Fiolka Reto
Opt Express. 2016 Dec 26;24(26):29556-29567. doi: 10.1364/OE.24.029556.
In Total Internal Reflection Fluorescence (TIRF) microscopy, the sample is illuminated with an evanescent field that yields a thin optical section. However, its widefield detection has no rejection mechanism against out-of-focus blur from scattered light that can compromise TIRF images. Here I demonstrate that via structured illumination, out-of-focus blur can be effectively suppressed in TIRF microscopy, yielding strikingly clearer images. The same mechanism can also be applied to oblique illumination schemes that extend the reach of TIRF microscopy beyond the basal surface of the cell. The two imaging modes are used to image a biosensor, clathrin coated vesicles and the actin cytoskeleton in different cell types with improved contrast.
在全内反射荧光（TIRF）显微镜中，样品由产生薄光学切片的倏逝场照明。然而，其宽场检测没有针对来自散射光的离焦模糊的抑制机制，这可能会损害TIRF图像。在此我证明，通过结构照明，在TIRF显微镜中可以有效抑制离焦模糊，从而产生明显更清晰的图像。相同的机制也可应用于倾斜照明方案，该方案将TIRF显微镜的检测范围扩展到细胞基底面之外。这两种成像模式用于对不同细胞类型中的生物传感器、网格蛋白包被小泡和肌动蛋白细胞骨架进行成像，对比度得到了改善。
