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首页> 外文期刊>Biochemistry >In Situ Single-Molecule Imaging with Attoliter Detection Using Objective Total Internal Reflection Confocal Microscopy
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In Situ Single-Molecule Imaging with Attoliter Detection Using Objective Total Internal Reflection Confocal Microscopy

机译：使用客观全内反射共聚焦显微镜进行Attoliter检测的原位单分子成像

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Confocal microscopy is widely used for acquiring high spatial resolution tissue sample images of interesting fluorescent molecules inside cells.The fluorescent molecules are often tagged proteins participating in a biological function.The high spatial resolution of confocal microscopy compared to wide field imaging conies from an ability to optically isolate and image exceedingly small volume elements made up of the lateral (focal plane) and depth dimensions.Confocal microscopy at the optical diffraction limit images volumes on the order of approx 0.5 femtoliter (10~(-15) L).Further resolution enhancement can be achieved with total internal reflection microscopy (TIRM).With TIRM,an exponentially decaying electromagnetic field (near-field) established on the surface of the sample defines a subdiffraction limit dimension that,when combined with conventional confocal microscopy,permits image formation from <7 attoL (10~(-18) L) volumes [Borejdo et al.(2006) Biochim.Biophys.Acta,in press].Demonstrated here is a new variation of TIRM,focused TIRM (fTIRM) that decreases the volume element to approx 3 attoL.These estimates were verified experimentally by measuring characteristic times for Brownian motion of fluorescent nanospheres through the volume elements.A novel application for TIRM is in situ single-molecule fluorescence spectroscopy.Single-molecule studies of protein structure and function are well-known to avoid the ambiguities introduced by ensemble averaging.In situ,proteins are subjected to the native forces of the crowded environment in the cell that are not present in vitro.The attoL fluorescence detection volume of TIRM permits isolation of single proteins in situ.Muscle tissue contains myosin at a approx 120 mu M concentration.Evidence is provided that >75% of the bleachable fluorescence detected with fTIRM is emitted by five chromophore-labeled myosins in a muscle fiber.
机译：共聚焦显微镜被广泛用于获取细胞内有趣的荧光分子的高空间分辨率组织样本图像。荧光分子通常是参与生物学功能的标记蛋白。与宽视野成像相比，共聚焦显微镜具有高空间分辨率光学隔离并成像由侧面（焦平面）和深度尺寸组成的极小体积的元素。在光学衍射条件下的孔镜检查将图像体积限制在约0.5飞升（10〜（-15）L）左右。进一步提高了分辨率借助全内反射显微镜（TIRM）可以实现。通过TIRM，在样品表面建立的指数衰减电磁场（近场）定义了亚衍射极限尺寸，当与常规共聚焦显微镜结合使用时，可以允许从体积小于7 attoL（10〜（-18）L）[Borejdo等人（2006）Biochim.Biophys.Acta，印刷中）。这里展示的是TIRM的新变化形式，即聚焦的TIRM（fTIRM），它使体积元素减少到大约3 attoL。这些估计值通过测量荧光纳米球通过体积元素的布朗运动的特征时间进行了实验验证。为了避免整体平均引入歧义，对蛋白质的结构和功能进行单分子研究是众所周知的。在原位，蛋白质要经受细胞内拥挤环境的自然力TIRM的attoL荧光检测体积允许原位分离单个蛋白质。肌肉组织中含有约120μM浓度的肌球蛋白。有证据表明，fTIRM检测到的可漂白荧光的> 75％是由五个发色团发出的肌纤维中标记的肌球蛋白。

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