Two-photon Brightness Research Articles

π-Expanded Dipyrrolonaphthyridinediones with Large Two-Photon Absorption Cross-Section Values.

A synthetic entry to novel dyes based on the dipyrrolonaphthyridinedione core was developed via the Heck reaction. These weakly fluorescent compounds bearing double bond linkages between the core and the peripheral units absorbed strongly in the far-red/NIR region and possessed large values of two-photon absorption (TPA) cross-sections (up to 5180 GM). Additionally, analogous dyes bearing triple bond linkages were also efficient TPA materials with relatively large two-photon absorption cross-sections (up to 2840 GM) as well as two-photon brightness (up to 1450 GM). The centrosymmetric nature of both of these families of dyes is responsible for the location of the maxima of two-photon absorption being at much higher energy than the ones corresponding to the double wavelength of the lowest-energy one-photon absorption. Theoretical calculations clarified that the enhancement of the TPA by the peripheral substitutions arose through different mechanisms depending on either the electron-donating or electron-withdrawing ability of a given substituent to the ambipolar core. The change in the electron distribution of HOMO and HOMO-1 by the push-pull effect was found to govern the strength of the lowest-energy TPA-allowed transition. Importantly, compounds from both series possessed a beneficial ratio of σ2/MW (1.6-9.8 GM/g).

A ratiometric two-photon probe for Ca2+ in live tissues and its application to spinal cord injury model.

Ratiometric imaging with a small-molecule probe is important for the in-situ quantitative analysis of chemical events. We developed a ratiometric two-photon fluorescent probe (SCa1-IREF) derived from dual dyes with different Stokes shifts. This probe has two identical windows: a Ca2+-sensing window and an internal reference window, with eliminated FRET interference. SCa1-IREF shows a marked change in the ratio upon response with Ca2+, significant two-photon brightness, considerable selectivity for Ca2+, and cell loading ability with low cytotoxicity. The ratiometric two-photon microscopy images revealed that this probe could directly and quantitatively estimate Ca2+ in live neurons and various tissues including rat spinal cord tissue. The studies of spinal cord injury model revealed that the Ca2+ level was significantly affected by elapsed time after injury. These results will provide useful applications for in-situ [Ca2+]i imaging and for the development of effective ratiometric probes.

Two-Photon Fluorescent Molybdenum Disulfide Dots for Targeted Prostate Cancer Imaging in the Biological II Window.

Molybdenum disulfide (MoS2) quantum dots (QDs) derived from this two-dimensional (2D) transition metal dichalcogenide are emerging zero-dimensional materials that possess very good optical properties. Bioimaging using light in the biological II window (950–1350 nm) is a next-generation approach that will allow clinicians to achieve deeper tissue imaging with better image contrast and reduced phototoxicity and photobleaching. This article reports the development of a water-soluble, zero-dimensional antibody-conjugated transition metal dichalcogenide MoS2 QD-based two-photon luminescence (TPL) probe for the targeted bioimaging of cancer cells in the biological II window. The data indicates that MoS2 QDs exhibit an extremely high two-photon absorption cross-section (σ = 58960 GM) and two-photon brightness (4.7 × 103 GM) because of the quantum confinement and edge effects. Experimental data show that anti-PSMA antibody-attached MoS2 QDs can be used for selective two-photon imaging of live prostate cancer cells using 1064 nm light because of the high two-photon brightness, very good photostability, and very good biocompatibility of these MoS2 QDs. The data demonstrate that the bioconjugated MoS2 QDs can distinguish targeted and nontargeted cells. This study illuminates the high two-photon brightness mechanism of MoS2 QDs and provides a zero-dimensional transition metal dichalcogenide-based selective TPL agent for high-efficiency live cell imaging.

Voltage-sensitive rhodol with enhanced two-photon brightness

We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using two-photon (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue.

New GFP Variants with Enhanced Two-Photon Brightness

New GFP Variants with Enhanced Two-Photon Brightness

Two-Photon Luminescence of Single Colloidal Gold Nanorods: Revealing the Origin of Plasmon Relaxation in Small Nanocrystals

The two-photon luminescence (TPL) of small 10 nm × 40 nm colloidal gold nanorods (GNR) is investigated at the single object level, combining polarization-resolved TPL and simultaneously acquired topography. A very high dependence of the TPL signal with both the nanorods longitudinal axis and the incident wavelength is observed, confirming the plasmonic origin of the signal and pointing to the limit of the analogy between GNRs and molecules. The maximum two-photon brightness of a single GNR is measured to be a few millions higher than the two-photon brightness of fluorescein molecules. The spectral analysis of the TPL evidence two emission bands peaks: in the visible (in direct connection with the gold band structure) and in the infrared (IR). In both bands, the TPL signal is observed to vary quadratically with the excitation beam; the signal emitted in either the visible or the IR exhibits, however, different polarization properties. We show that the important TPL observed in these small gold nanorods res...

Molecular-Based Fluorescent Nanoparticles Built from Dedicated Dipolar Thienothiophene Dyes as Ultra-Bright Green to NIR Nanoemitters.

Fluorescent Organic Nanoparticles (FONs), prepared by self-aggregation of dedicated dyes in water, represent a promising green alternative to the toxic quantum dots (QDs) for bioimaging purposes. In the present paper, we describe the synthesis and photophysical properties of new dipolar push-pull derivatives built from thieno[3,2-b]thiophene as a π-conjugated bridge that connects a triphenylamine moiety bearing various bulky substituents as electron-releasing moiety to acceptor end-groups of increasing strength (i.e., aldehyde, dicyanovinyl and diethylthiobarbiturate). All dyes display fluorescence properties in chloroform, which shifts from the green to the NIR range depending on the molecular polarization (i.e., strength of the end-groups) as well as a large two-photon absorption (TPA) band response in the biological spectral window (700–1000 nm). The TPA bands show a bathochromic shift and hyperchromic effect with increasing polarization of the dyes with maximum TPA cross-section reaching 2000 GM for small size chromophore. All dyes are found to form stable and deeply colored nanoparticles (20–45 nm in diameter) upon nanoprecipitation in water. Although their fluorescence is strongly reduced upon aggregation, all nanoparticles show large one-photon (up to 108 M−1·cm−1 in the visible region) and two-photon (up to 106 GM in the NIR) brightness. Interestingly, both linear and non-linear optical properties are significantly affected by interchromophoric interactions, which are promoted by the molecular confinement and modulated by both the dipolar strength and the presence of the bulky groups. Finally, we exploited the photophysical properties of the FONs to design optimized core-shell nanoparticles built from a pair of complementary dipolar dyes that promotes an efficient core-to-shell FRET process. The resulting molecular-based core-shell nanoparticles combine large two-photon absorption and enhanced emission both located in the NIR spectral region, thanks to a major amplification (by a factor of 20) of the core fluorescence quantum yield. These novel nanoparticles, which combine huge one-and two-photon brightness, hold major promise for in vivo optical bioimaging.

Dipolar Dyes with a Pyrrolo[2,3-b]quinoxaline Skeleton Containing a Cyano Group and a Bridged Tertiary Amino Group: Synthesis, Solvatofluorochromism, and Bioimaging.

Two strongly polarized dipolar chromophores possessing a cyclic tertiary amino group at one terminus of the molecule and a CN group at the opposite terminus were designed and synthesized. Their rigid skeleton contains the rarely studied pyrrolo[2,3-b]quinoxaline ring system. The photophysical properties of these regioisomeric dyes were different owing to differing π conjugation between the CN group and the electron-donor moiety. These dipolar molecules showed very intense emission, strong solvatofluorochromism, and sufficient two-photon brightness for bioimaging. One of these regioisomeric dyes, namely, 11-carbonitrile-2,3,4,5,6,7-hexahydro-1H-3a,8,13,13b-tetraazabenzo[b]cyclohepta[1,2,3-jk]fluorene, was successfully utilized in two-photon imaging of mouse organ tissues and showed distinct tissue morphology with high resolution.

Boron Difluoride Curcuminoid Fluorophores with Enhanced Two-Photon Excited Fluorescence Emission and Versatile Living-Cell Imaging Properties.

The synthesis of boron difluoride complexes of a series of curcuminoid derivatives containing various donor end groups is described. Time-dependent (TD)-DFT calculations confirm the charge-transfer character of the second lowest-energy transition band and ascribe the lowest energy band to a "cyanine-like" transition. Photophysical studies reveal that tuning the donor strength of the end groups allows covering a broad spectral range, from the visible to the NIR region, of the UV-visible absorption and fluorescence spectra. Two-photon-excited fluorescence and Z-scan techniques prove that an increase in the donor strength or in the rigidity of the backbone results in a considerable increase in the two-photon cross section, reaching 5000 GM, with predominant two-photon absorption from the S0-S2 charge-transfer transition. Direct comparisons with the hemicurcuminoid derivatives show that the two-photon active band for the curcuminoid derivatives has the same intramolecular charge-transfer character and therefore arises from a dipolar structure. Overall, this structure-relationship study allows the optimization of the two-photon brightness (i.e., 400-900 GM) with one dye that emits in the NIR region of the spectrum. In addition, these dyes demonstrate high intracellular uptake efficiency in Cos7 cells with emission in the visible region, which is further improved by using porous silica nanoparticles as dye vehicles for the imaging of two mammalian carcinoma cells type based on NIR fluorescence emission.

Enhanced two-photon brightness in molecular-based organic nanoparticles built from articulated-dipoles

New dyes built from two articulated push-pull dipolar moieties have been prepared and characterized as building blocks for the design of hyper-bright fluorescent organic nanoparticles (FONs). The two dipolar modules are coupled via a fluorene moiety that allows free rotation resulting in two remarkable conformations displaying dipolar and/or quadrupolar behavior. Whereas the dyes show little or no fluorescence in solution, a major enhancement of their luminescent properties as well as of their two-photon absorption (2 PA) response in the NIR region is achieved upon their self-aggregation in water. In contrast, earlier linear dipolar dyes yielding FONs upon self-aggregation in water evidenced a reduction of both reduced emission and peak 2 PA as compared to molecular dyes dissolved in low to medium polarity organic solvents. This striking difference demonstrates the potential of inner control of dipolar interactions within symmetrical and flexible molecular structures to promote combined emission and 2 PA enhancement. This work opens a fruitful bottom-up approach to complementary green and red-emitting molecular-based nanoparticles combining giant two-photon brightness (over 300 000 and 400 000 GM) as well as excellent chemical and colloidal stability. The remarkable chemical and colloidal stability of these molecular-based nanoparticles coupled with their brightness make them highly promising candidates for bioimaging applications.

Polar diketopyrrolopyrrole-imidazolium salts as selective probes for staining mitochondria in two-photon fluorescence microscopy.

Three rationally designed polar derivatives of diketopyrrolopyrrole consisting of 1,3-dimethylimidazolium cationic units and benzene, thiophene, or furan rings as π spacers were synthesized and thoroughly studied. The obtained salts are soluble in polar organic solvents and show satisfactory solubility in water, which makes them suitable for the applications in bioimaging. Photophysical measurements revealed that the obtained derivatives are characterized by strong absorption and good fluorescence quantum yields. The corresponding two-photon properties were also examined and showed that the synthesized salts exhibit large two-photon absorption cross-sections reaching 4000 GM (GM=Goeppert-Mayer unit, 1 GM=10(-50) cm(4) s photon(-1) ) and very high two-photon brightness values exceeding 2000 GM. It was demonstrated that these salts can be safely applied in two-photon fluorescence microscopy for selective staining of mitochondria in living cells.

Red-emitting DPSB-based conjugated polymer nanoparticles with high two-photon brightness for cell membrane imaging.

New red-emitting conjugated polymers have been successfully synthesized by incorporating classical two-photon absorption (TPA) units, electron-rich units, and a small amount of electron-deficient units along the polymer backbones. Water-dispersible nanoparticles (NPs) based on these polymers were also fabricated for applications in two-photon excitation fluorescence imaging of cell membrane. Through optimization of the polymer/matrix mass ratio and the initial feed concentration of the polymer solution, a high quantum yield (QY) of 24% was achieved for the red-emitting NPs in water. TPA cross section and two-photon action cross section values of these polymers at 750 nm reached up to 1000 GM and 190 GM per repeat unit in aqueous media, 2.5 × 10(5) GM and 4.7 × 10(4) GM per NP, respectively. Furthermore, these NPs displayed excellent photostability and biocompatibility. Their applications as two-photon excitation fluorescence probes for cell membrane imaging have been demonstrated in three different cell lines with excellent imaging contrast. These results demonstrated that these polymer NPs hold great potentials as excellent two-photon excitation fluorescence probes in various biological applications.

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, L-MAG0460, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0460. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca(2+)-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca(2+) imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.

Amplified two-photon brightness in organic multicomponent nanoparticles

A large amplification of the two-photon-induced luminescence is achieved in organic molecular-based core–shell nanoparticles.

Fluorenylethynylpyrene derivatives with strong two-photon absorption: influence of substituents on optical properties

A series of fluorenylethynylpyrene derivatives with large two-photon absorption cross-sections (≈250–2500 GM) and good fluorescence quantum yields (Φfl = 0.55–0.98) and with high two-photon brightness were synthesized.

Strategy towards large two-photon absorption cross-sections for diketopyrrolopyrroles

Decorating diketopyrrolopyrroles with strongly electron-rich heterocycles led to donor–acceptor–donor architectures possessing two-photon brightness 850–1900 GM.

Two-photon-induced fluorescence in new π-expanded diketopyrrolopyrroles.

Structurally unique π-expanded diketopyrrolopyrroles (EDPP) were designed and synthesized. Strategic placement of a fluorene scaffold at the periphery of a diketopyrrolopyrrole through tandem Friedel-Crafts-dehydration reactions resulted in dyes with supreme solubility. The structure of the dyes was confirmed by X-ray crystallography verifying a nearly flattened arrangement of the ten fused rings. Despite the extended ring system, the dye still preserved good solubility and was further functionalized by using Pd-catalyzed coupling reactions, such as the Buchwald-Hartwig amination. Photophysical studies of these new functional dyes revealed that they possess enhanced properties when compared with expanded DPPs in terms of two-photon absorption cross-section. It is further demonstrated that in addition to the initial diacetals, the final electrophilic cyclization step can also be applied to diketones. By placing two amine groups at peripheral positions of the resulting dyes, values of two-photon absorption cross-section on the level of 2000 GM around 1000 nm were achieved, which in combination with high fluorescence quantum yield (Φfl ), generated a two-photon brightness of approximately 1600 GM. These characteristics in combination with strong red emission (665 nm) make these new π-expanded diketopyrrolopyrroles of major promise as two-photon dyes for bioimaging applications. Finally, the corresponding N-alkylated DPPs displayed a solid-state fluorescence.

A new route towards fluorescent organic nanoparticles with red-shifted emission and increased colloidal stability

Suzuki–Miyaura cross-coupling reaction with a new boron reagent has been used to conveniently and efficiently synthetize a dipolar chromophore having an elongated π-conjugated system (i.e., bithiophene based), which displays a red-shifted emission while maintaining fluorescence. Bright orange emitting FONs made from this ‘naked’ dipole have been easily prepared and studied. Very interestingly, these FONs with red-shifted emission combine markedly enhanced colloidal and structural stability in water with giant one- and two-photon brightness. As such, they hold promises as fluorescent probes for bioimaging.

Non-classical donor–acceptor–donor chromophores. A strategy for high two-photon brightness

Rod-like π-expanded bis-imidazo[1,2-a]pyridines were designed and synthesized. Strategic placement of a central electron-poor unit at position C3 of the imidazo[1,2-a]pyridine core resulted in donor–acceptor–donor quadrupolar systems. We further demonstrated the applicability of the Ortoleva–King–Chichibabin tandem process in the preparation of complex imidazo[1,2-a]pyridines. The monomer model heterocycles, differing by substitution at C2 and C3, displayed luminescence properties marginally dependent on the substitution pattern and conjugation extension. The highest luminescence quantum yield (ϕflca. 0.2) is shown by the model compounds without a substitution at C3, whereas lower values are measured for the others. Quantum yields in toluene for the quadrupolar systems varied from ϕfl = 0.7 to ϕfl > 0.9. Apart from bis-imidazo[1,2-a]pyridine possessing two methyl groups, with ϕfl = 0.69 in toluene and 0.35 in dichloromethane, the luminescence properties were only slightly affected by the change in the solvent. Most of the fluorescence lifetimes ranged from 1–2 ns and the radiative rate constants reached values of ca. 6 × 108 s−1. Intense phosphorescence spectra at 77 K with lifetimes in the second range (τ = 0.6–1.3 s) are recorded for the monomers, whereas for the bis-imidazo[1,2-a]pyridines, phosphorescence spectra are detected only after enhancing intersystem crossing by heavy atoms. Non-linear optical properties of bis-imidazo[1,2-a]pyridines revealed two-photon absorption cross-sections (σ2) typically of the order of 500–800 GM. The remarkable fluorescence quantum yield, in combination with high a two-photon absorption cross-section, generated a two-photon brightness of ∼500 to 750 GM units within the biological window (700–800 nm).

Fluorescence Fluctuation Spectroscopy of Ternary Protein Interactions in Living Cells

Because many proteins contain multiple interaction sites, the formation of ternary or higher-order protein complexes are frequently encountered and play a critical role in many cellular processes. Fluorescence fluctuation spectroscopy (FFS) characterizes the brightness of fluorescently-labeled proteins, which provides a quantitative and noninvasive method to characterize protein interactions in living cells. Here we focus on extending two-photon brightness microscopy to the characterization of three interacting protein species, with each species carrying a differently colored tag. The fluorescence signal is split into three detection channels by optical filters. We discuss the choice of tags and filters that optimize the signal-to-noise ratio of resolving species, and the influence of spectral cross-talk on data analysis by time-integrated cumulants of the photon counts. While proof of principal experiments with dyes are useful, our main challenge remains the limited choice of fluorescence proteins that have simple photophysics and good color separation for cellular applications. We characterize different combinations of fluorescent proteins and evaluate their potential for three-color FFS studies of ternary protein interactions. This work has been supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (PHY 0346782).