Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, USA.
Phys Rev Lett. 2014 Jan 10;112(1):014302. doi: 10.1103/PhysRevLett.112.014302.
Subdiffraction optical microscopy allows the imaging of cellular and subcellular structures with a resolution finer than the diffraction limit. Here, combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for subdiffraction photoacoustic imaging of both fluorescent and nonfluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. The differential signal between the two excitations preserves the signal contribution mostly from the center of the excitation volume, and dramatically sharpens the lateral resolution. Moreover, due to the nonlinear nature of the signal, our method offers an inherent optical sectioning capability, which is lacking in conventional photoacoustic microscopy. By scanning the excitation beam, we performed three-dimensional subdiffraction imaging of varied fluorescent and nonfluorescent species. As any molecules have absorption, this technique has the potential to enable label-free subdiffraction imaging, and can be transferred to other optical imaging modalities or combined with other subdiffraction methods.
亚衍射光学显微镜允许对细胞和亚细胞结构进行成像，其分辨率优于衍射极限。在这里，我们结合基于吸收的光声效应和强度相关的光漂白效应，展示了一种简单的方法，用于对荧光和非荧光样品进行亚衍射光声成像。我们的方法基于双激发过程，其中第一激发脉冲部分地和非均匀地漂白在衍射受限激发体积中的分子，从而偏向于从第二激发脉冲撞击相同区域的信号贡献。两次激发之间的差分信号保留了主要来自激发体积中心的信号贡献，并显著提高了横向分辨率。此外，由于信号的非线性性质，我们的方法提供了固有光学切片能力，而传统的光声显微镜则缺乏这种能力。通过扫描激发光束，我们对不同的荧光和非荧光物质进行了三维亚衍射成像。由于任何分子都具有吸收，因此该技术有可能实现无标记的亚衍射成像，并且可以转移到其他光学成像模式或与其他亚衍射方法结合使用。
