Field-dependent photophysics - video

Below you can see a video of a raw STORM movie that I acquired in the lab a few months ago on a standard, inverted laser fluorescence microscope. The sample consists of AlexaFluor 647-labeled tubulin in Cos7 cells at a frame rate of 100 fps. (I have slowed things down a bit to more easily see what's going on.) Go ahead and watch the video now, I'll discuss a little bit about what you're seeing aftwards.

In the beginning of the movie you can see the moment when the laser beam first hits the sample. Since the illumination geometry is just widefield epi-illumination, the laser beam has a Gaussian-like irradiance profile. (As explained in the link, irradiance is the amount of energy the laser beam carries per differential unit area. It is often referred to as intensity, though intensity actually refers to a different quantity in the field of radiometry.) This means that the fluorophores in the middle of the beam are exposed to a higher irradiance than those at the edges.

The effect of the laser beam profile for STORM is quite clear when watching the video. The photoswitching rates of the fluorophores to the long-lived dark state are much higher in the center of the beam than at the edges. This leads to a field-dependent density of molecules in the fluorescence emitting state, which will ultimately affect one's ability to precisely and accurately localize the single molecule emissions. If you look closely, you can also see that the brightness of the fluorophores is also higher near the beam center, meaning that these molecules will be localized more precisely because we recorded more photons from them.

All of these features mean that one must take care when performing quantitative analyses on STORM datasets, because the density, precision, and accuracy of the single molecule position estimates will vary across the field of view.