Many experimental biological techniques utilize hollow glass needles called micropipettes to perform fluid extraction, cell manipulation, and electrophysiological recordings. For electrophysiological recordings, micropipettes are typically fabricated immediately before use using a “pipette puller”, which uses open-loop control to heat a hollow glass capillary while applying a tensile load. Variability between manufactured micropipettes requires a highly trained operator to qualitatively inspect each micropipette; typically this is achieved by viewing the pipette under 40–100× magnification in order to ensure that the tip has the correct shape (e.g., outer diameter, cone angle, taper length). Since laboratories may use hundreds of micropipettes per week, significant time demands are associated with micropipette inspection. Here, we have automated the measurement of micropipette tip outer diameter and cone angle using optical microscopy. The process features repeatable constraint of the micropipette, quickly and automatically moving the micropipette to bring its tip into the field of view, focusing on the tip, and computing tip outer diameter and cone angle measurements from the acquired images by applying a series of image processing algorithms. As implemented on a custom automated microscope, these methods achieved, with 95% confidence, ±0.38 μm repeatability in outer diameter measurement and ±5.45° repeatability in cone angle measurement, comparable to a trained human operator. Accuracy was evaluated by comparing optical pipette measurements with measurements obtained using scanning electron microscopy (SEM); optical outer diameter measurements differed from SEM by 0.35 ± 0.36 μm and optical cone angle measurements differed from SEM by −0.23 ± 2.32°. The algorithms we developed are adaptable to most commercial automated microscopes and provide a skill-free route to rapid, quantitative measurement of pipette tip geometry with high resolution, accuracy, and repeatability. Further, these methods are an important step toward a closed-loop, fully-automated micropipette fabrication system.

许多实验生物学技术都使用称为微量移液器的中空玻璃针来进行液体提取，细胞操作和电生理记录。对于电生理记录，通常在使用前立即使用“移液器拉拔器”制造微量移液器，该移液器使用开环控制来加热中空玻璃毛细管，同时施加拉伸载荷。制造的微量移液器之间的差异需要训练有素的操作员定性检查每个微量移液器。通常，这是通过在40-100倍的放大倍数下观察移液器来实现的，以确保尖端具有正确的形状（例如，外径，锥角，锥度）。由于实验室每周可能使用数百个微量移液器，因此大量的时间需求与微量移液器的检查相关。这里，我们使用光学显微镜自动测量了微量移液器吸头的外径和锥角。该过程具有可重复使用的微量移液器约束，可快速自动移动微量移液器以使其尖端进入视野，聚焦于尖端，并通过应用一系列图像处理来从获取的图像中计算尖端的外径和锥角测量值算法。如在定制的自动显微镜上实施的那样，这些方法可以达到95％的置信度，外径测量的可重复性为±0.38μm，锥角测量的可重复性为±5.45°，可与受过训练的人工操作者媲美。通过将光学移液器测量结果与使用扫描电子显微镜（SEM）获得的测量结果进行比较来评估准确性；光学外径测量值与SEM相差0。35±0.36μm和光学锥角测量值与SEM相差−0.23±2.32°。我们开发的算法适用于大多数商用自动显微镜，并提供了一种无需技巧的方法即可快速，定量地测量移液器吸头的几何形状，并具有高分辨率，准确性和可重复性。此外，这些方法是朝着闭环，全自动微量移液器制造系统迈出的重要一步。