Straub Benedikt B, Lah David C, Schmidt Henrik, Roth Marcel, Gilson Laurent, Butt Hans-Jürgen, Auernhammer Günter K
Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany.
Rev Sci Instrum. 2020 Mar 1;91(3):033706. doi: 10.1063/1.5122311.
We present a new flexible high speed laser scanning confocal microscope and its extension by an astigmatism particle tracking velocimetry (APTV) device. Many standard confocal microscopes use either a single laser beam to scan the sample at a relatively low overall frame rate or many laser beams to simultaneously scan the sample and achieve a high overall frame rate. The single-laser-beam confocal microscope often uses a point detector to acquire the image. To achieve high overall frame rates, we use, next to the standard 2D probe scanning unit, a second 2D scan unit projecting the image directly onto a 2D CCD-sensor (re-scan configuration). Using only a single laser beam eliminates crosstalk and leads to an imaging quality that is independent of the frame rate with a lateral resolution of 0.235 µm. The design described here is suitable for a high frame rate, i.e., for frame rates well above the video rate (full frame) up to a line rate of 32 kHz. The dwell time of the laser focus on any spot in the sample (122 ns) is significantly shorter than those in standard confocal microscopes (in the order of milli- or microseconds). This short dwell time reduces phototoxicity and bleaching of fluorescent molecules. The new design opens up further flexibility and facilitates coupling to other optical methods. The setup can easily be extended by an APTV device to measure three dimensional dynamics while being able to show high resolution confocal structures. Thus, one can use the high resolution confocal information synchronized with an APTV dataset.
我们展示了一种新型的灵活高速激光扫描共聚焦显微镜及其通过像散粒子跟踪测速仪（APTV）装置的扩展。许多标准共聚焦显微镜要么使用单个激光束以相对较低的整体帧率扫描样品，要么使用多个激光束同时扫描样品以实现高整体帧率。单激光束共聚焦显微镜通常使用点探测器来获取图像。为了实现高整体帧率，除了标准的二维探头扫描单元外，我们还使用了第二个二维扫描单元，将图像直接投影到二维CCD传感器上（重新扫描配置）。仅使用单个激光束可消除串扰，并产生与帧率无关的成像质量，横向分辨率为0.235微米。这里描述的设计适用于高帧率，即适用于远高于视频帧率（全帧）直至32kHz行速率的帧率。激光聚焦在样品中任何点上的驻留时间（122纳秒）明显短于标准共聚焦显微镜中的驻留时间（毫秒或微秒量级）。这种短驻留时间减少了荧光分子的光毒性和漂白。新设计提供了进一步的灵活性，并便于与其他光学方法耦合。该装置可以很容易地通过APTV装置进行扩展，以测量三维动力学，同时能够显示高分辨率的共聚焦结构。因此，可以使用与APTV数据集同步的高分辨率共聚焦信息。
