conventional wide field fluorescence microscopy imaging  (Nikon)

Journal: Nucleic Acids Research

Article Title: Specific killing and resensitization of pathogenic Escherichia coli strains carrying bla CTX-M-15 β-lactamase using targeted-antibacterial-plasmids (TAPs)

doi: 10.1093/nar/gkaf1466

Figure Lengend Snippet: Visualization and model of TAP activity. ( A ) Fluorescence microscopy images of time-lapse experiments in a microfluidic chamber, showing TAPs’ transfer between a donor carrying the RP4 helper and the indicated TAP, and the E2 recipient strains. Real-time monitoring allows the visualization of the effect of TAP acquisition in the absence and presence of exposure to Ctx treatment. Arrows indicate Ctx S TAP donors producing both red and green fluorescence (red D), and Ctx R recipients producing no fluorescence (white R). TAP acquisition is reflected by the production of green fluorescence in transconjugant cells (green T). Time in minutes, with 0 min corresponding to the time of Ctx injection. Scale bar 1 μm. ( B ) Model for TAP activity against strains carrying bla CTX-M-15 . The donor strain transfers a TAP carrying cas9 or dcas9 genes through the conjugation machinery provided by the helper plasmid. The recipient cell having acquired the TAP produces the components of the CRISPR system. The Cas9 or the dCas9 protein is recruited to the targeted bla CTX-M-15 gene by the complementary gRNA. The Cas9 induces a DSB, while the dCas9 inhibits the expression of the bla CTX-M-15 gene. The subsequent effect strictly depends on the chromosomic or plasmidic location of the bla CTX-M-15 gene. TAP-Cas9 targeting the chromosome leads to cell death. TAP-Cas9 targeting a plasmid results in plasmid loss, which can lead to cell death if some TA systems are activated. TAP-dCas9 targeting the chromosome or a plasmid does not affect viability but results in resensitization of the recipient to the drug.

Article Snippet: Ctx was added and cells were imaged every 10 min for 3 h. Conventional wide-field fluorescence microscopy imaging was carried out on an Eclipse Ti2-E microscope (Nikon), equipped with x100/1.45 oil Plan Apo Lambda phase objective, ORCA-Fusion digital CMOS camera (Hamamatsu), and using NIS software for image acquisition.

Techniques: Activity Assay, Fluorescence, Microscopy, Injection, Conjugation Assay, Plasmid Preparation, CRISPR, Expressing

Journal: bioRxiv

Article Title: PEPCy: Photostable fluoromodules for live cell, super-resolution microscopy of surface proteins

doi: 10.1101/2024.07.03.601615

Figure Lengend Snippet: Applications of PEPCy tags for imaging cell surface receptors. A . Schematic of membrane protein tracking experiment using TIRF microscopy. AlphaFold predicted structures of PEPCy tags. B . Normalized probability distributions of photons detected per trajectory from single particle analyses between PEPCy and HT targeted to their cognate dyes. (N = 1000-1300 single tracks per sample). C. Super-resolution microscopy of Intimin fused to PEPCy3 expressed on E.coli outer membrane. Schematic of the fusion is shown followed by diffraction-limited DIC and fluorscence images of the cells expressing the same. Maximum intensity projections (MIP) compared with a 2D reconstruction of single molecule localization data. Inset compares a cluster observable in the MIP that is resolved into two clusters in the super-resolved image. D . Time-lapse confocal microscopy of HeLa cells expressing PEPCy3-B2AR labeled using Cy3. Numbers on the left of each image denote the time in minutes. Isoproterenol was added at t = 0 and imaged were acquired every 30s for 1 hour. E . Schematic of simultaneous two color tracking of PEPCy3-B2AR and PEPCy5-B2AR-Ala on the same cell. F . Single molecule trajectories of PEPCy3 (left) and PEPCy5 (right) on a HEK cell, color coded by median trajectory speed. All scale bars in the figure unless mentioned otherwise are 5 µ m. A and E were created with BioRender.com .

Article Snippet: Prior to single molecule imaging PEPCy3-Cy3 signal was confirmed by diffraction limited wide-field epifluorescence microscopy, performed on a Nikon Eclipse Ti2 microscope equipped with a super apochromat objective (PlanApo, 100x, 1.45 N.A., oil immersion, Nikon) and scientific cMOS camera (Prime95B, Photometrix).

Techniques: Imaging, Membrane, Microscopy, Single Particle, Super-Resolution Microscopy, Expressing, Confocal Microscopy, Labeling