Dr. Alex Small

Cal Poly Pomona

“Optics Beyond the Diffraction Limit: How to see tiny things in cells”

Recent experimental work has shown that it is possible to beat the diffraction limit in fluorescence microscopy, and obtain images of cells with nanometer resolution. These techniques usually involving randomly switching molecules on and off, so that only a fraction are in an “activated” (i.e. light-emitting) state at any given time. Although each molecule still produces a diffraction-limited image, its image does not overlap any other molecules, so the molecular position can be inferred with noise-limited accuracy. As the density of activated molecules increases, a faster experiment becomes possible, but the possibility of errors increases. We have formulated a statistical model of this process, and found that there is a maximum possible activation probability, and hence a maximum activation rate. Interestingly, this maximum activation probability is related to the performance of the algorithms used to analyze the data. We have also proven the existence of an optimal image acquisition scheme in the case of bleaching, with an error rate that can be predicted from the imaging speed and the algorithm performance. Finally, we have begun bench-marking common image analysis algorithms, and are using insights from our model to develop image analysis algorithms with comparable performance and better speed.