Incorporation of sensing modalities into de novo designed fluorescence-activating proteins.

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca for real-time fluorescence reporting.

De novo design of a fluorescence-activating β-barrel.

The regular arrangements of β-strands around a central axis in β-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with β-barrels. Here we show that accurate de novo design of β-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain.

Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM.

Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores.

Twinkle, twinkle little star: photoswitchable fluorophores for super-resolution imaging.

Photoswitchable fluorescent probes are key elements of newly developed super-resolution fluorescence microscopy techniques that enable far-field interrogation of biological systems with a resolution of 50 nm or better. In contrast to most conventional fluorescence imaging techniques, the performance achievable by most super-resolution techniques is critically impacted by the photoswitching properties of the fluorophores. Here we review photoswitchable fluorophores for super-resolution imaging with discussion of the fundamental principles involved, a focus on practical implementation with available tools, and an outlook on future directions.