An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy.

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in cellular media due to strong cross-talk between energetically separated detection channels.

Sensitized luminescence from water-soluble LaF3:Eu nanocrystals via partially-capped 1,10-phenanthroline: time-gated emission and multiple lifetimes.

Water dispersible citrate-capped LaF3:Eu(5%) nanocrystals (NCs) have been partially surface-functionalized by 1,10-phenanthroline (phen) via a ligand exchange method to produce novel water dispersed citrate/phen-capped LaF3:Eu(5%) NCs in which citrate ligands preserve the water dispersibility of the NCs and phen ligands act as sensitizers of surface Eu(3+)-dopant sites. The partial ligand exchange and the formation of water dispersed NCs have been monitored by (1)H NMR spectroscopy, as well as luminescence measurements at different time intervals during the reaction. These NCs display a distinct phen-sensitized Eu(3+)-emission profile with enhanced intensity in water as compared to the emission profile and intensity obtained upon direct excitation. Time-resolved (or time-gated) emission spectroscopy (TRES) has been used to probe PL dynamics of Eu(3+)-sites of LaF3:Eu(5%) NCs by taking advantage of selectively sensitizing surface Eu(3+)-dopant sites by phen ligands as well as by exciting all the Eu(3+)-sites in the NCs upon direct excitation. TRES upon direct excitation of the citrate-capped LaF3:Eu(5%) NCs reveals that Eu(3+)-dopants occupy at least three different sites, each with a different emission profile and lifetime, and emission from purely interior Eu(3+)-sites has been resolved due to their long lifetime as compared to the lifetime of purely surface and near surface Eu(3+)-sites. In contrast, the phen-sensitized emission from citrate/phen-capped LaF3:Eu(5%) NCs displays similar emission profiles and lifetimes in TRES measurements, which reveal that phen truly sensitizes purely surface dopant sites of the NCs in water, all of which have nearly the same local environment. The phen-sensitized Eu(3+)-emission of the NCs in water remains stable even upon addition of various buffer solutions at physiological pH, as well as upon addition of water-miscible organic solvents. Furthermore, the two-photon excitation (λex. = 720 nm) of these water-soluble phen-capped NCs produces bright red Eu(3+) emission, which reveals that these NCs are promising for potential applications in biological imaging.

Colloidal CsPbBr3 Perovskite Nanocrystals: Luminescence beyond Traditional Quantum Dots.

Traditional CdSe-based colloidal quantum dots (cQDs) have interesting photoluminescence (PL) properties. Herein we highlight the advantages in both ensemble and single-nanocrystal PL of colloidal CsPbBr3 nanocrystals (NCs) over the traditional cQDs. An ensemble of colloidal CsPbBr3 NCs (11 nm) exhibits ca. 90 % PL quantum yield with narrow (FWHM=86 meV) spectral width. Interestingly, the spectral width of a single-NC and an ensemble are almost identical, ruling out the problem of size-distribution in PL broadening. Eliminating this problem leads to a negligible influence of self-absorption and Förster resonance energy transfer, along with batch-to-batch reproducibility of NCs exhibiting PL peaks within ±1 nm. Also, PL peak positions do not alter with measurement temperature in the range of 25 to 100 °C. Importantly, CsPbBr3 NCs exhibit suppressed PL blinking with ca. 90 % of the individual NCs remain mostly emissive (on-time >85 %), without much influence of excitation power.

Europium-doped LaF3 nanocrystals with organic 9-oxidophenalenone capping ligands that display visible light excitable steady-state blue and time-delayed red emission.

Visible light excitable and color tunable ∼5% Eu(3+)-doped LaF3 nanocrystals (NCs), containing 9-oxidophenalenone ligands bound to the surface as visible light sensitizers for Eu(3+) dopants, have been synthesized by a facile solution-based method. The crystalline phase structure, size, composition, morphology and luminescence properties of the NCs are characterized using X-ray diffraction, high-resolution transmission electron microscopy, energy dispersive X-ray spectroscopy, and steady-state and time-resolved emission spectroscopy. The results show that these NCs are very small in size (<10 nm), display high degree of crystallinity and have pure tysonite structure of LaF3 with P3[combining macron]c1 space group. The visible light excitation of the capping ligands triggers an alternate display of steady-state, short-lived blue emission (τ < 1 ns) and time-delayed, long-lived sensitized red Eu(3+) emission (τ = 0.41 ms), allowing photoluminescence chromacity tuning as a function of delay time within a specific inorganic composition. The visible light sensitization of the dopant Eu(3+) sites proves more efficient than direct excitation of 5% Eu(3+)-doped LaF3 NCs capped by citrate ligands. The dopant Eu(3+) ions are well protected from non-radiative deactivation through high-energy vibrations of the organic capping ligands which is proved by the long lifetime of the sensitized Eu(3+) emission. The time-resolved emission spectra collected over a period of several milliseconds reveal that the dopant Eu(3+) ions occupy at least three different sites in the NC host. It is further inferred that the sensitized Eu(3+) emission primarily comes from surface dopant sites and sites just underneath the surface of the NCs. We propose that some of the interior Eu(3+) sites also display sensitized emission, which are indirectly populated via Eu(3+) → Eu(3+) energy migration from surface-sensitized Eu(3+) sites of the NCs.

The correlation between f-f absorption and sensitized visible light emission of luminescent Pr(III) complexes: role of solvents and ancillary ligands on sensitivity.

The electronic absorption, excitation and sensitized visible light emission studies of three praseodymium (III) complexes: [Pr(fod)(3)(bpy)], [Pr(fod)(3)(phen)] and [Pr(fod)(3)(bpm)](n) (fod = anion of 6,6,7,7,8,8,8- heptafluoro-2,2-dimethyl-3,5-octanedione; bpy=2,2'-bipyridyl, phen=1,10-phenanthroline, bpm=2,2'-bipyrimidine) in a series of non-aqueous solvents is presented. The f-f absorption transitions of Pr(III) are environment sensitive which is reflected by the change in the intensity (oscillator strength) and band shape (stark splitting) upon change in the solvent and/or the ligands. The sensitization of intense Pr(III) emission, in the visible region, of the complexes in solution upon excitation into the ligand centered π→π* absorption band is remarkable. The planar phen has pronounced impact and increases considerably the emission intensity of Pr(III) luminescence than the flexible bpy while bpm has been found least effective in promoting the emission intensity. The intensity of the f-f absorption and sensitized emission are correlated with the nature of the solvents. The donor solvent pyridine enhances the emission intensity of the [Pr(fod)(3)(phen)] drastically and of [Pr(fod)(3)(bpy)] marginally while the luminescent intensity of [Pr(fod)(3)(bpm)](n) is decreased. The combined photophysical studies demonstrate that entry of the solvent molecule(s) to inner coordination sphere (complex-solvent interaction) is governed by the structure and basicity of the ancillary heterocyclic ligand attached to the Pr(III) complex. The strong donor DMSO transforms the three complexes into a similar species, [Ln(fod)(3)(DMSO)(2)], which results in similar electronic absorption and emission properties of the complexes in this solvent. The results demonstrate that highly luminescent praseodymium chelates can be designed with ligands containing suitable energy levels and their emission properties can be further modulated through suitable ancillary ligands and donor solvents, thus opening perspectives for applications in electroluminescent devices and luminescent probes.

Hypersensitivity in the luminescence and 4f-4f absorption properties of mono- and dinuclear EuIII and ErIII complexes based on fluorinated ß-diketone and diimine/ bis-diimine ligands.

The results of our investigation on the sensitized luminescence properties of three Eu(III) ß-diketonate complexes of the form [Eu(2)(fod)(6)(µ-bpm)], [Eu(fod)(3)(phen)] and [Eu(fod)(3)(bpy)] and 4f-4f absorption properties of their Er(III) analogues ( fod = anion of 6,6,7,7,8,8,8- heptafluoro-2,2-dimethyl-3,5-octanedione, bpm = 2,2'-bipyrimidine, phen = 1,10-phenanthroline and bpy = 2,2'-bipyridyl) in a series of non-aqueous solvents are presented. The Eu(III) complexes are highly luminescent and their luminescence properties (intensity and band shape) are sensitive to the changes in the inner coordination sphere of the Eu(III) ion. The luminescence intensity of the mononuclear complexes in pyridine is drastically decreased. The coordination structure of the complexes in pyridine is transformed into a more symmetrical one which results into a slow radiative rate of the emission from the complexes. The ancillary ligands, phen and bpy are found better co-sensitizers as compared to the bpm to sensitize Eu(III)-luminescence. The 4f-4f absorption properties (oscillator strength and band shape) of the Er(III) complexes demonstrate that (4)G(11/2) ← (4)I(11/2) and (2)H(11/2) ← (4)I(15/2) hypersensitive transitions of Er(III) are very sensitive in some coordinating solvents which reflects complex-solvent interaction in solution. The hypersensitive transitions of [Er(fod)(3)(phen)] remain unaffected in any of the solvents and this complex retains its bulk composition in solution. The erbium complexes as well as the Er(fod)(3) chelate are invaded by DMSO. This solvent enters the inner coordination sphere by replacing heterocyclic ligand and the complexes acquire similar structure [Er(fod)(3)(DMSO)(2)] in this solvent. The results reveal that the luminescence and absorption properties of lanthanide complexes in solution can be controlled by tuning the coordination structure through ancillary ligands and donor solvents. This work shall prove useful in designing new biological applications with such probes.