DHL细胞中5-ALA代谢PpIX的双光子荧光光谱

双光子激发生物组织荧光，激发光仅作用于焦点区域，对生物样品的光漂白性和光毒性都很小，因而双光子荧光显微技术已成为细胞生物学研究的一种新技术。文章采用波长为820 nm飞秒激光激发孵育有5-ALA的DHL细胞，在激光扫描显微镜的Lambda模式中获得单个DHL细胞的双光子荧光光谱，并测量DHL细胞内积聚的卟啉九(PpIX)特征荧光值。获得了浓度分别为2, 4和10 mmol·L-1的5-ALA溶液中，细胞代谢的PpIX含量随孵育时间的变化情况。DHL细胞内积聚的PpIX处于动态变化过程，并呈现出两阶段性的特点：细胞内积聚的PpIX含量随着孵育时间增长而增加，在3 h附近达到最大值，随后随着孵育时间增长反而下降。结果表明，基于激光扫描显微的双光子荧光光谱可成为DHL细胞等白血病细胞摄取5-ALA并生成PpIX的动力学研究的有效方法。

Two-Photon Excitation Fluorescence of 5-ALA Induced PpIX in DHL Cells

Two-photon fluorescence microscopy is a novel imaging technique, which is primarily sensitive to a specimen’s response coming from an in-focus plane, thus has low photo-bleaching and photo-damage to biological samples. 5-ALA induced production of PpIX in DHL cells was excited by 820 nm femtosecond laser; two-photon excitation fluorescence of single cell was obtained in Lambda mode of laser scanning confocal microscope. The specific fluorescence intensity of PpIX which accumulated in DHL cells was measured at 2，4 and 10 mmol·L-1 concentration of 5-ALA with different incubation time, which reflected the kinetics of 5-ALA accumulated in DHL cells. Accumulation of PpIX in DHL cells was a dynamic change process. Biphasic alterations of PpIX accumulation were noted: PpIX content enhanced with the increasing time and reached the maximal value around 3 h, however PpIX content decreased in the subsequent incubation time. Results indicate that two-photon fluorescence based on laser scanning microscope can be a useful technology for studying the kinetics of 5-ALA induced PpIX production in DHL cells and other leukemia cells.