California Institute of Technology, Bioengineering, Pasadena, California 91125, USA.
J Biomed Opt. 2011 Nov;16(11):116009. doi: 10.1117/1.3647570.
Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.
光学切片提供生物组织的三维（3D）信息。然而，大多数采用光学切片的成像技术要么速度较慢，要么对活组织有害。在这里，我们提出了一种用于宽场多光子显微镜的简单设计，该设计以合理的帧率提供光学切片，并且激光剂量具有生物相容性。光学切片的基本原理是基于扩散器的时间聚焦。从理论上推导并实验证明了与共聚焦显微镜相当的轴向分辨率。为了在不增加激光功率的情况下实现合理的帧率，我们在设置中使用了低重复率超快激光放大器。在对活上皮细胞簇中表达的荧光蛋白进行的 3D 成像中，我们展示了与荧光显微镜相当的帧率。在本报告中，我们的设计具有展示潜力，可广泛用于具有与激光扫描显微镜相当的轴向分辨率的视频帧率活组织和胚胎成像。
