亚硒酸盐对丝状真菌脂滴的影响,采用先进的非线性显微镜技术监测“体内”无标签

Tanja Pajić, N. Todorović, Dunja Stefanovic, M. Rabasović, A. Krmpot, M. Živić
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摘要

采用三次谐波(THG)显微镜测量脂滴(LD)并定量分析亚硒酸盐对LD的影响。非线性激光扫描显微镜(NLSM)采用超短激光脉冲进行成像。THG显微镜是NLSM的方式。强THG信号只能从折射率不均匀的区域观测到。生物样品中的这些区域是脂水界面,到目前为止,细胞中最亮的特征是ld。因此,THG显微镜是对活的未固定细胞的ld进行成像的合适方法,不需要额外的标记。在体内观察了1 mM亚硒酸盐对丝状真菌细胞从孢子到指数末持续时间(27 ~ 30 h)的生物学效应。我们测量了一种模式真菌黑藻的脂滴密度和大小分布。使用内部构建的显微镜框架,辅以Yb KGW激光器(1040 nm, 200 fs脉冲),通过Hoya玻璃紫外滤光片(峰值在340 nm)在透射臂上通过PMT进行检测。从对照菌丝和Se+4处理菌丝的THG图像中测定了LD的大小和数量,结果表明Se+4处理菌丝的LD密度比对照提高了60%以上。Se+4处理对平均LD尺寸分布变化不大。结果表明,1 mM亚硒酸盐处理可能引起丝状真菌的细胞应激反应。
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THE EFFECTS OF SELENITE ON FILAMENTOUS FUNGI LIPID DROPLETS MONITORED „IN VIVO“ LABEL FREE USING ADVANCED NONLINEAR MICROSCOPY TECHNIQUE 2021ICCBIKG (2021)
Third Harmonic Generation (THG) microscopy was employed as a method of choice for lipid droplet (LD) measurements and quantification of the effect of selenite on LDs. Nonlinear laser scanning microscopy (NLSM) employs ultra-short laser pulses for imaging. THG microscopy is the modality of NLSM. Strong THG signals can only be observed from regions with non- uniformities with respect to their refractive index. Such regions in biological samples are lipid-water interfaces, and by far the brightest features in cells are LDs. For that reason, THG microscopy is the appropriate method for imaging of LDs from live unfixed cells, without the need for additional labeling. The biological effects of spore- to- end- of- exponential- phase duration (27 – 30 h) of exposure to 1 mM selenite were monitored in vivo on the cells of filamentous fungi in liquid culture. We measured the lipid droplet density and size distribution in a model fungi Phycomyces blakesleeanus. The in-house built microscope frame complemented with Yb KGW laser (1040 nm, 200 fs pulses) was used, while detection was enabled in the transmission arm by PMT through the Hoya glass UV filter (peak at 340 nm). From THG images of control and Se+4–treated hyphae, LD size and number were measured, showing that LD density was increased by more than 60% in Se+4–treated hyphae, compared to control. The average LD size distribution seemed slightly changed by Se+4 -treatment. The obtained results suggest that 1 mM selenite treatment probably induces cellular stress response in filamentous fungi.
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