Keck Advanced Microscopy Laboratory and Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA.
J Microsc. 2010 Feb;237(2):136-47. doi: 10.1111/j.1365-2818.2009.03315.x.
Live imaging in cell biology requires three-dimensional data acquisition with the best resolution and signal-to-noise ratio possible. Depth aberrations are a major source of image degradation in three-dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide-field fluorescence microscope that incorporates a large-throw deformable mirror to simultaneously focus and correct for depth aberration in three-dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2-fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.
活细胞生物学成像需要以最佳分辨率和信噪比进行三维数据采集。深度像差是三维显微镜图像退化的主要原因，会导致样品深处的分辨率和强度显著下降。这些像差是由于样品折射率与浸液折射率不匹配造成的。我们构建了一台宽场荧光显微镜，它包含一个大行程变形镜，可在三维成像中同时聚焦并校正深度像差。我们用油浸透镜对水中和甘油中的荧光珠进行成像，展示了校正后的点扩散函数和信号强度提高了 2 倍。我们将这台新显微镜应用于生物样品成像，结果显示出更清晰的图像和改进的反卷积效果。
