Haga Takanobu, Sonehara Tsuyoshi, Sakai Tomoyuki, Anazawa Takashi, Fujita Takeshi, Takahashi Satoshi
Central Research Laboratory, Hitachi, Ltd., Tokyo, Japan.
Rev Sci Instrum. 2011 Feb;82(2):023701. doi: 10.1063/1.3524570.
We developed a total-internal-reflection (TIR) fluorescence microscopy using three dichroic mirrors and four charge-coupled devices (CCDs) to detect simultaneously four colors of single-molecule (SM) fluorophores. Four spectrally distinct species of fluorophores (Alexa 488, Cy3, Cy5, or Cy5.5) were each immobilized on a different fused silica slide. A species of fluorophores on the slide was irradiated simultaneously, by two excitation beams from an Ar ion laser (488 and 514.5 nm) and a diode laser (642 nm) through TIR on the slide surface. Fluorescence emitted from the fluorophores was spectrally resolved into four components by the dichroic mirrors, and four images were generated from them simultaneously and continuously, with the four CCDs at a rate of 10 Hz. A series of images was thus obtained with each CCD. Fluorescence spots for a species were observed mainly in the series of images recorded by its respective-color CCD. In the first image in the series, we picked out the spots as continuous pixel regions that had the values greater than a threshold. Then we selected only those spots that exhibited single-step photobleaching and regarded them as SM fluorescence spots. Pixel values of SM fluorescence spots widely differed. Some SM fluorophores had pixel values smaller than the threshold, and were left unpicked. Assuming the pixel values of SM fluorescence spots differed with a Gaussian profile, we estimated the ratios of unpicked fluorophores to be less than 20% for all the species. Because of the spectral overlaps between species, we also observed cross-talk spots into CCDs other than the respective-color CCDs. These cross-talk SM fluorescence spots can be mistaken for correct species. We thus introduced the classification method and classified SM fluorescence spots into correct species in accordance with two kinds of four-dimensional signal vectors. The error rates of fluorophore classification were estimated to be less than 3.2% for all the species. Our system is suitable for the biological studies that desire to simultaneously monitor the four colors of SM fluorophores.
我们开发了一种全内反射（TIR）荧光显微镜，它使用三个二向色镜和四个电荷耦合器件（CCD）来同时检测单分子（SM）荧光团的四种颜色。四种光谱不同的荧光团（Alexa 488、Cy3、Cy5或Cy5.5）分别固定在不同的熔融石英载玻片上。载玻片上的一种荧光团通过载玻片表面的全内反射，同时被来自氩离子激光器（488和514.5 nm）和二极管激光器（642 nm）的两束激发光束照射。荧光团发出的荧光通过二向色镜在光谱上被分解为四个成分，并通过四个CCD以10 Hz的速率同时连续地从它们生成四个图像。这样就为每个CCD获得了一系列图像。一种荧光团的荧光斑点主要在其各自颜色的CCD记录的一系列图像中观察到。在该系列的第一张图像中，我们将斑点挑选为具有大于阈值的值的连续像素区域。然后我们只选择那些表现出单步光漂白的斑点，并将它们视为SM荧光斑点。SM荧光斑点的像素值差异很大。一些SM荧光团的像素值小于阈值，因此未被挑选出来。假设SM荧光斑点的像素值呈高斯分布，我们估计所有种类未被挑选出的荧光团的比例小于20%。由于不同种类之间存在光谱重叠，我们还观察到除各自颜色的CCD之外的其他CCD上出现串扰斑点。这些串扰的SM荧光斑点可能会被误认为是正确的种类。因此，我们引入了分类方法，并根据两种四维信号向量将SM荧光斑点分类为正确的种类。估计所有种类的荧光团分类错误率均小于3.2%。我们的系统适用于希望同时监测SM荧光团四种颜色的生物学研究。
