期刊
ACS NANO
卷 8, 期 12, 页码 12418-12427出版社
AMER CHEMICAL SOC
DOI: 10.1021/nn505096t
关键词
super-resolution microscopy; second harmonic generation; BaTiO3 nanocrystals; single-copy detection; mRNA quantification; diagnostics
类别
资金
- NSF [1249315]
- Showalter Trust Grant
- W.M. Keck Foundation
- SIRG fellowship from the Purdue Center for Cancer Research [NIH-NCI P30 CA023168]
- CTSI NIH-NCI grant
- Ismail Travel Grant from Purdue University
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1249315] Funding Source: National Science Foundation
Cell-specific information on the quantity and localization of key mRNAs at single copy sensitivity in single cells is critical for evaluating basic cellular process, disease risk, and efficacy of therapy. Quantification of overexpressed mRNAs beyond the diffraction limit is constrained by the optical property of the probes and microscopy techniques. In this report, nanosized barium titanium oxide (BaTiO3, BTO) crystals were utilized as probes for mRNA quantification by a second harmonic super-resolution microscopy (SHaSM). The SHaSM was able to detect a single copy of the human epidermal growth factor receptor 2 (Her2) mRNA at a resolution of 55.6 nm with the ability to resolve multiple mRNA copies in a diffraction-limited spot. Her2 mRNA per cell was counted in SK-BR-3, MCF-7, and HeLa cell lines as 595 +/- 79.1, 38.9 +/- 8.26, and 1.5 +/- 2.8, respectively. Our single-cell quantification results were validated with the fluorescence in situ hybridization studies and quantitative PCR, showing better specificity and selectivity over current single-molecule approaches for transcript detection. The SHaSM is expected to have an upper limit of resolving similar to 10(4) transcripts in a single cell with the ability to monitor intracellular transcriptional dynamics at video rate. The developed approach has strong potential in clinical research and in the early diagnosis of life-threatening diseases such as cancer.
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