Technology

WHAT IS DNA-PAINT?

DNA-PAINT (DNA-Point Accumulation for Imaging in Nanoscale Topography) is a localization based super-resolution microscopy method that uses short, dye-labeled single stranded DNA (ssDNA strands) that transiently bind to their complements:

Imager strand – a dye-labeled DNA oligo present in imaging buffer
Docking strand – a DNA oligo complementary to the imager strand that is delivered to the target of interest via binders (e.g. antibodies, single domain antibodies)

When a dye-labeled imager transiently hybridizes to a docking strand in DNA-PAINT imaging buffer, fluorescent signal or “blinking” occurs. To create a super-resolution image, a camera captures blinking over a period of time (10 – 50 min) and the resulting image is post-processed using Picasso software.

WHY USE DNA-PAINT?

Ultra resolution! Resolve 10 – 15 nm distances in cells.
Unlimited target multiplexing! In DNA-PAINT, spectrally compatible dyes aren’t needed to image multiple targets at a time. Instead, you can simply couple different docking strands (e. different DNA sequences) to your target binders and image each target sequentially using the same laser line with its corresponding imager strand.
Target Counting! The predictable nature of DNA binding kinetics allows you to quantify how many proteins are present in cells (qPAINT).

WHERE TO START?

You need 3 things to do DNA-PAINT:

Simple TIRF Microscope – like the ZEISS Elyra 7, Nikon N-STORM, ONI Nanoimager, abbelight SAFe 180, Bruker VUTARA VXL microscope, Andor Dragonfly or similar system found in your imaging facility.
DNA-PAINT compatible binder – any antibody or single-domain antibody that binds to your target protein and is coupled to a docking strand. Find a DNA-PAINT compatible binder here at Massive Photonics! We have a wide range of antibodies and single domain antibodies. Order your custom antibody or single-domain antibody (PRODUCTS page).
Picasso data analysis software – a free software developed for DNA-PAINT image analysis (Download Picasso at Github). Find out how to use Picasso in Nature Protocols 12, 1198–1228 (2017)

RESI

WHAT IS RESI?

Resolution Enhancement by Sequential Imaging (RESI) is a paradigm shifting DNA-PAINT super-resolution microscopy technique that enables sub nanometer resolution (Nature 617, 711–716 (2023)).

How does RESI work?

RESI takes advantage of the ability of DNA-PAINT to encode target identities through DNA sequences. To visualize multiple targets in a single DNA-PAINT experiment, called multiplexing, each target is imaged sequentially with orthogonal docking and imager strands. RESI uses the same multiplexing principle for a single target species, creating sparser and more isolated localizations. In each imaging round, the center of each localization group is determined to create a resolution enhancement. Resolution enhancement scales with the number of imaging rounds performed.

Experimentally, RESI can be easily performed by immunolabeling a target species with the same single-domain antibody binder conjugated to orthogonal DNA-PAINT docking sequences. The sample then undergoes multiple imaging and washing rounds until all sequences are imaged.

Why use RESI?

RESI is for researchers who want to see their target of interest at single protein resolution in cells or investigate biomolecule interactions that occur below 10 nm. RESI is an easy-to-implement DNA-PAINT microscopy technique that can be used on any DNA-PAINT compatible microscope.

Which Massive Photonics products can be used for RESI?

Currently, our MASSIVE-RESI product contains anti-GFP sdABs (single-domain antibodies) that have been tested and verified for successful RESI imaging. However, most other sdABs should work for RESI. Feel free to contact us if you’re interested in using another sdAB.

How do I analyze RESI data?

Good news! RESI data analysis is fully integrated in the open-source DNA-PAINT analysis software Picasso (Download Picasso at Github). Detailed user instructions are available at the Read The Docs (Read The Docs RESI) and in the RESI publication (Nature 617, 711–716 (2023)).

Can I use my current DNA-PAINT microscope set-up for RESI?

Yes! RESI doesn’t require any microscope upgrades to work.