Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology (KTH), Albanova University Center, 106 91, Stockholm, Sweden.
Methods Appl Fluoresc. 2021 Jan 11;9(1). doi: 10.1088/2050-6120/abcbed.
In stimulated emission depletion (STED) imaging, the excitation and depletion laser beams extend well beyond the focal plane in the imaged sample. We investigated how photobleaching resulting from this irradiation can affect STED images, by acquiring 3D images of fluorescent polystyrene beads using a 2D STED microscope, and applying different Z pixel sizes, scanning speeds, resulting in different laser light doses. While higher STED beam irradiances can increase the spatial resolution, they can also significantly increase photobleaching and thereby reduce signal-to-background levels. In 2D STED imaging, based on a single scan within the focal plane, scan parameters can often be selected to avoid photobleaching effects. Upon 3D optical sectioning experiments however, using the same scan parameters, additional cumulative effects of photobleaching may appear, due to the extension of the excitation and depletion laser beams beyond the focal planes being scanned. Apart from a reduction in signal-to-background levels, such photobleaching can lead to an apparent shift of the axial localization of the objects in the images, but also to an increased resolution in the axial dimension. These findings, confirmed by simulations based on a simplified model for photobleaching, suggests some caution in volumetric STED imaging experiments, but also a possibility for enhanced axial resolution in such experiments.
在受激发射损耗（STED）成像中，激发和耗尽激光束在被成像样品的焦平面之外延伸得很好。我们通过使用二维 STED 显微镜获取荧光聚苯乙烯珠的 3D 图像，并应用不同的 Z 像素大小和扫描速度，从而导致不同的激光光剂量，研究了这种辐照导致的光漂白如何影响 STED 图像。虽然更高的 STED 光束辐照度可以提高空间分辨率，但它们也会显著增加光漂白，从而降低信号与背景的水平。在二维 STED 成像中，基于焦平面内的单次扫描，可以选择扫描参数以避免光漂白效应。然而，在进行三维光学切片实验时，使用相同的扫描参数，由于激发和耗尽激光束在被扫描的焦平面之外延伸，可能会出现额外的累积光漂白效应。除了信号与背景水平降低之外，这种光漂白会导致图像中物体的轴向定位出现明显偏移，同时在轴向维度上也会提高分辨率。这些发现通过基于光漂白简化模型的模拟得到了证实，这表明在体积 STED 成像实验中需要谨慎，但也为这种实验中提高轴向分辨率提供了可能性。
