Central Research Laboratory, Hitachi, Ltd., Kokubunji-shi, Tokyo, Japan.
J Fluoresc. 2013 May;23(3):591-7. doi: 10.1007/s10895-013-1208-8. Epub 2013 Mar 8.
A prism-based imaging system for simultaneously detecting four species of single-molecule (SM) fluorophores was developed. As for the detection method, four spectrally distinct species of BigDye fluorophores were bound to 50-nm-diameter gold nanoparticles (AuNPs) to form AuNP/BigDye complexes. Four species of complexes were randomly immobilized on different fused-silica slides. BigDyes were excited by an argon-ion-laser (excitation wavelengths: 488 and 514.5 nm) beam through total internal reflection on the slide surface. SM fluorescence emitted from a complex was spectrally dispersed through a prism to form an SM spot elongated in the spectral direction on a charge-coupled device. A scattered light spot generated by the AuNP of the same complex under 594-nm laser illumination was used as a wavelength reference, and the SM fluorescence spectrum was obtained from the pixel-intensity pattern of the elongated SM spot. Peak locations of fluorescence spectra of all the observed SM spots were obtained, and their histograms were distinctly separated according to species. SM spots can thus be classified as one of four species according to their peak locations. By statistically analyzing the histograms, the classification accuracy was estimated to be above 93.8 %. The number of pixels in the spectral direction required for classifying four species of SM fluorophores was estimated to be 10. As for the conventional system (which uses two excitation lasers), 15 pixels are required. Using BigDyes as the four fluorophores (which consist of donors linked to acceptors and can be excited by just an argon-ion laser) is the reason that such a small number of pixels was achieved. The developed system can thus detect 1.5 times more SM fluorophores per field of view; that is, its throughput is 1.5 times higher. The approach taken in this study, namely, using BigDye with a prism-type system, is effective for increasing the throughput of DNA microarray-chip analysis and SM real-time DNA sequencing.
一种基于棱镜的成像系统，用于同时检测四种单分子（SM）荧光团。对于检测方法，将四种光谱上不同的 BigDye 荧光团结合到 50nm 直径的金纳米粒子（AuNP）上，形成 AuNP/BigDye 复合物。四种复合物随机固定在不同的熔融硅片上。BigDyes 通过激光在幻灯片表面的全内反射激发氩离子激光（激发波长：488nm 和 514.5nm）。从复合物发射的 SM 荧光通过棱镜分光，在电荷耦合器件上形成沿光谱方向拉长的 SM 点。在 594nm 激光照射下，同一复合物的 AuNP 产生的散射光点用作波长参考，从拉长的 SM 点的像素强度图案中获得 SM 荧光光谱。获得所有观察到的 SM 点的荧光光谱的峰位置，并根据物种明显分离其直方图。根据峰位置，可以将 SM 点分类为四种物种之一。通过对直方图进行统计分析，估计分类准确率高于 93.8%。分类四种 SM 荧光团所需的光谱方向的像素数估计为 10。对于传统系统（使用两个激发激光），需要 15 个像素。使用 BigDyes 作为四种荧光团（由与受体相连的供体组成，仅用氩离子激光即可激发）是实现如此少像素数的原因。因此，开发的系统可以在每个视场中检测到 1.5 倍的 SM 荧光团；也就是说，它的吞吐量提高了 1.5 倍。本研究采用的方法，即使用带有棱镜型系统的 BigDye，可有效提高 DNA 微阵列芯片分析和 SM 实时 DNA 测序的通量。
