Pant Shilpa, Duan Yubo, Xiong Fei, Chen Nanguang
Department of Biomedical Engineering, National University of Singapore, 117583, Singapore.
Biomed Opt Express. 2017 Nov 17;8(12):5698-5707. doi: 10.1364/BOE.8.005698. eCollection 2017 Dec 1.
Multi-dimensional fluorescence imaging of live animal models demands strong optical sectioning, high spatial resolution, fast image acquisition, and minimal photobleaching. While conventional laser scanning microscopes are capable of deep penetration and sub-cellular resolution, they are generally too slow and causing excessive photobleaching for volumetric or time-lapse imaging. We demonstrate the performance of an augmented line-scan focal modulation microscope (aLSFMM), a high-speed imaging platform that affords above video-rate imaging speed by the use of line scanning. Exceptional background rejection is accomplished by combining a confocal slit with focal modulation. The image quality is further improved by merging the information from simultaneously acquired focal modulation and confocal images. Such a hybrid imaging scheme makes it possible to use very low power excitation light in high-speed imaging, and therefore leads to reduced photobleaching that is desirable for three-dimensional (3D) and four-dimensional (4D) in vivo image acquisition.
对活体动物模型进行多维荧光成像需要强大的光学切片能力、高空间分辨率、快速图像采集以及最小化的光漂白。虽然传统的激光扫描显微镜能够实现深度穿透和亚细胞分辨率，但它们通常速度太慢，在进行体积成像或延时成像时会导致过度的光漂白。我们展示了增强型线扫描焦点调制显微镜（aLSFMM）的性能，这是一个高速成像平台，通过使用线扫描实现高于视频速率的成像速度。通过将共焦狭缝与焦点调制相结合，实现了出色的背景抑制。通过合并同时采集的焦点调制图像和共焦图像的信息，进一步提高了图像质量。这种混合成像方案使得在高速成像中可以使用非常低功率的激发光，因此减少了光漂白，这对于三维（3D）和四维（4D）体内图像采集是非常理想的。
