When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark‐field and more recently laser‐based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark‐field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark‐field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal‐to‐noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high‐speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules.

中文翻译：

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使用干涉反射显微镜对单个微管进行无标记高速宽视野成像

在体外研究微管时，当纯化的微管蛋白的数量太低而无法进行有效的荧光标记或担心标记会破坏功能时，需要对单个微管进行无标记成像。用于观察未标记微管的常用技术，例如视频增强微分干涉对比度、暗场和最近的基于激光的干涉散射显微镜，存在许多缺点。微分干涉对比度图像的对比度取决于微管的方向，暗场对杂质和光学错位高度敏感。此外，所有这些技术都需要昂贵的光学元件，例如诺马斯基棱镜、暗场聚光镜、激光器和激光扫描仪。在这里，我们表明，使用干涉反射显微镜可以对单个微管进行高速和高对比度成像，而没有上述缺点。干涉反射显微镜实施起来很简单，只需要在荧光显微镜中加入 50/50 镜子而不是二向色镜，并且在适当的显微镜设置下，具有与微分干涉对比度和荧光相似的信噪比。我们通过每秒 100 帧的动态微管高速成像和跟踪展示了干涉反射显微镜的实用性。总之，干涉反射显微镜的光学质量属于其他显微镜技术的范围，根据所使用的度量，其光学质量低于某些技术，但优于其他技术，并且只需对显微镜进行最小的修改，即可用于研究未标记微管的动力学。