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 super-resolution 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 super-resolution images.
Highlights
The crowded, intracellular environment is highly dynamic
There are a wide variety of microfluidic chip designs that have already been demonstrated for optical microscopy and/or immunostaining of bacteria and mammalian cells [12, 13, 23], many of which utilize a multi-layered design with on-chip valves [12, 24]
Direct implementation of established protocols in these devices for cell seeding, immunostaining, and stochastic optical reconstruction microscopy (STORM) imaging buffers resulted in very low cell concentrations, poor labeling densities, and complete absence of the photoswitching of fluorophores that is necessary for STORM
Summary
The crowded, intracellular environment is highly dynamic. Visualizing a specific subcellular process requires high spatial and high temporal resolution in combination with a molecular marker that highlights the structure of interest. Most cellular dynamics occur at much faster timescales (millisecond) and achieving both nanoscale spatial and millisecond temporal resolution is still very challenging using super-resolution microscopy methods [10] To circumvent this problem, recently, we developed an all-optical correlative imaging approach that combines time-lapse live-cell microscopy with STORM to achieve both high temporal resolution and high spatial resolution, respectively [11]. We developed an all-optical correlative imaging approach that combines time-lapse live-cell microscopy with STORM to achieve both high temporal resolution and high spatial resolution, respectively [11] This approach has enabled us to study cargo transport dynamics at the level of single microtubules, revealing how microtubule intersections impact motorprotein mediated transport. The technique requires precise delivery and removal of fluid from a sample that remains on the microscope stage for the duration of the experiment, a procedure that, when performed manually, is imprecise, labor-intensive, and time consuming
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
