An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.
An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.
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DOI：
10.3389/fpls.2014.00140
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发表时间：
2014
影响因子：
5.6
通讯作者：
Love J
中科院分区：
文献类型：
--
作者：
Littlejohn GR;Mansfield JC;Christmas JT;Witterick E;Fricker MD;Grant MR;Smirnoff N;Everson RM;Moger J;Love J
植物叶片是光学复杂的，这使得它们难以通过光学显微镜成像。因此，需要仔细的样品制备，使研究人员能够最大限度地利用荧光蛋白标记，细胞染料和显微镜技术和技术创新的进步所获得的信息。我们以前已经表明，安装在无毒，非荧光全氟化碳（PFC），全氟萘烷（PFD）的叶子增强了光学特性的叶子，对生理的影响最小。在这里，我们评估使用的PFC，PFD，和全氟perhydrophenanthrene（PP11）在体内植物叶片成像使用四种先进的显微镜模式：激光扫描共聚焦显微镜（LSCM），双光子荧光显微镜，二次谐波发生显微镜，和受激拉曼散射（SRS）显微镜。对于测试的每种成像模式，与将样品安装在水中相比，当将叶片安装在PFD或PP11中时，我们观察到改善的信号。使用基于自相关的图像分析技术，定量评估LSCM图像随深度的恶化，我们表明，PP11优于PFD作为安装介质，使采集更清晰的图像更深入到组织。此外，我们表明，SRS显微镜可用于直接在叶肉中的PFC图像，从而很容易划定的“负空间”内的叶子，这可能有重要的影响，叶片发育的研究。直接比较共振和非共振SRS显微照片表明，PFC在活体植物中不形成胞内聚集体。我们的结论是，PFCs作为安装介质的应用程序大大提高了先进的显微镜图像质量的活叶肉和叶维管束细胞。
Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the “negative space” within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.
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影响因子：
7.4
作者：
SCHONHERR, J;BUKOVAC, MJ
通讯作者：
BUKOVAC, MJ
影响因子：
9.4
作者：
Littlejohn, George R.;Gouveia, Joao D.;Love, John
通讯作者：
Love, John
影响因子：
3.3
作者：
Nandakumar, P.;Kovalev, A.;Volkmer, A.
通讯作者：
Volkmer, A.
影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
影响因子：
3.6
作者：
Brown, RM;Millard, AC;Campagnola, PJ
通讯作者：
Campagnola, PJ