Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms
Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms
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DOI：
10.1128/aem.68.2.901-909.2002
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发表时间：
2002-02-01
影响因子：
4.4
通讯作者：
Lawrence, JR
中科院分区：
文献类型：
--
作者：
Neu, TR;Kuhlicke, U;Lawrence, JR
在生物膜和其他研究中使用两质子激光扫描显微镜(2P-LSM)的一个主要限制是缺乏对潜在荧光染料的激发-发射响应的透彻了解。为了使用2P-LSM，必须评估针对一系列生物膜成分的各种荧光染料和探针的实用性。这项研究中测试的荧光染料包括经典的核酸特异染色，如丫啶橙(AO)和4‘，6’-二氨基-2-苯基吲哚(DAPI)，以及最近发展起来的染色。此外，对生物膜外聚合物(EPS基质成分)进行了特异性染色。用钛/蓝宝石激光在760~900 nm波长范围内以10 nm步长进行双光子激发。研究发现，光养生物(蓝藻和绿藻)的自发荧光在整个激发范围内都产生了强烈的信号。此外，产甲烷细菌的辅酶F-420相关的自体荧光可以用来获得密集聚集体的图像(激发波长，780 nm)。核酸特异的荧光染料的发射信号强度不同。例如，对于激发波长从780到900 nm的AO，强度是相似的，但对于较窄的范围，DAPI的强度更高，为780到810 nm。在选择性激发、褪色、多重染色和单光子-双光子联合研究中，最近发展起来的核酸特异性荧光染料无论是用于活的还是固定的样品都被证明是更适合的。针对蛋白质和糖偶联物的探针允许对聚合物生物膜成分进行双光子成像。观察到硫系荧光白M2R(780~800 nm)和SyproOrange(880~900 nm)的选择性激发发射。此外，还检测了荧光标记的刀豆蛋白A凝集素，并在780到800 nm的波长范围内提供了可接受的双光子发射信号。最后，CellTracker，一种适用于微生物真核细胞的长期标记的荧光色素，被发现在770到810 nm的波长处有很强的发射。如果荧光染料具有相同的双光子激发截面，则它们适合于多重染色和多通道记录。一般来说，如果使用合适的激发波长和荧光染料，双光子激光显微镜可以获得比传统的单光子激光显微镜更高分辨率的厚生物膜样品的图像。由于2P-LSM在生物膜等光散射类组织材料中具有更高的分辨率和极局域激发，是传统单光子激发共聚焦激光扫描显微镜的一种有价值的补充。但要充分发挥非线性激发作用在界面微生物生态学研究中的作用，还需要进一步的方法发展和基础研究。
A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4',6'-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F-420-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.