长时程双光子成像技术

双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。

Long-time two-photon imaging technology

Two-photon imaging technology is widely used for dynamic 3D imaging of live cells due to its superior properties. However, severe photobleaching of fluorescence molecular on focal plane caused by short light pulse with extreme high optical power greatly affects long-term two-photon imaging. This paper proposes a method called Optimized Lighting -Two Photon(OL-TP)for the problem of two-photon fluorescence bleaching. With this method we obtain the high and low thresholds of the two-photon image through pre-scanning, optimize the duration of light in different areas of the image with preset high and low thresholds, and reconstruct the OL-TP image by using the fluorescence information and the illumination time information recorded during the scanning.Therefore, both the signal-to-noise ratio and fluorescence bleaching are guaranteed. The reconstructed OL-TP image is almost the same as the conventional two-photon image, though the signal-to-noise ratio is slightly reduced, the image is not distorted. For the 110 nm fluorescent ball samples, 30 normal two-photon images and optimized light two-photon images were successively taken. By the 30th image, the OL-TP imaging technology reduces 28.86% of photobleaching compared with standard two-photon images. All in all, OL-TP greatly reduces the photobleaching of two-photon imaging by optimizing the illumination time, enabling the two-photon fluorescence microscope to better observe biospecimen for long periods of time.