As mentioned above, polystyrene (PS) beads were used as markers for the co-localization of the single-molecule trajectories and TEM images. The beads must be small, to pre- vent a strong influence on the porous system within the thin films, but big enough to be well observable in the white light transmission images of the wide-field microscope. A size of about280nm was found to be optimal. Figure 6.4a shows a side-view scanning
Figure 6.4: Scanning electron micrographs (SEM) of the coated Si3N4 membrane. (a) Side
view of the film. At this position, near the edge of the membrane, the mesoporous film is about
200nm thick and the concentration of PS beads is too high for single-molecule tracking. (b) Plan-view of an intermediate region, close to the centre of the membrane. The concentration of PS beads is well suited for single-molecule measurements and the retrieval of identical regions in TEM and optical microscopy. (c) Plan-view at higher magnification of the region marked with a rectangle in (b). Courtesy of A. Zürner, Bein group, LMU Munich.
electron micrograph (Phillips XL40 ESEM scanning electron microscope) of the film. The polystyrene beads are embedded into the film and covered with a thin layer of mesoporous silica, i.e. that they are enclosed by the mesoporous film. At this position the mesoporous silica film on the 30nm Si3N4 membrane is about 200nm thick. This image was taken in a region close to the edge of the Si3N4 membrane window, there- fore the concentration of beads is too high here for single-molecule tracking and the mesoporous film is too thick for TEM imaging. As the coating solution was deposited into the interior of the window, the solution was accumulated at the edges during spin-coating and therefore the film was thicker in this region. In the centre region of the membrane the concentration of PS was in the perfect regime for the overlay of single-molecule trajectories from optical microscopy and detailed electron microscopy pictures of the structure. Panels (b) and (c) of Figure 6.4 show images taken in this re- gion, with around three to five particles per10µm2. In this region the film was found to be thin enough for both TEM and optical microscopy (see below).
All measurements with the optical microscope were done prior to the electron mi- croscopy, as the ultra-high vacuum in the TEM will probably evaporate partly the sol- vent out of the pores and therefore change the environment in which the molecules diffuse. In order to obtain an overview of the bead concentration, and thus the cor- rect film thickness, three edges of the membrane window and the centre region with lowest PS bead concentration were mapped in white light transmission images of
62.5µm×62.5µm prior to single-molecule fluorescence imaging. These images were overlaid using automatic image cross correlation in Adobe Photoshopr(Version CS2) by the ’Photomerge’ tool resulting in the map shown in Figure 6.5. The black square indicates the size of one of these images. In panel (b) an extract of the map of white- light transmission images is shown. At higher magnification a distinct pattern of
Figure 6.5: White light transmission and TEM images of the mesoporous film, showing the embedded polystyrene beads. (a)Map of white light transmission images of the whole membrane window (500µm × 500µm). The size of one white light transmission image is
62.5µm×62.5µm (dashed square). (b)Zoom of the white light transmission map, showing the well separated PS beads in the region marked by the orange box in (a).(c)Magnification of a region with a distinct structure. (d)Map of TEM images at low resolution (LM 50); the size of one image is indicated by the white, dashed square. The image is rotated that the central region, with the right PS concentration and film thickness is at the same orientation as in (a). (e)Medium resolution TEM (LM 200) of the region highlighted with the yellow box in (d). (f) Magnification of the same characteristic formation of beads as in (c).
polystyrene beads can be resolved (c). Subsequent to optical microscopy, the sample was analysed with TEM. Again, the first step was mapping of the whole membrane with low resolution (50×), depicted in Figure 6.5d. This image was used to obtain an estimate of the orientation of the sample in the electron microscope with respect to the wide-field images. In this figure the image is already rotated such that the two pictures have the same relative orientation. The thin central region was then mapped at a mag- nification high enough to distinguish single PS beads (200×), shown in panel (e). On closer inspection of the white-light transmission image (b) and the intermediate reso- lution TEM (e), the identical arrangement of PS beads could be found in both. A zoom on this pattern is depicted in panels (c) and (f). Starting from this pattern, identical re- gions anywhere on the membrane window with suitable PS bead concentration could be recognized in wide-field and TEM images.
The whole centre region was thus investigated first by wide-field fluorescence mi- croscopy and single-molecule tracking. The regions in which highly structured single- molecule trajectories could be found were subsequently mapped with high resolution TEM images. This procedure is described in the following paragraphs.
6.3 Single-Molecule Tracking
For the single-molecule experiments the fluorescent dye molecules were excited with a HeNe Laser at633nm, and movies of up to 1000 images were recorded to follow their diffusion. Since the films were much thinner than the focal depth of the microscope objective used (> 1µm), images contain data from molecules at all heights inside and on the surface of the sample, and the molecules remained in focus during the complete duration of the movie. Image series were acquired with temporal resolutions of100ms,
200ms or400ms per frame, but finally an exposure time of200ms was found to be most appropriate for single-molecule tracking. As the Si3N4 membranes give a relatively high and inhomogeneous background signal, the background was subtracted frame- to-frame from the movies prior to tracking of the molecules.
One exemplary fluorescence image from a movie is shown in Figure 6.6a. The white square boxes indicate the regions that were subsequently mapped with TEM images. As described for the sol-gel samples in the previous chapter, fitting the positions of the molecules from frame to frame resulted in the single-molecule trajectories. In order to overlay these trajectories on the TEM images, the positions of the polystyrene beads had to be determined from the same movie. Therefore the laser shutter was closed after
Figure 6.6: Wide-field microscopy images.(a) One exemplary fluorescence image of the wide- field movie, which is an extract from a complete30µm×30µm image. The molecules whose trajectories are depicted in Figures 6.10 and 6.12 are highlighted by an arrow. (b) Sum of the last 240 white light transmission images of the film, where the laser was shut. The PS beads are visible as black circles. The white square boxes in both images indicate the regions that were subsequently mapped with TEM. The upper square box indicates the region that was mapped with TEM images for Figure 6.10 and the lower one the region of the trajectory in Figure 6.12.
imaged in transmission under white light illumination. Their positions were fitted in inverted images with the same gaussian fit routine as the single molecules. Mean and standard deviation of the fitted PS bead positions were calculated and tabulated. Fig- ure 6.6b shows the sum of all the white light transmission images that were recorded at the end of the movie. As there are no diffusing particles in those images, summing up all the images increases the image contrast and the PS beads can be better visualized.