3D visualization of additive occlusion and tunable full-spectrum fluorescence in calcite

Abstract

From biomineralization to synthesis, organic additives provide an effective means of controlling crystallization processes. There is growing evidence that these additives are often occluded within the crystal lattice. This promises an elegant means of creating nanocomposites and tuning physical properties. Here we use the incorporation of sulfonated fluorescent dyes to gain new understanding of additive occlusion in calcite (CaCO₃), and to link morphological changes to occlusion mechanisms. We demonstrate that these additives are incorporated within specific zones, as defined by the growth conditions, and show how occlusion can govern changes in crystal shape. Fluorescence spectroscopy and lifetime imaging microscopy also show that the dyes experience unique local environments within different zones. Our strategy is then extended to simultaneously incorporate mixtures of dyes, whose fluorescence cascade creates calcite nanoparticles that fluoresce white. This offers a simple strategy for generating biocompatible and stable fluorescent nanoparticles whose output can be tuned as required.

Figure 1. GREEN/calcite host–guest composites.

(a,f,k) Representative SEM micrographs of GREEN/calcite composite crystals precipitated under the conditions (a–e=[Ca²⁺]=[CO₃²⁻]=25 mM and [GREEN]=0.1 mM; f–j=[Ca²⁺]=[CO₃²⁻]=5 mM and [GREEN]=0.1 mM; and k–o=[Ca²⁺]=[HCO₃⁻]=3.5 mM and [GREEN]=0.1 mM). (b,g,l) Three-dimensional (3D) representation of the morphologies of the crystals imaged in c,h,m, with approximate faces labelled, and the growth sectors coloured in green. The + and − labels denote obtuse and acute step morphologies, respectively. (c,h,m) Confocal fluorescence micrographs of composite crystals grown under the conditions. (d,i,n) Orthogonal views (XY, YZ and XZ) of composites obtained from z-stacked confocal micrographs, showing the distribution of dye in 3D (z+ direction is away from the substrate, XY is the imaging plane) Colour scale: blue (low intensity) to red (high intensity); black, no signal; White, detector saturation. (e,j,o) Line profiles (e) and intensity histograms (j,o) corresponding to the lines or regions shown on the orthogonal view images in (d,i,n) respectively. Scale bars, 15 μm (a); 10 μm (f); 25 μm (k); 20 μm (c,h,m).

Figure 2. Three-dimensional distribution of GREEN in GREEN/calcite composites.

SEM (a) and optical (b) micrographs of a GREEN/calcite composite grown under conditions [Ca²⁺]=[CO₃²⁻]=5 mM and [GREEN]=0.1 mM oriented with crystallographic c axis on a glass slide. (c,d,e) z-stacks obtained by fluorescence confocal microscopy rendered into three dimensions (3D) with accompanying model. The rendered 3D model was viewed as shown in the optical image (c), orthogonally to {104} face (d) and down the crystallographic a axis (e). The occupied zones dominated by growth at acute steps are shaded green on modelled images, and correspond well to confocal images. Scale bars, 20 μm (a–e).

Figure 3. Changing GREEN distribution with changing initial [Dye].

(a,f) Representative SEM micrographs of GREEN/calcite composites grown from different conditions (a–e=[Ca²⁺]=[HCO₃⁻]=3.5 mM and [GREEN]=0.01 mM; f–j=[Ca²⁺]=[HCO₃⁻]=3.5 mM and [GREEN]=0.2 mM). (b,g) Three-dimensional (3D) representation of composites as imaged by CFM, with approximate faces labelled, and growth sectors coloured in green. (c,h) Confocal fluorescence micrographs of composites from the same conditions. (d,i) Orthogonal views images (XY, YZ and XZ) of composites obtained from z-stacked confocal micrographs effectively detailing the distribution of dye in 3D (z+ direction is away from the substrate, XY is the imaging plane). Colour scale: blue (low intensity) to red (high intensity); black, no signal; white, detector saturation. (e,j) Image analyses in the form of line profiles (e) and intensity histograms (j) corresponding to lines or regions as denoted on orthogonal view images in (d,i), respectively. Scale bars, 20 μm (a,f,c,h).

Figure 4. Effect of occlusion on photophysical properties of fluorescent dyes.

(a) Excitation (dotted) and emission (solid) spectra for aqueous solutions of BLUE (blue), GREEN (green) and RED (red). (b–d) Emission spectra of dye/calcite composites of different initial concentrations of dyes in [Ca²⁺]=[CO₃²⁻]=5 mM experiments (b) BLUE, (c) GREEN and (d) RED. For clarity, the spectra deriving from different starting concentrations of dyes are indicated in the legends.

Figure 5. FLIM analysis of GREEN/calcite composites.

Samples prepared under (a–d) [Ca²⁺]=[CO₃²⁻]=10 mM and (e–h) 2.5 mM conditions were characterized by (a,e) optical and (b,f) CFM. (c,g) FLIM analysis of the same plane as that in the confocal image revealed regions of differing fluorescence lifetime. (d,h) Global fluorescence decays obtained from the regions of interest labelled in (c,g) yielded fluorescence lifetimes (d) 1=4.1 ns and 2=3.2 ns, and (h) 1=2.9 ns, 2=2.3 ns and 3=3.1 ns. Scale bars, 10 μm (a–c,e–g).

Figure 6. Direct comparison of local GREEN environment by FLIM.

(a) Optical micrograph of ACC after extended exposure to reaction solution. (b) CFM reveals calcite embedded amongst bulk ACC, with greater fluorescence intensity associated with the crystalline phase compared with the amorphous. (c) FLIM micrograph revealing differences in fluorescent lifetime between crystalline (i and ii) compared with amorphous (iii). (d) Global fluorescence decays obtained for regions of interest i (black), ii (red) and iii (blue) in c yielding fluorescence lifetime τ=3.9, 3.9 and 5.2 ns, respectively. Scale bars, 20 μm (a–c).

Figure 7. White calcite.

(a) A mixture of blue, green and red light emitted from crystals containing BLUE, GREEN and RED, respectively, would yield a full-spectrum white light. (b) The fluorescence cascade concept (i), where ultraviolet radiation excites a blue fluorescent dye, which in turn excites a green fluorescent dye, which finally excites a red fluorescent dye. With appropriate proportions of each dye, an equal intensity of blue, green and red light will be emitted, yielding white light. (c) Optical and confocal fluorescence micrographs of a sample calcite/dye composite containing all three dyes. (d) z-stack of confocal fluorescence micrographs rendered in three dimensions at obtained and rotated through 90° along y axis; Orthogonal views plot of calcite containing all three dyes and line profiles of each colour as denoted.

Figure 8. Fluorescent dye/calcite composite nanoparticles.

(a) Representative TEM micrograph of calcite nanoparticles occluding GREEN and selected-area electron diffraction pattern (inset). (b) Powder X-ray diffraction analysis of calcite nanoparticles demonstrating wide-line broadening associated with small crystalline domain size (τ=53 nm). (c–f) photographs of ethanolic suspensions of fluorescent calcite nanoparticles containing (c) BLUE, (d) GREEN, (e) RED and (f) dye mixture under normal light (top) and ultraviolet light (365 nm, bottom). (g) Emission spectra of ethanolic suspensions of calcite nanoparticles occluding BLUE (blue, λ_ex=360 nm), GREEN (green, λ_ex=430 nm), RED (orange, λ_ex=512 nm) and dye mixture (black, λ_ex=360 nm; section at ∼700–760 nm removed due to signal from excitation light at 2λ_ex). Scale bar, 100 nm (a).