Two-Color Picosecond Time-resolved Infrared Super-resolution Microscopy and its Application to Cell

基于瞬时荧光检测红外光谱的红外显微镜能够突破衍射极限。简而言之，可调谐的红外光与波长固定为略长于可见光吸收带的可见光共线引入。如果红外光的频率与振动能级不共振，则不会出现荧光，因为可见光与吸收带不匹配。当红外频率与振动能级共振时，由红外激发产生的振动激发分子可以吸收可见光，并产生瞬时荧光。在这里，红外激发的瞬时荧光只出现在红外和可见光重叠的空间区域。该重叠区域可以小于红外光的衍射限，并且其尺寸可以提高到可见光的衍射限。在本工作中，我们成功地观察到了罗丹明6G/氯仿-D1溶液中小于衍射限的瞬时荧光图像。我们还报道了红外超分辨显微镜在罗丹明-6G掺杂荧光小球中的应用。观察到了直径为15μ的微珠的瞬时荧光图像，其红外图像的空间分辨率为6.8μm半高宽。由于红外光的理论衍射极限约为16.6TFDm半高宽，可见μ-IR图像的空间分辨率小于红外衍射极限，红外显微镜达到了超分辨率。此外，空间分辨率仅由可见光决定，而不是由红外光决定。通过使用这种红外超分辨显微镜，我们可视化了拟南芥根细胞的结构和振动弛豫动力学。

The IR microscopy based on the transient fluorescence detected IR spectroscopy is capable of breaking the diffraction limit. Briefly, a tunable IR light is co-linearly introduced with a visible light of which the wavelength is fixed to slightly longer than the visible absorption band. If the frequency of the IR light does not resonant to the vibrational level, no fluorescence will appear because the visible light does not match the absorption band. When the IR frequency is resonant to the vibrational level, the vibrationally excited molecule generated by the IR excitation can absorb the visible light, and gives the transient fluorescence. Here, the transient fluorescence due to IR excitation appears only the spatial region where both IR and visible lights are overlapped. This overlapped region can be smaller than the diffraction limit of IR light and its size is improved to the diffraction limit of visible light.In this work, we succeeded in observation of the transient fluorescence image that is smaller than the diffraction limit in rhodamine 6G/chloroform-d1 solution. We also reported the application of an IR super-resolution microscopy to a rhodamine-6G doped fluorescence bead as a microscopic target. The transient fluorescence image of a 15-μm-diameter bead is clearly observed, and the observed spatial resolution of TFD-IR image is 6.8 μm FWHM. Since the theoretical diffraction limit for the IR light is about 16.6 μm FWHM, it is clear that the spatial resolution of TFD-IR image is smaller than the IR diffraction limit, and the super-resolution is achieved for IR microscopy. In addition, the spatial resolution is determined only by visible light, not IR light. By using this infrared super-resolution microscopy, we visualized the structure and vibrational relaxation dynamics in Arabidopsis root cells.