Photon event distribution sampling: an image formation technique for scanning microscopes that permits tracking of sub‐diffraction particles with high spatial and temporal resolutions

• Published in: Journal of microscopy (Oxford) Vol. 241; no. 1; pp. 54 - 68
• Main Authors: LARKIN, J.D., PUBLICOVER, N.G., SUTKO, J.L.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-01-2011
• Subjects: Image formation; non‐pixel based sampling; object recognition; PEDS; quantitative image analysis; scanning microscopy
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1111/j.1365-2818.2010.03406.x

Summary
In photon event distribution sampling, an image formation technique for scanning microscopes, the maximum likelihood position of origin of each detected photon is acquired as a data set rather than binning photons in pixels. Subsequently, an intensity‐related probability density function describing the uncertainty associated with the photon position measurement is applied to each position and individual photon intensity distributions are summed to form an image. Compared to pixel‐based images, photon event distribution sampling images exhibit increased signal‐to‐noise and comparable spatial resolution. Photon event distribution sampling is superior to pixel‐based image formation in recognizing the presence of structured (non‐random) photon distributions at low photon counts and permits use of non‐raster scanning patterns. A photon event distribution sampling based method for localizing single particles derived from a multi‐variate normal distribution is more precise than statistical (Gaussian) fitting to pixel‐based images. Using the multi‐variate normal distribution method, non‐raster scanning and a typical confocal microscope, localizations with 8 nm precision were achieved at 10 ms sampling rates with acquisition of ∼200 photons per frame. Single nanometre precision was obtained with a greater number of photons per frame. In summary, photon event distribution sampling provides an efficient way to form images when low numbers of photons are involved and permits particle tracking with confocal point‐scanning microscopes with nanometre precision deep within specimens.