed αβ integrin targeting two RGDS tetrapeptide residues is tethered at the macrocycle of MIMs-peptide bioconjugate via Cu(I)-catalyzed click chemistry on the Wang resin, and mitochondria targeting lipophilic cationic TPP functionality is conjugated at the axle dye. Living carcinoma cell selective active targeting, subsequently cell penetration, mitochondrial imaging, including the ultrastructure of cristae, and real-time tracking of malignant mitochondria by MIMs-peptide bioconjugate (RGDS)-Mito-MIMs-TPP are established by stimulated emission depletion (STED) super-resolved fluorescence microscopy.
View Article and Find Full Text PDFLately, important advancements in visualizing RNAs in fixed and live cells have been achieved. While mRNA imaging techniques are well-established, the development of effective methods for studying non-coding RNAs (ncRNAs) in living cells are still challenging but necessary, as they show a variety of functions and intracellular localizations, including participation in highly dynamic processes like phase-transition, which is still poorly studied in vivo. Addressing this issue, we tagged two exemplary ncRNAs with the fluorescent RNA (fRNA) Pepper.
View Article and Find Full Text PDFObtaining super-resolved images at the mesoscale through super-resolution radial fluctuations.
J Biomed Opt
December 2024
University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, United Kingdom.
Significance: Current super-resolution imaging techniques allow for a greater understanding of cellular structures; however, they are often complex or only have the ability to image a few cells at once. This small field of view (FOV) may not represent the behavior across the entire sample, and manual selection of regions of interest (ROIs) may introduce bias. It is possible to stitch and tile many small ROIs; however, this can result in artifacts across an image.
View Article and Find Full Text PDFFortunate molecules boost signal to background ratio and localization precision in correlation based single molecule localization microscopy.
Commun Biol
December 2024
Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore, 560012, India.
Single-molecule localization microscopy (SMLM) can decipher fine details that are otherwise impossible using diffraction-limited microscopy. Often, the reconstructed super-resolved images suffer from noise, strong background and are prone to false detections that may impact quantitative imaging. To overcome these limitations, we propose a technique (corrSMLM) that recognizes and detects fortunate molecules (molecules with long blinking cycles) from the recorded data.
View Article and Find Full Text PDFSingle-molecule orientation-localization microscopy: Applications and approaches.
Q Rev Biophys
December 2024
Preston M. Green Department of Electrical and Systems Engineering, Washington University in St. Louis, St. Louis, MO, USA.
Single-molecule orientation-localization microscopy (SMOLM) builds upon super-resolved localization microscopy by imaging orientations and rotational dynamics of individual molecules in addition to their positions. This added dimensionality provides unparalleled insights into nanoscale biophysical and biochemical processes, including the organization of actin networks, movement of molecular motors, conformations of DNA strands, growth and remodeling of amyloid aggregates, and composition changes within lipid membranes. In this review, we discuss recent innovations in SMOLM and cover three key aspects: (1) biophysical insights enabled by labeling strategies that endow fluorescent probes to bind to targets with orientation specificity; (2) advanced imaging techniques that leverage the physics of light-matter interactions and estimation theory to encode orientation information with high fidelity into microscope images; and (3) computational methods that ensure accurate and precise data analysis and interpretation, even in the presence of severe shot noise.
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