De novo design of proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

Deuteration of heptamethine cyanine dyes enhances their emission efficacy.

The design of bright short-wave infrared fluorophores remains a grand challenge. Here we investigate the impact of deuteration on the properties in a series of heptamethine dyes, the absorption of which spans near-infrared and SWIR regions. We demonstrate that it is a generally applicable strategy that leads to enhanced quantum yields of fluorescence, longer-lived singlet excited states and suppressed rates of non-radiative deactivation processes.

Spectrally Selective Time-Resolved Emission through Fourier-Filtering (STEF).

We demonstrate a method for separating and resolving the dynamics of multiple emitters without the use of conventional filters. By directing the photon emission through a fixed path-length imbalanced Mach-Zehnder interferometer, we interferometrically cancel (or enhance) certain spectral signatures corresponding to one emissive species. Our approach, Spectrally selective Time-resolved Emission through Fourier-filtering (STEF), leverages the detection and subtraction of both outputs of a tuned Mach-Zehnder interferometer, which can be combined with time-correlated single photon counting (TCSPC) or confocal imaging to demix multiple emitter signatures. We develop a procedure to calibrate out imperfections in Mach-Zehnder interferometry schemes. Additionally, we demonstrate the range and utility of STEF by performing the following procedures with one measurement: (1) filtering out laser scatter from a sample, (2) separating and measuring a fluorescence lifetime from a binary chromophore mixture with overlapped emission spectra, (3) confocally imaging and separately resolving the standard fluorescent stains in bovine pulmonary endothelial cells and nearly overlapping fluorescent stains on RAW 264.7 cells. This form of spectral balancing can allow for robust and tunable signal sorting.

Decay-Associated Fourier Spectroscopy: Visible to Shortwave Infrared Time-Resolved Photoluminescence Spectra.

We describe and implement an interferometric approach to decay-associated photoluminescence spectroscopy, which we term decay-associated Fourier spectroscopy (DAFS). In DAFS, the emitted photon stream from a substrate passes through a variable path length Mach-Zehnder interferometer prior to detection and timing. The interferometer encodes spectral information in the intensity measured at each detector enabling simultaneous spectral and temporal resolution. We detail several advantages of DAFS, including wavelength-range insensitivity, drift-noise cancellation, and optical mode retention. DAFS allows us to direct the photon stream into an optical fiber, enabling the implementation of superconducting nanowire single photon detectors for energy-resolved spectroscopy in the shortwave infrared spectral window (λ = 1-2 µm). We demonstrate the broad applicability of DAFS, in both the visible and shortwave infrared, using two Förster resonance energy transfer pairs: a pair operating with conventional visible wavelengths and a pair showing concurrent acquisition in the visible and the shortwave infrared regime.