Photon Event Distribution Sampling (PEDS): Image Formation & High Precision Particle Localization in Scanning Microscopy

Abstract

At the limit of resolution of light microscopy, high magnification images are conventionally formed using relatively large pixels because acquiring images with smaller pixels results in lost signal too great to be beneficial. At resolution near the diffraction limit, the measured position of a photon has an uncertainty on the same order of size as these large pixels, decreasing the accuracy with which the specimen is imaged. A novel method of forming images using a scanning microscope called photon event distribution sampling (PEDS) is described herein. Using probability density functions, the uncertainty of the measured position of a photon is accounted for in the final image, which produces images with greater accuracy and facilitates more efficient image acquisition. The benefits of PEDS are accomplished without any deleterious effects, including loss in signal, allowing even small advantages in spatial accuracy to be realized. Additionally, the small pixels associated with PEDS retain subtle spatial information that is beneficial in assessing the nature of the distribution of photons in an image. When implemented to localize sub-diffraction particles, PEDS produces results with precision as good as, and at low photon counts better than, what is currently reported in the literature, and is capable of doing so with fewer photons than has been previously reported. Furthermore, PEDS is implemented using a simple confocal laser scanning microscope, with components commonly available, and has potential to realize high precision tracking of single molecules and super-resolution imaging deep within specimens.