Photoactivated Localization Microscopy, or PALM imaging, is a super-resolution technique that relies on the random activation of fluorophores to spatially resolve molecular details (Betzig et al. 2006). In brief, upon activation with the adequate laser, a sparse number of fluorophores emit for a short period before bleaching. Because fluorophores are only activated at the same time in small numbers until all have emitted, it is possible to localize and track single molecules over time.
Cells release different types of small-sized membrane vesicles (from 30nm to 1000nm) as a way of communicating with other cells and remove unwanted cell contents; these vesicles are collectively known as extracellular vesicles (EVs). Here we share how we have optimized the imaging of purified EVs to study their size, content and behavior using super-resolution microscopy.
We have all been there, after spending a day or two fixing and staining your samples, the images don't look as good as expected, the signal is not optimal, and no obvious conclusion can be drawn from the data. We have put together some tips to help you optimize your immunofluorescence staining protocol and increase the signal-to-noise ratio in your samples. If you are unsure about how the technique works, we recommend you read our immunofluorescence guide first.
When preparing a sample for STORM microscopy, one of the most important factors to consider is the choice of fluorophore. The ability to accurately localize single molecules is dependent on the use of fluorescent molecules that stochastically switch between a fluorescent “on” state and a dark “off” state – a photophysical behavior that is often referred to as blinking.