When applied to fixed samples, Single-Molecule Localization Microscopy (SMLM) gives rise to high-resolution images of biological structures. Reconstructing images of immobile structures is, however, not the only application of this powerful technology, and gaining access to the movement of single molecules can be equally informative. Single-particle tracking (SPT) has always raised interest in the microscopy community, and even more so since the discovery of single-molecule techniques.
Single Particle Tracking
What is SPT?
In fluorescence microscopy, the principle of tracking is to follow the motion of a fluorescent entity over time in living cells. This entity can be a group of molecules, a full structure or, in the case of Single-Particle Tracking (SPT), a single molecule. SPT aims to monitor the movement of individual molecules, and to determine their trajectory, speed, diffusion coefficient etc.
Although FRAP can provide dynamic information at the population level, it cannot guarantee that individual molecules in the population behave the same way. SPT, however, addresses this issue by reconstructing the trajectory (or “track”) of each molecule individually.
How does SPT work?
In order to track the movement of single molecules in time, it is essential to, first, identify single molecules with confidence. Using Single-Molecule Localization (SMLM) techniques, has, therefore, been the natural choice for SPT since the discovery of PALM and STORM (two SMLM techniques; you can read more about PALM and about STORM). PALM and STORM rely on the use of photoactivatable, photoconvertible, or photoswitchable probes. What these probes have in common is that they can be controlled to emit light in a random and isolated manner, so that, at any given time, it is possible to determine the position of single molecules with high precision (typically 10-15 nm).
In SPT, the same probes are used, but in living cells instead of fixed cells. During an acquisition, single molecules are identified and tracked for several frames until they stop emitting light. The output is not an image of a biological structure, but a map of the trajectories of single molecules. This map can then be used to determine if the target molecules move fast or slow depending on where they are in the cell, whether there are two distinct populations, one fast-diffusing and one immobile, measure the diffusion coefficient, etc.
Which probes can be used for SPT?
There are several elements to consider when choosing a probe for SPT. First, the probe needs to be photoactivatable, photoconvertible or photoswitchable to enable tracking of single molecules. Second, the probe and staining protocol must be compatible with living cells.
Traditionally, fusion proteins that are photoactivatable (PA) or photoconvertible (PC) are chosen for SPT. Because they are genetically encoded in the strain used, they require no staining with an external cell-permeable probe, thereby greatly simplifying the procedure and reducing non-specific signal to almost nothing. With recent genome editing technique, it is even possible to tag endogenous proteins, limiting artefacts related to protein overexpression. mEOS, PAmCHERRY, and DENDRA2 are examples of PA and PC fusion proteins that have been commonly used in SPT.
While it can be possible to use photoswitchable STORM dyes, it is not as common. First, the standard STORM staining protocol including photoswitchable probes (such as AF647, CF680 etc.) is immunofluorescence, which is incompatible with living cells. Second, most of these probes require the use of a photoswitching buffer and of high laser power, both of which are toxic for cells. However, a few photoswitchable probes and staining techniques happen to be compatible with living cells and, therefore, SPT. For example, the SNAP tag and Halo tag systems allow staining of target proteins in living cells, and several SNAP- and Halo- compatible fluorophores are photoswitchable, such as TMR, or JF dyes designed by the Lavis Lab in Janelia Farm. These staining procedures are usually quite straightforward and the imaging that follows fairly simple; however, the downside is that there can be a lot of non-specific staining, adding noise to the final data.
How to acquire SPT data?
SPT raw data are similar to standard single molecule acquisitions. The main differences are the exposure time (which needs to be short to follow fast dynamics but long enough to guarantee sufficient signal) and the laser power (high enough for single molecule imaging but not too high to avoid photobleaching and phototoxicity). The main challenge in SPT is then to analyze these data to reconstruct tracks.
How to analyse SPT data?
The goal of an SPT algorithm is to connect the dots from frame to frame. Typically, a standard SPT algorithm takes all tracks at frame t and all dots at frame t+1 and calculates the probability of assigning each track to each dot. This probability can be calculated based on several different parameters: proximity, motion speed, brightness… Afterwards, the algorithm chooses the solution that maximizes the overall probability.
Often, track reconstruction is challenging and implies to make assumptions on the data. Low density samples can be straight-forward, as a molecule detected in the same perimeter in consecutive frames is likely to be the same. However, in high-density samples, tracks are difficult to reconstruct, and close examination of the data is required before any interpretation is possible.
