Time-resolved microscopy for imaging lanthanide luminescence in living cells.
Time-resolved microscopy for imaging lanthanide luminescence in living cells.
复制标题
DOI：
10.1002/cyto.a.20964
复制
发表时间：
2010-12
期刊：
影响因子：
3.7
通讯作者：
Miller, Lawrence W.
中科院分区：
文献类型：
--
作者：
Gahlaut, Nivriti;Miller, Lawrence W.
时间分辨发光（TRL）显微镜能够对来自镧系配位化合物或其他具有长发射寿命的探针的信号进行成像，从而消除生物样本中短寿命（<100纳秒）的自发荧光背景。然而，镧系配合物单位时间内发射的光子数比常规荧光探针少得多，这使得在与活细胞实验相关的探针浓度下难以快速获取高质量图像。本文介绍了一种TRL显微镜的研制和特性，该显微镜采用发光二极管（LED，发射波长λem = 365纳米）进行脉冲落射照明，并使用增强型电荷耦合器件（ICCD）相机进行门控宽场检测。用铕螯合物浸渍的微球从短寿命荧光背景抑制、光子收集效率、图像对比度和信噪比（SNR）等方面对仪器性能进行了评估。在ICCD的时间分辨率极限（66.7毫秒）内对200纳米的微球进行了成像，且自发荧光被完全抑制。在约1秒的采集时间内检测到了含有约400个螯合分子的40纳米微球。通过胞饮小泡的渗透裂解方法，将一种发光的铽配合物Lumi4 - Tb®以估计为300纳摩尔的浓度引入到培养细胞的细胞质中。在短至333毫秒的采集时间内获取了负载铽配合物的活细胞的时间分辨图像，并评估了增加曝光时间和帧叠加对图像对比度和信噪比的影响。性能分析表明，TRL显微镜具有足够的灵敏度和精度，能够在生理相关的实验条件下对活细胞中的镧系发光进行高分辨率的定量成像。
Time-resolved luminescence (TRL) microscopy can image signals from lanthanide coordination complexes or other probes with long emission lifetimes, thereby eliminating short-lifetime (< 100 ns) autofluorescence background from biological specimens. However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, making it difficult to rapidly acquire high quality images at probe concentrations that are relevant to live cell experiments. This article describes the development and characterization of a TRL microscope that employs a light-emitting diode (LED, λem = 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection. Europium chelate-impregnated microspheres were used to evaluate instrument performance in terms of short-lifetime fluorescence background rejection, photon collection efficiency, image contrast and signal-to-noise ratio (SNR). 200 nm microspheres were imaged within the time resolution limit of the ICCD (66.7 ms) with complete autofluorescence suppression. 40 nm microspheres containing ~400 chelate molecules were detected within ~1 s acquisition times. A luminescent terbium complex, Lumi4-Tb®, was introduced into the cytoplasm of cultured cells at an estimated concentration of 300 nM by the method of osmotic lysis of pinocytic vesicles. Time-resolved images of the living, terbium complex-loaded cells were acquired within acquisition times as short as 333 ms, and the effects of increased exposure time and frame summing on image contrast and SNR were evaluated. The performance analyses show that TRL microscopy is sufficiently sensitive and precise to allow high-resolution, quantitative imaging of lanthanide luminescence in living cells under physiologically relevant experimental conditions.
登录
查看更多内容
影响因子：
3.2
作者：
deHaas, RR;vanGijlswijk, RPM;Tanke, HJ
通讯作者：
Tanke, HJ
影响因子：
3.7
作者：
Connally, Russell;Jin, Dayong;Piper, James
通讯作者：
Piper, James
影响因子：
14.8
作者：
Gruenwald, David;Shenoy, Shailesh M.;Singer, Robert H.
通讯作者：
Singer, Robert H.
影响因子：
6.2
作者：
Ghose, Sraboni;Trinquet, Eric;Mathis, Gerard
通讯作者：
Mathis, Gerard
DOI：
10.1002/cyto.990160302
发表时间：
1994-07-01
期刊：
CYTOMETRY
影响因子：
--
作者：
CONDRAU, MA;SCHWENDENER, RA;ANLIKER, M
通讯作者：
ANLIKER, M