Optically Activated Delayed Fluorescence.

We harness the photophysics of few-atom silver nanoclusters to create the first fluorophores capable of optically activated delayed fluorescence (OADF). In analogy with thermally activated delayed fluorescence, often resulting from oxygen- or collision-activated reverse intersystem crossing from triplet levels, this optically controllable/reactivated visible emission occurs with the same 2.2 ns fluorescence lifetime as that produced with primary excitation alone but is excited with near-infrared light from either of two distinct, long-lived photopopulated dark states.

Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery.

Excitation of few-atom Ag cluster fluorescence produces significant steady-state dark state populations that can be dynamically optically depopulated with long wavelength coillumination. Modulating this secondary illumination dynamically repopulates the ground state, thereby directly modulating nanodot fluorescence without modulating background. Both fast and slow modulation enable unmodulated background to be quantitatively removed in fluorescence correlation spectroscopy (FCS) through simple correlation-based averaging.

Optically Modulated Photoswitchable Fluorescent Proteins Yield Improved Biological Imaging Sensitivity.

Photoswitchable fluorescent proteins (PS-FPs) open grand new opportunities in biological imaging. Through optical manipulation of FP emission, we demonstrate that dual-laser modulated synchronously amplified fluorescence image recovery (DM-SAFIRe) improves signal contrast in high background through unambiguous demodulation and is linear in relative fluorophore abundance at different points in the cell. The unique bright-to-dark state interconversion rates of each PS-FP not only enables discrimination of different, yet spectrally indistinguishable FPs, but also allows signal rejection of diffusing relative to bound forms of the same PS-FP, rsFastLime.

Tailoring cyanine dark states for improved optically modulated fluorescence recovery.

Cyanine dyes are well-known for their bright fluorescence and utility in biological imaging. However, cyanines also readily photoisomerize to produce nonemissive dark states. Co-illumination with a secondary, red-shifted light source on-resonance with the longer wavelength absorbing dark state reverses the photoisomerization and returns the cyanine dye to the fluorescent manifold, increasing steady-state fluorescence intensity.

Optically modulated fluorescence bioimaging: visualizing obscured fluorophores in high background.

Fluorescence microscopy and detection have become indispensible for understanding organization and dynamics in biological systems. Novel fluorophores with improved brightness, photostability, and biocompatibility continue to fuel further advances but often rely on having minimal background. The visualization of interactions in very high biological background, especially for proteins or bound complexes at very low copy numbers, remains a primary challenge.

Optically modulatable blue fluorescent proteins.

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination.

DNA-Templated Molecular Silver Fluorophores.

Conductive and plasmon-supporting noble metals exhibit an especially wide range of size-dependent properties, with discrete electronic levels, strong optical absorption, and efficient radiative relaxation dominating optical behavior at the ~10-atom cluster scale. In this Perspective, we describe the formation and stabilization of silver clusters using DNA templates and highlight the distinct spectroscopic and photophysical properties of the resulting hybrid fluorophores. Strong visible to near-IR emission from DNA-encapsulated silver clusters ranging in size from 5-11 atoms has been produced and characterized.

Modulated fluorophore signal recovery buried within tissue mimicking phantoms.

Optically modulated fluorescence from ∼140 nM Cy5 is visualized when embedded up to 6 mm within skin tissue mimicking phantoms, even in the presence of overwhelming background fluorescence and scatter. Experimental and finite element analysis (FEA)-based computational models yield excellent agreement in signal levels and predict biocompatible temperature changes. Using synchronously amplified fluorescence image recovery (SAFIRe), dual-laser excitation (primary laser: λ = 594 nm, 0.

Signal Discrimination Between Fluorescent Proteins in Live Cells by Long-wavelength Optical Modulation.

Fluorescent proteins (FPs) have revolutionized molecular and cellular biology; yet, discrimination over cellular autofluorescence, spectral deconvolution, or detection at low concentrations remain challenging problems in many biological applications. By optically depopulating a photoinduced dark state with orange secondary laser co-excitation, the higher-energy green AcGFP fluorescence is dynamically increased. Modulating this secondary laser then modulates the higher-energy, collected fluorescence; enabling its selective detection by removing heterogeneous background from other FPs.

All-optical fluorescence image recovery using modulated Stimulated Emission Depletion.

Fluorescence modulation for selective recovery of desired fluorescence signals has to date required careful fluorophore selection combined with repeated optical recovery from long-lived photoinduced dark states. Adapting an all-optical scheme, modulated Stimulated Emission Depletion generalizes such modulation schemes by eliminating the need for dark state residence by directly optically depopulating the emissive state at any externally applied frequency. Using two overlapped Gaussian laser spots with the depletion beam being intensity-modulated, fluorescence modulation is readily achieved with a depletion ratio governed by the intensity of the depleting laser.

Optical modulation and selective recovery of Cy5 fluorescence.

Fluorescence modulation offers the opportunity to detect low-concentration fluorophore signals within high background. Applicable from the single-molecule to bulk levels, we demonstrate long-wavelength optical depopulation of dark states that otherwise limit Cy5 fluorescence intensity. By modulated excitation of a long-wavelength Cy5 transient absorption, we dynamically modulate Cy5 emission.

Correlated Single Quantum Dot Blinking and Interfacial Electron Transfer Dynamics.

The electron transfer (ET) dynamics from core/multi-shell (CdSe/CdS(3ML)ZnCdS(2ML)ZnS(2ML)) quantum dots (QDs) to adsorbed Fluorescein (F27) molecules have been studied by single particle spectroscopy to probe the relationship between single QD interfacial electron transfer and blinking dynamics. Electron transfer from the QD to F27 and the subsequent recombination were directly observed by ensemble-averaged transient absorption spectroscopy. Single QD-F27 complexes show correlated fluctuation of fluorescence intensity and lifetime, similar to those observed in free QDs.

Optically enhanced, near-IR, silver cluster emission altered by single base changes in the DNA template.

Few-atom silver clusters harbored by DNA are promising fluorophores due to their high molecular brightness along with their long- and short-term photostability. Furthermore, their emission rate can be enhanced when co-illuminated with low-energy light that optically depopulates the fluorescence-limiting dark state. The photophysical basis for this effect is evaluated for two near-infrared-emitting clusters.

FRET-enabled optical modulation for high sensitivity fluorescence imaging.

Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightens and optically modulates higher energy donor emission.

Electron transfer-induced blinking in Ag nanodot fluorescence.

Various single-standed DNA-encapsulated Ag nanoclusters (nanodots) exhibit strong, discrete fluorescence with solvent polarity-dependent absorption and emission throughout the visible and near-IR. All species examined, regardless of their excitation and emission energies, show similar µs single-molecule blinking dynamics and near IR transient absorptions. The polarity dependence, µsec blinking, and indistinguishable µsec-decaying transient absorption spectra for multiple nanodots suggest a common charge transfer-based mechanism that gives rise to nanodot fluorescence intermittency.

Synchronously amplified fluorescence image recovery (SAFIRe).

Fluorescence intermittency severely limits brightness in both single molecule and bulk fluorescence. Herein, we demonstrate that optical depopulation of organic fluorophore triplet states opens a path to significantly increased sensitivity by simultaneously increasing brightness and greatly reducing background through synchronously detected fluorescence modulation. Image recovery is achieved through selective fluorescence enhancement via modulating a secondary laser excitation at much lower energy than the observed emission in order to depopulate the long-lived triplet states.

Optically modulated fluorophores for selective fluorescence signal recovery.

Fluorescence imaging in biological sciences is hindered by significant depth-dependent signal attenuation and highly fluorescent backgrounds. We have developed optically modulated near-IR-emitting few-atom Ag nanodots that are selectively and dynamically photobrightened upon simultaneous excitation with a secondary laser, enabling high-sensitivity image extraction to reveal only the demodulated fluorophores. Image demodulation is demonstrated in high-background environments to extract weak signals from completely obscuring background emission.

Water-soluble Ag nanoclusters exhibit strong two-photon-induced fluorescence.

Water-soluble ssDNA-encapsulated Ag clusters exhibit large two-photon cross sections reaching 50 000 GM, with high quantum yields in the red and near-IR. Three distinct, spectrally pure, several atom clusters emitting at 660, 680, or 710 nm have been created with two-photon cross sections rivaling those of much larger water-soluble semiconductor quantum dots. Their stability, biocompatibility, and small size offer the promise of nontoxic, sensitive high-resolution biological imaging.

Oligonucleotide-stabilized Ag nanocluster fluorophores.

Single-stranded oligonucleotides stabilize highly fluorescent Ag nanoclusters, with emission colors tunable via DNA sequence. We utilized DNA microarrays to optimize these scaffold sequences for creating nearly spectrally pure Ag nanocluster fluorophores that are highly photostable and exhibit great buffer stability. Five different nanocluster emitters have been created with tunable emission from the blue to the near-IR and excellent photophysical properties.

Strongly emissive individual DNA-encapsulated Ag nanoclusters as single-molecule fluorophores.

The water-soluble, near-IR-emitting DNA-encapsulated silver nanocluster presented herein exhibits extremely bright and photostable emission on the single-molecule and bulk levels. The photophysics have been elucidated by intensity-dependent correlation analysis and suggest a heavy atom effect of silver that rapidly depopulates an excited dark level before quenching by oxygen, thereby conferring great photostability, very high single-molecule emission rates, and essentially no blinking on experimentally relevant time scales (0.1 to >1,000 ms).