A Platform for 3D Tracking of Single Molecules in Living Cells

In the last years, a wide range of microscopy techniques able to localize fluorescent dyes with a few nanometers accuracy have been developed, opening new avenues for super-resolution techniques such as STORM and PALM. Despite their power in pushing for higher spatial resolution all these techniques show some limitations when applied to the study of proteins inside a living cell. Some are limited by the small penetration depth of the fluorescence excitation (TIRF configuration), some other by the low temporal resolution, while looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). We developed an apparatus which combines different microscopy techniques in order to satisfy all the technical requirements for a nanometer accuracy 3D tracking of fluorescent single molecules inside living cells. To account for the optical sectioning of thick samples we built up a HILO (Highly Inclined and Laminated Optical sheet) microscopy system through which we can excite the sample in a widefield (WF) configuration by a thin sheet of light that is able to follow the molecule up and down along the z axis spanning the entire thickness of the cell with a SNR much higher than traditional WF microscopy. Since protein dynamics inside a cell involve all three dimensions we included a method to measure the x, y, and z coordinates with nanometre accuracy, exploiting the properties of the point-spread-function of out-of-focus quantum dots bound to the protein of interest. Finally, a feedback system stabilizes the microscope from thermal drifts, assuring accurate localization during the entire duration of the experiment. (Fundings from Italian Ministry for Education, University and Research in the framework of the Flagship-Project NANOMAX).