Background: Plant cryptochromes are blue light sensors forming an exceptionally stable flavin neutral radical as a signaling state. Results: Blockage of proton transfer to flavin severely reduces the lifetime of the radical. Conclusion: The proton donor aspartic acid acts as an intrinsic stabilizer of the signaling state. Significance: The role of a key structural element is identified, distinguishing plant cryptochromes from other members of the family.

The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M−1cm−1, mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths.

Red-emitting fluorescent proteins (RFPs) with fluorescence emission above 600 nm are advantageous for cell and tissue imaging applications for various reasons. Fluorescence from an RFP is well separated from cellular autofluorescence, which is in the green region of the spectrum, and red light is scattered less, which allows thicker specimens to be imaged. Moreover, the phototoxic response of cells is lower for red than blue or green light exposure. Further red-shifted FP variants can be obtained by genetic modifications causing an extension of the conjugated π-electron system of the chromophore, or by placing amino acids near the chromophore that stabilize its excited state or destabilize its ground state. We have selected the tetrameric RFP eqFP611 from Entacmaea quadricolor as a lead structure and discuss several rational design trials to generate RFP variants with improved photochemical properties.

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Nitrogen-Vacancy (NV) colour centres in diamond is a rapidly progressing field with promising applications ranging from quantum information to bioimaging and sensing.1 The ability to manipulate NV electron spin optically under the ambient condition is the main driving force behind developments in nanoscale sensing techniques. In this work, we present investigations on using single NV spin to monitor diffusion dynamics of proteins. We investigate effects of spin labels interacting with NV spin and analyse protein diffusion and oligomerization. For these studies, we use a home built single NV pulsed optically-detected-magnetic-resonance (ODMR) setup capable of handling aqueous samples. The NV spin state initialized by optical pumping and transition between states are manipulated using microwaves. Coherent control of the spin states and pulsed protocols designed to sense fluctuations were used to probe spin coherences and monitor the diffusion dynamics. Figure 1 (a,b) shows the schematic of our setup and confocal fluorescence image of NV centers created~5nm close to the surface of a diamond. Some unspecific surface adhesions tend to cause problems with reliable spin coherence signals. Lipid bilayer coated surfaces could minimize unwanted adsorption while the adhesion of the membrane to the surface is challenging. We will present some results on surface treatments that promote adhesion on diamond surfaces. Surfaces treated with oxygen, argon plasma and wet chemical routes were analysed. The result indicates differences when the molecules dispersed in chloroform and aqueous buffer respectively and dried under ambient conditions. This provides routes to achieve monolayer of lipids on the diamond surface. The lipid bilayer films serve as a platform for investigating protein diffusion, dynamics and oligomerization at a single molecule level using NV-spin sensors.