A microfluidic platform for correlative live-cell and super-resolution microscopy

Abstract

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of superresolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and superresolution images. Funding: This project was financially supported by Fundació CELLEX Barcelona, by the Systems Microscopy Network of Excellence consortium (call identifier number FP-7- HEALTH.2010.2.1.2.2) and by ERC-Starting grant (grant agreement number 337191-MOTORS) and the Plan Nacional Grant from the Spanish Ministry (Ministerio de Economia y Competitividad FIS2012-37753) to M.L. J.T was supported by the Whitaker International Fellows and Scholars Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.