A new paper published this week in Angewandte Chemie marries the nanoscale fluorescence imaging technique DNA-PAINT, developed by Professor Ralf Jungmann’s team at the Max Planck Institute of Biochemistry and the Ludwig Maximilian University, Germany, with the highly specific molecular targeting of Affimer technology performed by Dr Darren Tomlinson’s lab at the University of Leeds, UK to create new reagents for super-resolution cellular imaging.
Super-resolution microscopy offers high resolution imaging of the complex 3D cellular architectures, well below the classical diffraction limit. However, achieving this promised nanoscale level of vision into cellular structures has proven difficult due to the dearth of suitable small reagents available for use. Antibodies that provide the required high target specificity for super-resolution imaging are too large in size, resulting in reporting of the probe location not the target location, which can be as much as 15 nm away from the target of interest.
Ideal probes for super-resolution imaging must be highly target specific and small in size. The small size is important to reduce linkage error seen with antibodies and allow for high-density labelling with minimal steric hindrance. Beyond probe size, it is equally important to be able to efficiently develop any reagents against a large target library and that the probe is functional for both intra- and extracellular targets. Affimer reagents meet all these criteria and so offer a solution to fill this needs gap in super-resolution imaging reagents.
In DNA-PAINT, the apparent “switching” between bright and dark states of dye molecules used for super-resolution reconstruction is achieved by the transient interaction of a dye-labelled imager strands with their complementary docking strands linked to the target-specific Affimer protein. For this study an actin-specific Affimer was site-specifically labelled with DNA using maleimide chemistry to ensure a quantitative 1:1 stochiometric labelling.
To allow fast diffraction-limited imaging prior to the acquisition of DNA-PAINT images, an Atto488 dye was conjugated to the DNA-PAINT docking strand used to label the Affimer. When imaged in labelled fixed Cos7 cells 3D DNA-PAINT microscopy of the actin cytoskeleton shows a clear improvement over the initial diffraction-limited imaging, demonstrating the efficiency and specificity of Affimer labelling.
Using the DNA-labelled Affimer the researchers compared the super-resolution imaging of actin in Cos7 cells with phalloidin, a commonly used small molecule for actin-labelling and imaging. Comparable actin staining patterns were seen with both reagents in these cells. Furthermore, analysis of the width of thirteen actin fibres using each labelling technique revealed very similar results of 18 nm with Affimer labelling and 13 nm using phalloidin.
Demonstrating the versatility of Affimer labelling, Jungmann’s team explored both copper-catalysed cycloaddition of the DNA docking strand to the Affimer binder and directly labelling the Affimer protein with Alexa647. Michelle Peckham’s lab applied direct stochastic optical reconstruction microscopy (dSTORM) using these reagents to show highly similar results, indicating the wide applicability of Affimer reagents as super-resolution imaging reagents.
Evaluation of the achievable 3D resolution of the actin network in Cos7 cells confirmed two distinct layers of actin present within the cells. Each of the layers had an apparent thickness of 40 nm and were separated by approximately 130 nm.
The small size and quantitative labelling ability of DNA-PAINT labelling in combination with Affimer reagents offer ideal imaging probes for super-resolution imaging. Future applications using these reagents could include absolute quantitative microscopy approaches and multiplexed target detection using orthogonal Affimer binders coupled to distinct DNA sequences.