PAINT Imaging

Why is DNA-PAINT useful?

The labeling strategy used in PAINT leads to very specific targeting of your protein of interest. The main advantages of DNA-PAINT are:

– Increased resolution
– Higher localization precision
– Lack of bleaching due to continuously replenishing fluorophores
– Multiplexing

Inherent to the transition binding-induced blinking, PAINT offers several benefits, such increased resolution and the lack of photobleaching. The duration of each blink depends on the DNA sequence, which determines the off-rate of the binding. A longer blink means a brighter spot that can be detected by microscopes like the Nanoimager with higher localization precision, leading to images of higher resolution. In addition, the fluorophores are constantly replaced by new imaging strands through binding kinetics, which prevents them from being permanently bleached like in dSTORM where these are fixed to the epitopes. 

Some remarkable experiments leveraging PAINT as a technique over other super-resolution techniques have also been performed. For instance, multiplexing 3D imaging in cells using DNA-PAINT has been shown. This takes advantage of the serial imaging of different epitopes tagged with separate docking strands, while the imager strands for each epitopes is flown into the sample in a sequential manner. A recent in vitro study revealed that by using the kinetic fingerprint between the docking and imager strands, one can image up to 124 colors in one sample!

By using probes whose photophysics can be calibrated, it is also possible to perform quantitative experiments. With this technique, called qPAINT, one can extract the number of docking strands, and thus targets, from a region imaged using SMLM, assuming a constant binding rate.

Interestingly, PAINT can also be used in combination with other SMLM techniques. Read our previous Application note showing how multimodal super-resolution imaging provides the opportunity to access further information of subcellular structures by using different labeling strategies. Together with PAINT staining, one can use genetically encoded fluorescent proteins to target other molecules, hardly accessible with PAINT labeling.

Although PAINT has the advantage of facilitating longer imaging time, higher resolution, and multiple labeling, it also comes with a drawback. The imaging sessions tend to be longer as the positional sampling rate depends on the hybridization kinetics, which are slower than dSTORM dye blinking. Some recent developments have successfully demonstrate the ability to speed this process up by 100x, either by optimizing the DNA sequences to increase kinetic rates, using computational methods, or supplementing ethylene carbonate to the imaging buffer.

Figure 2. Two-color imaging using PALM and PAINT sequentially in the same sample. RNAP tagged to PAmCherry1 (cyan) and JF646-Hoechst transiently labeling the bacterial nucleoid (magenta). Samples provided by Dr. Christoph Spahn, Max Planck Institute for ​​Terrestrial Microbiology, Marburg, Germany.

Figure 3. Clathrin coated pits imaged on the Nanoimager labeled with a rabbit anti-clathrin heavy chain primary antibody, and detected with the Massive Photonics “massive sdAb-2-plex” kit for DNA-PAINT imaging in fixed COS-7 cells. Localizations are colored by frame index, showing how localizations are overall evenly distributed over the acquisition.