El teatro en la escuela, tres tendencias en su enseñanza

ticles we expect a spatial correlation between the nanoparticles and the fluorescence signal. This correlation is shown by superimposing TEM images and widefield fluorescence images of the same region of a dried MPD-SiO2 sample (Figure 5.3).

To prepare the sample, the MPD-SiO2 nanoparticles were dispersed in methanol. Fluorescent

polymer beads (175±5 nm, Invitrogen; Karlsruhe, Germany) were added as spatial markers as they

are visible both in widefield and TEM microscopy and therefore enable the recognition of the same sample region by both techniques. The suspension was transferred onto an optical transparent 25 nm thick Si3N4membrane (Plano) which is suitable as sample carrier for widefield fluorescence imaging

and TEM imaging. After air drying, the membrane was mounted onto the widefield fluorescence microscope. Fluorescence images of the perylene species and the fluorescent polymers beads were taken by alternated excitation of the sample with 633 nm and 488 nm laser light respectively. The fluorescence was collected via a 60x Nikon Plan APO water immersion objective in epifluorescence mode. The consecutive illumination together with separation of fluorescence emission below and above 645 nm in separate detection channels allowed distinguishing the fluorescence signal of the MPD-SiO2 nanoparticles and the fluorescent polymer beads. The images were captured with an electron multiplier charge coupled device camera (iXon DV884, Andor Technology, Belfast, UK). After fluorescence imaging, the membrane was air-dried and transferred to the TEM (JEM 2011). The region examined by fluorescence microscopy was localized via the polymer beads. As the resolution of TEM images is approximately four orders of magnitude better than the resolution of fluorescence widefield images, a region of interest within the whole fluorescence image was chosen (Figure 5.3 a) and multiple, partly overlapping TEM images were acquired within this region. The single TEM images were combined and a map of the region was generated (Figure 5.3 b) utilizing the CorelDraw software (Corel Cooperation). The superposition of the fluorescence images and the TEM map reveals the correlation between nanoparticles and fluorescent dye (Figure 5.3 c). Regions with nanoparticles clearly show fluorescence signals. The fluorescence intensity directly correlates with the amount of nanoparticles visible, whereas regions without nanoparticles show no fluorescence. Regions with very high fluorescence intensities were not mapped by TEM due to the high number of nanoparticles in these regions.

These measurements show that the MPD has attached to or incorporated into the silica nanopar- ticles.

5.4 Fluorescence emission spectra

Information about the fluorescence characteristics of the silica nanoparticles and the free dyes was obtained by looking at their fluorescence emission spectra. As shown in Figure 5.4, the emission spectra of the free dyes and the nanoparticles have the same characteristic shape whereas the fluorescence maxima are dependent on the solvent and/or the nanoparticle type. The solvents were chosen according to solubility. The reactive perylene dyes MPD and BPD are soluble in ethanol and methanol but not in pure water. Therefore the spectra of MPD and BPD were measured in methanol and a mixture of methanol:water = 2:1. As illustrated in Figure 5.4 a) (MPD) and 5.4 b)

Figure 5.4: Normalized fluorescence spectra of the functionalized dyes a) MPD and b) BPD and of the syn-

thesized nanoparticles c) MPD-SiO2 and d) BPD-SiO2, as well as e) core-shell nanoparticles, resulting from excitation with 488 nm. The spectra were measured in water, methanol, or mixtures of both, respectively.

5.5 Fluorescence anisotropy

(BPD) the fluorescence maximum responds to solvent polarity. With increasing solvent polarity from methanol to methanol:water = 2:1 the fluorescence maxima shift from 537 nm to 541 nm (MPD) and from 535 nm to 539 nm (BPD) respectively. This solvent polarity dependent shift to higher wavelengths is caused by the higher stabilization of the S1 excited state compared to the S0 ground state in polar solvents. It shows that perylene has a stronger dipolar moment in the excited state than in the ground state, resulting in the decrease in energy of S1.

Unlike the free dyes, the labeled nanoparticles can be dispersed in pure water leading to a fluores- cent suspension. This is another proof that the dye molecules have attached to the nanoparticles. The solubility of nanoparticles in water or aqueous solutions like cell media is an important require- ment for live-cell imaging as organic solvents and alcohol are toxic for cells. The fluorescence spectra of MPD-SiO2and BPD-SiO2 were measured in methanol:water = 1:1 and water. The attachment

of MPD or BPD to the nanoparticles does not change the shape of the fluorescence emission bands of the dyes (Figures 5.4 c and 5.4 d). Similar to the free dye in solution, the increase of solvent polarity leads to a fluorescence maximum shift to longer wavelengths. MPD-SiO2shifts from 550 nm

to 554 nm and BPD-SiO2 from 539 nm to 550 nm. This strongly indicates that the dye molecules

of MPD-/BPD-SiO2 are in contact with the solvent and therefore mainly located at the surface of

the particles.

The core-shell nanoparticles behave differently. Compared to all spectra shown above, the fluores- cence emission spectra of perylene-core nanoparticles is shifted approx. 20 nm to shorter wavelengths in water and methanol:water = 1:1. Note that no solvent dependent shift is present in the spectra of the core-shell nanoparticles (Figure 5.4 e). This leads to the conclusion that the dyes are located inside the core of the nanoparticle and therefore shielded from the solvent by the silica shell. Under these conditions the local environment of the perylene molecules consists mainly of silica and is therefore different from the aqueous environment of MPD-/BPD-SiO2 . This results in the 20 nm

hypsochromic shift of the fluorescence maxima described above.

5.5 Fluorescence anisotropy

To confirm the localization of the dye inside or on the surface of the nanoparticle, we studied the ability of the dye to rotate in solution. Dye molecules that are incorporated into the structure of the nanoparticle are expected to be less flexible than dye molecules that are attached to the surface of the nanoparticle via a linker. The rotation of dye molecules is linked to an angular displacement of their absorption and emission dipole moments which lie along specific directions within their structure. This leads to a directional dependence of the polarization of the fluorescence, the fluorescence anisotropy. To probe the fluorescence anisotropy, the sample is excited with linearly polarized light. The fraction of molecules within the sample which happen to have their transition dipole moment aligned in parallel to the electric field vector of the light is excited preferentially resulting in a partially oriented excited state population. During the timeframe between excitation and subsequent emission of light, the dye molecules can rotate and the excited state population therefore partially looses its parallel alignment. The intensity of the emitted light is measured in

Figure 5.5: Fluorescence anisotropy data of BPD, as an example for a free dye, and of the various la-

beled nanoparticles depending on solvent polarity. The anisotropy values of the unbound perylene dye BPD (squares), BPD-SiO2 (triangles), MPD-SiO2 (circles), and core-shell nanoparticles (stars) in different solvents are shown (see legend).

5.5 Fluorescence anisotropy

parallel and in perpendicular orientation with respect to the polarization of the excitation light. The normalized difference between these intensities is called the anisotropy r (see equation 3.3.1) and mirrors the flexibility of the dye. For details please see Section 3.3.

The lowest anisotropy to be expected of the dyes was estimated by measuring the anisotropy of free BPD as an example for free perylene. According to solubility we determined the anisotropy in varying methanol:water mixtures with increasing polarity; ranging from pure methanol to

methanol:water = 3:2. The resulting anisotropy values of BPD are below 0.05 (Figure 5.5, squares) in all applied solvents. These values are typical for the free motion of dye molecules in solution. Nevertheless the anisotropy of BPD fluorescence increases with increasing solvent polarity. The latter is accompanied by an increase in solvent viscosity, explaining the gradually diminished mobility of BPD.

The anisotropy values of BPD-SiO2 nanoparticles (Figure 5.5, triangles), MPD-SiO2nanoparticles

(Figure 5.5, circles) and the core-shell nanoparticles (Figure 5.5, stars) were determined in water and methanol:water = 1:1. All nanoparticles show anisotropy values above 0.36 when dissolved in water (filled symbols). This indicates that the dye molecules are rather immobile. As perylene is insoluble in water we think that the dye is sticking tightly to the surface of the nanoparticles leading to a comparably slow movement.

The anisotropy values change drastically when the nanoparticles are dissolved in a 1:1 mixture of methanol and water. The anisotropy of BPD-SiO2 nanoparticle fluorescence decreases strongly

to a value of 0.25 (Figure 5.5, half filled triangle) in response to the solvent polarity variation and the thereby increased solubility of perylene. We think this strong drop of anisotropy can only be explained if BPD is mainly located on the surface on the nanoparticles and has eventually formed dye chains by condensation of several BPD molecules. The movement of these chains is less restricted by the attachment to the surface of the nanoparticle than are single dye molecules. Thus the fluorescence anisotropy of the BPD-SiO2 nanoparticles responds strongly to the improved perylene

solubility.

In the case of MPD-SiO2 nanoparticles (Figure 5.5, circles), the anisotropy values are less af-

fected by the water to methanol:water = 1:1 solvent change than BPD-SiO2. As MPD has only

one reactive site the formation of dye chains is impossible. Therefore all MPD molecules are at- tached to the nanoparticles via a very short linker which inhibits strong movements of the dye molecules. The decrease of the fluorescence anisotropy is not very pronounced but it is still twice as much as the corresponding fluorescence anisotropy decrease of the core-shell nanoparticles (Fig- ure 5.5, stars). Therefore we think that the MPD molecules are mainly located on the surface of the MPD-SiO2 nanoparticles.

Concerning the core-shell nanoparticles (Figure 5.5, stars), the influence of the solvent change from water to methanol:water = 1:1 is even less than for MPD-SiO2 nanoparticles. This supports