Hillsley Alexander, Stein Johannes, Tillberg Paul W, Stern David L, Funke Jan
HHMI Janelia Research Campus, Ashburn, Virginia 20147, United States.
Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts 02215, United States.
Nano Lett. 2025 Apr 16;25(15):6059-6068. doi: 10.1021/acs.nanolett.4c06304. Epub 2025 Apr 4.
We address the problem of inferring the number of independently blinking fluorescent light emitters, when only their combined intensity contributions can be observed. This problem occurs regularly in light microscopy of objects smaller than the diffraction limit, where one wishes to count the number of fluorescently labeled subunits. Our proposed solution directly models the photophysics of the system, as well as the blinking kinetics of the fluorescent emitters as a fully differentiable hidden Markov model, estimating a posterior distribution of the total number of emitters. We show that our model is more accurate and increases the range of countable subunits by a factor of 2 compared to current state-of-the-art methods. Furthermore, we demonstrate that our model can be used to investigate the effect of blinking kinetics on counting ability and therefore can inform optimal experimental conditions.
当只能观察到荧光发射体的组合强度贡献时，我们着手解决推断独立闪烁荧光发射体数量的问题。在小于衍射极限的物体的光学显微镜观察中，这个问题经常出现，在这种情况下，人们希望对荧光标记的亚基数量进行计数。我们提出的解决方案直接对系统的光物理过程以及荧光发射体的闪烁动力学进行建模，将其作为一个完全可微的隐马尔可夫模型，估计发射体总数的后验分布。我们表明，与当前的最先进方法相比，我们的模型更准确，并且可计数亚基的范围增加了两倍。此外，我们证明我们的模型可用于研究闪烁动力学对计数能力的影响，因此可为优化实验条件提供依据。
