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Aptamer Probes Labeled with Lanthanide-Doped Carbon Nanodots Permit Dual-Modal Fluorescence and Mass Cytometric Imaging.

ABSTRACT: High-dimensional imaging mass cytometry (IMC) enables simultaneous quantification of over 35 biomarkers on one tissue section. However, its limited resolution and ultralow acquisition speed remain major issues for general clinical application. Meanwhile, conventional immunofluorescence microscopy (IFM) allows sub-micrometer resolution and rapid identification of the region of interest (ROI), but only operates with low multiplicity. Herein, a series of lanthanide-doped blue-, green-, and red-fluorescent carbon nanodots (namely, B-Cdots(Ln₁ ), G-Cdots(Ln₂ ), and R-Cdots(Ln₃ )) as fluorescence and mass dual-modal tags are developed. Coupled with aptamers, B-Cdots(¹⁵⁹ Tb)-A10-3.2, G-Cdots(¹⁶⁵ Ho)-AS1411, and R-Cdots(¹⁶⁹ Tm)-SYL3C dual-functional aptamer probes, which are then multiplexed with commercially available Maxpar metal-tagged antibodies for analyzing clinical formalin-fixed, paraffin-embedded (FFPE) prostatic adenocarcinoma (PaC) tissue, are further synthesized. The rapid identification of ROI with IFM using fluorescence signals and subsequent multiplexed detection of in situ ROI with IMC using the same tissue section is demonstrated. Dual-modal probes save up to 90% IMC blind scanning time for a standard 3.5 mm × 3.5 mm overall image. Meanwhile, the IFM provides refined details and topological spatial distributions for the functional proteins at optical resolution, which compensates for the low resolution of the IMC imaging.

SUBMITTER: Yu Y

PROVIDER: S-EPMC8693039 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083(T). Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of E. coli should facilitate a more rational selection of probe target sites for other species as well.

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Aptamer Probes Labeled with Lanthanide-Doped Carbon Nanodots Permit Dual-Modal Fluorescence and Mass Cytometric Imaging.

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