Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy
Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy
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DOI：
10.1021/ac026333r
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发表时间：
2003-05-01
影响因子：
7.4
通讯作者：
Sauer, M
中科院分区：
文献类型：
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作者：
Knemeyer, JP;Herten, DP;Sauer, M
单个mRNA分子标记的显微注射，单荧光标记的寡（dT）43聚体分子在活细胞中的准自然环境，即细胞培养基中的检测，证明。单链寡核苷酸在5 '端用红色吸收恶嗪衍生物（MR 121）标记，并通过在635 nm处发射的脉冲激光二极管以64 MHz的重复率激发。光谱分辨荧光寿命成像显微镜（SFLIM）对未经处理的活3 T3小鼠成纤维细胞显示自发荧光信号主要存在于细胞质中，荧光寿命类似于1.3 ns，发射最大值类似于665 -670 nm。因此，表现出2.8ns的荧光寿命和685 nm的荧光发射最大值的单个MR 121标记的寡核苷酸分子的荧光信号可以容易地与自发荧光区分开。使用微量移液管将MR 121标记的寡核苷酸显微注射到活的3 T3小鼠成纤维细胞的细胞质或细胞核中。由于微量移液器在尖端末端的内径为500 +/- 200 nm-与所应用的检测体积的直径相当-因此可以对经由微量移液器递送到细胞中的分子的数量进行计数。此外，所提出的技术，使定量检测和时间分辨识别活细胞中的单个分子作为其特征发射最大值和荧光寿命的结果。从单分子研究中获得的结果首次证明，10-30%的显微注射的oligo（0）43-mer分子不能在细胞核内自由扩散，而是被束缚在转录、剪接或聚腺苷酸化机制的固定元件上。
The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition, rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of similar to1.3 ns and emission maximums of similar to665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30% of the microinjected oligo(0) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.