Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-stokes Raman scattering microscopy
Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-stokes Raman scattering microscopy
复制标题
DOI：
10.1529/biophysj.105.074534
复制
发表时间：
2006-07-15
影响因子：
3.4
通讯作者：
Xie, X. Sunney
中科院分区：
文献类型：
--
作者：
Nan, Xiaolin;Potma, Eric O.;Xie, X. Sunney
利用相干反斯托克斯拉曼散射(CARS)显微镜实现了对未染色活细胞内细胞器运输的非微扰监测。为了避免激光辐射对细胞器运输可能产生的干扰，我们首先考察了不同的光照条件。利用基于细胞形态变化的新的光损伤判据，确定了相应波长的脉冲能量阈值和平均功率阈值。在比阈值温和得多的激发条件下，我们能够在不干扰细胞的情况下，用CARS显微镜实时监测类固醇生成小鼠肾上腺皮质(Y-1)细胞中脂滴(LD)细胞器的运动。粒子跟踪分析揭示了LDS沿微管的次扩散和主动传输。有趣的是，LD的主动转运只存在于培养的Y-1细胞中，这种形态变化与类固醇的生成有关，这表明后者可能参与了LD的主动转运。用CARS和双光子荧光显微镜对LDS和线粒体的同时成像清楚地表明，高LD运动性可以促进两个细胞器之间的相互作用。这些观察表明，CARS显微镜是研究活细胞动态过程的一种强大的非侵入性成像工具。
Nonperturbative monitoring of intracellular organelle transport in unstained living cells was achieved with coherent anti-Stokes Raman scattering (CARS) microscopy. To avoid possible interference with the organelle transport introduced by laser radiation, we first examined different illumination conditions. Using a new photodamage criterion based on morphological changes of the cells, we determined the threshold values of both pulse energy and average power at relevant wavelengths. Under excitation conditions much milder than the threshold levels, we were able to monitor the motions of lipid droplet (LD) organelles in steroidogenic mouse adrenal cortical (Y-1) cells with CARS microscopy in real time without perturbations to the cells. Particle tracking analyses revealed subdiffusion as well as active transport of LDs along microtubules. Interestingly, LD active transport is only present in Y-1 cells that rounded up in culture, a morphological change associated with steroidogenesis, suggesting possible involvements of LD active transport in the latter. Simultaneous imaging of LDs and mitochondria with CARS and two-photon fluorescence microscopy clearly showed that interactions between the two organelles could be facilitated by high LD motility. These observations demonstrate CARS microscopy as a powerful noninvasive imaging tool for studying dynamic processes in living cells.