QuickPALM - 3D real-time photoactivation nanoscopy image processing in ImageJ

Authors: Ricardo Henriques, Mickael Lelek, Eugenio F Fornasiero, Flavia Valtorta, Christophe Zimmer, Musa M Mhlanga
Technologies: QuickPALM
Paper published in Nature methods, January 2010
Publisher: Nature Publishing Group US New York

QuickPALM - 3D real-time photoactivation nanoscopy image processing in ImageJ
DOI: 10.1038/nmeth0510-339

The manuscript describes the development of QuickPALM, a real-time photoactivation nanoscopy image processing algorithm for three-dimensional (3D) super-resolution imaging using the widely used ImageJ software. The algorithm combines the classical Högbom 'CLEAN' method for spot finding with a modified center of mass algorithm for computing spot position and shape parameters. It also includes features for 3D reconstruction, drift correction, and real-time acquisition control. The method achieves a typical processing time of 30-50 ms per image with a weak dependence on the number of spots per image, making it of the same order as common electron-multiplying charge-coupled device (EMCCD) maximum frame rates. The software package should facilitate the conversion of many laser-excitation widefield or TIRF microscopes into powerful super-resolution microscopes. The authors also discuss the potential of QuickPALM when combined with the µManager open source package for microscope hardware control and acquisition and a custom laser control system. The method was tested on microtubules in mouse neurons labeled with a primary antibody to neural β-III-tubulin and a secondary Cy5-conjugated antibody, achieving a resolution of 40 nm in x and y dimensions and 50 nm in the z dimension over a 1 µm depth.