Center for Biological Physics, Department of Physics, Arizona State University, Tempe, Arizona 85287, USA.
J Chem Phys. 2019 Mar 21;150(11):114108. doi: 10.1063/1.5083869.
One way to achieve spatial resolution using fluorescence imaging-and track single molecules-is to use wide-field illumination and collect measurements over multiple sensors (camera pixels). Here we propose another way that uses confocal measurements and a single sensor. Traditionally, confocal microscopy has been used to achieve high temporal resolution at the expense of spatial resolution. This is because it utilizes very few, and commonly just one, sensors to collect data. Yet confocal data encode spatial information. Here we show that non-uniformities in the shape of the confocal excitation volume can be exploited to achieve spatial resolution. To achieve this, we formulate a specialized hidden Markov model and adapt a forward filtering-backward sampling Markov chain Monte Carlo scheme to efficiently handle molecular motion within a symmetric confocal volume characteristically used in fluorescence correlation spectroscopy. Our method can be used for single confocal volume applications or incorporated into larger computational schemes for specialized, multi-confocal volume, optical setups.
实现荧光成像空间分辨率并跟踪单个分子的一种方法是使用宽场照明，并在多个传感器（相机像素）上收集测量值。在这里，我们提出了另一种使用共焦测量并使用单个传感器的方法。传统上，共焦显微镜用于以牺牲空间分辨率为代价来实现高时间分辨率。这是因为它利用很少的传感器，通常只有一个，来收集数据。然而，共焦数据编码空间信息。在这里，我们表明可以利用共焦激发体积的形状不均匀性来实现空间分辨率。为了实现这一点，我们制定了一个专门的隐马尔可夫模型，并采用前向滤波-后向采样马尔可夫链蒙特卡罗方案来有效地处理荧光相关光谱中典型使用的对称共焦体积内的分子运动。我们的方法可用于单个共焦体积应用，也可纳入更大的计算方案中，以用于专门的多共焦体积光学设置。
