Department of Optics and Photonics, National Central University, No. 300, Zhongda Rd., Zhongli Dist., Taoyuan City, 32001, Taiwan.
College of Photonics, National Yang Ming Chiao Tung University, No.301, Sec.2, Gaofa 3rd Rd., Guiren Dist., Tainan City, 71150, Taiwan.
Angew Chem Int Ed Engl. 2024 Jul 1;63(27):e202404942. doi: 10.1002/anie.202404942. Epub 2024 May 21.
Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.
基于时间聚焦多光子激发（TFMPE）和单波长激发的单分子定位显微镜（SMLM）用于可视化厚标本中自发闪烁荧光标记亚细胞结构的三维（3D）分布，具有纳米级空间分辨率。为了消除离焦区域中未定位分子的光漂白效应，以提高 3D SMLM 成像中光子预算的利用率，具有单波长 TFMPE 的 SMLM 实现了自发闪烁荧光团的宽场和轴向限制的双光子激发（TPE）。然后通过在可调谐激发波长下进行 TFMPE 成像来进行闪烁荧光团的 TPE 光谱测量，从而获得用于在 SMLM 期间从单个闪烁事件中增加检测到的光子数的最佳 TPE 波长。随后，记录闪烁荧光团的 TPE 荧光，以获得癌细胞中微管的二维 TFMPE-SMLM 图像，其定位精度为 18±6nm，整体成像分辨率约为 51nm，这是基于奈奎斯特分辨率和定位精度的贡献来估计的。结合像散成像，该系统能够对具有阿尔茨海默病病理特征的 5XFAD 转基因小鼠脑组织切片进行 3D TFMPE-SMLM 成像，揭示神经毒性淀粉样β肽沉积物的分布。
