Photoactivated localization microscopy

In each image of the sequence, the position of a fluorophore is calculated with a precision typically greater than the diffraction limit - in the typical range of a few to tens of nm - and the resulting information of the position of the centers of all the localized molecules is used to build up the super-resolution PALM or STORM image.

The localization precision ${\displaystyle \sigma }$ can be calculated according to the formula:

${\displaystyle \sigma ={\sqrt {\left({\frac {s_{i}^{2}+{\frac {a^{2}}{12}}}{N}}\right)\cdot \left({\frac {16}{9}}+{\frac {8\pi s_{i}^{2}b^{2}}{a^{2}N^{2}}}\right)}}}$

where N is the number of collected photons, a is the pixel size of the imaging detector, ${\displaystyle b^{2}}$ is the average background signal and ${\displaystyle s_{i}}$ is the standard deviation of the point spread function.[10] The requirement of localizing at the same time multiple fluorophores simultaneously over an extended area determines the reason why these methods are wide-field, employing as a detector a CCD, EMCCD or a CMOS camera.

The requirement for an enhanced signal-to-noise ratio to maximize localization precision determines the frequent combination of this concept with widefield fluorescent microscopes allowing optical sectioning, such as total internal reflection fluorescence microscopes (TIRF) and light sheet fluorescence microscopes.

Strategies for multicolor localization microscopy. Left: spectral separation. Center: multiple activators (STORM). Right: Ratiometric imaging

The peculiar photophysical properties of the fluorophores employed in PALM/STORM super resolution imaging pose both constraints and opportunities for multicolor imaging. Three strategies have emerged so far: excitation of spectrally separated fluorophores using an emission beamsplitter,[12] using of multiple activators/reporters in STORM mode[13][14] and ratiometric imaging of spectrally close fluorophores.[15]

Although originally developed as 2D (x,y) imaging methods, PALM and STORM have quickly developed into 3D (x,y,z) capable techniques. To determine the axial position of a single fluorophore in the sample the following approaches are currently being used: modification of the point spread function to introduce z-dependent features in the 2D (x,y) image (the most common approach is to introduce astigmatism in the PSF); multiplane detection, where the axial position is determined by comparing two images of the same PSF defocused one with respect to the other; interferometric determination of the axial position of the emitter using two opposed objectives and multiple detectors;[7] use of temporal focusing to confine the excitation/activation; use of light sheet excitation/activation to confine to a few hundred nanometers thick layer arbitrarily positioned along the z-plane within the sample.

The requirement for multiple cycles of activation, excitation and de-activation/bleaching would typically imply extended periods of time to form a PALM/STORM image, and therefore operation on a fixed sample. A number of works have been published as early as 2007 [16] performing PALM/STORM on live cells. The ability to perform live super-resolution imaging using these techniques ultimately depends on the technical limitations of collecting enough photons from a single emitter in a very short time. This depends both on the photophysical limitations of the probe as well as on the sensitivity of the detector employed. Relatively slow (seconds to tens of seconds) processes such as modification in the organization of focal adhesions have been investigated by means of PALM,[17] whereas STORM has allowed imaging of faster processes such as membrane diffusion of clathrin coated pits or mitochondrial fission/fusion processes. A promising application of live cell PALM is the use of photoactivation to perform high-density single-particle tracking (sptPALM [18]), overcoming the traditional limitation of single particle tracking to work with systems displaying a very low concentration of fluorophores.

While traditional PALM and STORM measurements are used to determine the physical structure of a sample, with the intensities of fluorescent events determining the certainty of the localization, these intensities can also be used to map fluorophore interactions with nanophotonic structures. This has been performed on both metallic (plasmonic) structures, such as gold nanorods,[19][20] as well as semiconducting structures, such as silicon nanowires.[21] These approaches can either be used for fluorophores functionalized on the surface of the sample of interest (as for the plasmonic particle studies mentioned here), or randomly adsorbed onto the substrate surrounding the sample, allowing full 2D mapping of fluorophore-nanostructure interactions at all positions relative to the structure.[21]

These studies have found that, in addition to the standard uncertainty of localization due to the point spread function fitting, self-interference with light scattered by nanoparticles can lead to distortions or displacements of the imaged point spread functions,[20][21] complicating the analysis of such measurements. These may be possible to limit, however, for example by incorporating metasurface masks which control the angular distribution of light permitted into the measurement system.[22]