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Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

机译：增强型模拟器软件，用于图像验证和解释，用于多模式定位超分辨率荧光显微镜

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摘要

Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.)of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour,polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.
机译：诸如单分子定位之类的光学超分辨率技术已成为光学显微镜领域最动态发展的领域之一。这些技术通常提供具有亚衍射空间分辨率的固定细胞或组织的图像，甚至可以在适当的情况下用于活细胞成像。定位技术基于点扩散函数（PSF）与随机激发的相同荧光分子的测量图像的精确拟合。这些技术需要控制开，关和漂白状态之间的速率，将活性荧光分子的数量保持在最佳值，因此可以在空间和时间上分别检测其衍射受限的图像。由于参数众多（有时未知），因此只能随机处理成像系统。例如，染料分子的旋转掩盖了偏振相关的PSF形状，并且仅观察到平均分布-通常由高斯函数估计。开发了TestSTORM软件以生成用于传统定位显微镜的图像堆栈，其中定位意味着精确确定分子的空间位置。但是，可以将发出的光子的其他光学特性（偏振，光谱等）用于进一步监视本地环境的化学和物理特性（粘度，pH等）。图像堆栈生成程序已通过多项新功能升级，例如：多色，偏振相关的PSF，内置3D可视化，结构化背景。这些功能使该程序成为优化成像和样品制备条件的理想工具。

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