East Carolina University, Department of Physics, Greenville, North Carolina 27858-4353, USA.
J Biomed Opt. 2010 Sep-Oct;15(5):056010. doi: 10.1117/1.3494567.
Raman tweezers and quantitative differential interference contrast (DIC) microscopy are combined to monitor the dynamic germination of individual bacterial spores of Bacillus species, as well as the heterogeneity in this process. The DIC bias phase is set properly such that the brightness of DIC images of individual spores is proportional to the dipicolinic acid (DPA) level of the spores, and an algorithm is developed to retrieve the phase image of an individual spore from its DIC image. We find that during germination, the rapid drop in both the intensity of the original DIC image and the intensity of the reconstructed phase image precisely corresponds to the release of all DPA from that spore. The summed pixel intensity of the DIC image of individual spores adhered on a microscope coverslip is not sensitive to the drift of the slide in both horizontal and vertical directions, which facilitates observation of the germination of thousands of individual spores for long periods of time. A motorized stage and synchronized image acquisition system is further developed to effectively expand the field of view of the DIC imaging. This quantitative DIC technique is used to track the germination of hundreds or thousands of individual spores simultaneously.
拉曼镊子和定量微分干涉对比（DIC）显微镜相结合，用于监测单个芽孢杆菌属细菌孢子的动态萌发过程，以及该过程中的异质性。DIC 偏置相设置得恰到好处，使得单个孢子的 DIC 图像的亮度与孢子的二吡啶羧酸（DPA）水平成正比，并且开发了一种算法，可从 DIC 图像中提取单个孢子的相位图像。我们发现，在萌发过程中，原始 DIC 图像的强度和重建相位图像的强度迅速下降，这与该孢子中所有 DPA 的释放精确对应。附着在显微镜盖玻片上的单个孢子的 DIC 图像的像素强度总和对载物台在水平和垂直方向上的漂移不敏感，这便于长时间观察数千个单个孢子的萌发。进一步开发了一个带电机的载物台和同步图像采集系统，以有效地扩展 DIC 成像的视场。这种定量 DIC 技术用于同时跟踪数百个或数千个单个孢子的萌发。
