Tracing of microtubules in fixed fibroblasts

Actin filaments in vitro, as presented in the previous subsection, are a filamentous structure, but do not come with the typical challenges of cell imaging: The density of the structure can be adapted, since it is governed by the chosen dilution during preparation, and the labeling back-ground is negligible. This is caused by the absence of other structures as well as the restriction of the actin filaments to a thin layer only. Also the properties of the SiR-actin dye are beneficial

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0 50 Figure 4.19:Tracing ofα-tubulin in fixed fibroblasts (dye: Abberior STAR 635P): (a) In the confocal overview, acquired withPexc = 1.19µW, a pixel size of 200 nm and pixel dwell time of 10µs, the area chosen for the closeup in (b), imaged withPexc= 3.4µW, a pixel size of 20 nm and pixel dwell time of 30µs, is indicated. The starting position and direction for the filament tracing are indicated in (b), with the results depicted in (d-i). The acquisition parameters are chosen asP_exc= 10.7µW,P_STED= 35 mW, a pixel size of 20 nm and a pixel dwell time of 10µs per pattern direction. The tracing is performed for 19 pixels per line for blocks of 5 lines each. (c) Maximum-value reconstruction of the raw data in (d-i).

The scale bar indicates a length of 10µm in (a) and 1µm in all other images.

in this case: It specifically binds to F-actin and increases its fluorescence intensity more than 100-fold [LRD⁺14] upon binding, yielding a highly specific staining with low labeling back-ground. Especially the low background signal obviously facilitates imaging and in particular filament tracing which relies on detecting differences in signal. Nevertheless, the same tracing algorithm is shown to successfully trace filaments also in the challenging conditions offered by the cell cytoplasm environment.

Figure 4.19(a) shows a confocal overview of microtubules stained in fixed fibroblasts. The area chosen for the closeup in figure 4.19(b) as well as for the underlying grid for the tracing algo-rithm is highlighted. From the chosen starting position, the tracing is performed over several µm, with the results displayed in figure 4.19(d-i) for the six different depletion pattern orienta-tions. The corresponding maximum-value reconstruction is shown in figure 4.19(c). The band scanned around the structure of interest is again clearly visible, and the algorithm correctly identifies the course of the filament despite the noticeable background (cf. figure 4.19(b)), proving its suitability for the imaging of biological structures.

As has been shown so far, the employed algorithm correctly traces the desired structure, but no conclusion about the quality of the resulting image or the obtainable resolution was drawn.

For this, the results of the tracing algorithm are compared to a conventional tomoSTED ac-quisition, as presented in subsection 4.1.2. Figure 4.20 depicts such a comparison of both acquisition modes. In figure 4.20(a), a confocal scan is displayed, highlighting a line pro-file employed for comparing the resolution between the reconstructed tomoSTED images (b) and (c) and the reconstructed tracing images (e) and (f) employing maximum-value recon-struction and Richardson-Lucy deconvolution, respectively. The profile plot in figure 4.20(d), obtained by averaging over five neighboring lines, yields the same observed structure size in the maximum-value reconstruction for both the tomoSTED acquisition and the filament tracing.

The comparison of the resulting reconstructed images does not present any sign of artifacts or distortions caused by the selective scanning performed during the tracing routine.

Hence, this tracing method successfully yields not only a localization of the desired structure, but also enables super-resolution imaging without the need of scanning the whole field of view.

Two further examples of the tracing algorithm are displayed in figure 4.21(a-d) and (e-h). In figure 4.21(a) and (e), a confocal overview of the cell is shown, indicating the areas for the close-ups in (b) and (f), respectively, with the starting positions for the tracing algorithm highlighted therein. Both maximum-value reconstruction (cf. figure 4.21(c,g)) and Richardson-Lucy de-convolution (cf. figure 4.21(d,h)) of the raw data are displayed. In figure 4.21(c,d), the tracing algorithm is performed twice for two different starting positions within the same field of view, demonstrating the ability of iteratively scanning all structures of interest within a certain area.

However, a more elaborate decision algorithm needs to be employed at positions of crossing filaments as discussed in chapter 5 in order to avoid multiple scanning of the same filament.

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Figure 4.20: Comparison between tomoSTED acquisition and filament tracing. (a) Confocal scan of α-tubulin stained in fixed fibroblasts (dye: Abberior STAR 635P) acquired withP_exc= 2.0µW, a pixel size of 20 nm and pixel dwell time of 30µs. The scale bar indicates a length of 2µm. (b) Maximum-value reconstruction and (c) Richardson-Lucy deconvolution (10 iteration steps, αRch = 0.0001) of a standard tomoSTED acquisition with pixel-wise switching of six pattern orientations, acquired with Pexc= 3.4µW,PSTED = 48 mW, a pixel size of 20 nm and pixel dwell time of 10µs per direction. The same acquisition parameters are chosen for the filament tracing with the starting position indicated in (b). The tracing is performed for 19 pixels per line for blocks of 5 lines each. The maximum-value reconstruction and Richardson-Lucy deconvolution of the data acquired by the tracing algorithm are displayed in (e) and (f), respectively. Line profiles (averaged over five neighboring lines) of a filament, as indicated in (a), drawn for the confocal scan as well as the maximum-value reconstruction of both the standard tomoSTED acquisition and the tracing result are shown in (d).

Figure 4.21(c,d) moreover demonstrates that the algorithm can also handle short disruptions of the filament at the image border, simply continuing the scan hereafter.

The second example, depicted in figure 4.21(g,h), shows two further characteristics of the algo-rithm. First, the algorithm is designed to follow a single filament, meaning that the number of pixels per line is chosen to cover the width of a single filament. For two filaments next to each other, as identified by the STED acquisition in figure 4.21(g,h) and not immediately apparent from the confocal scan in figure 4.21(f), the scanned lines will be centered around one of the two filaments. This might leave the other one only partly scanned, as seen in the right part of figure 4.21(g,h). Further adaptations of the algorithm, as outlined in chapter 5, will resolve this issue, leading to a full scan of both filaments. The second characteristic is immediately visible considering the ending filament in the left part of figure 4.21(g,h). Since no signal is detected in the continuation of this filament, the algorithm will gradually yield a turn of the direction, causing a re-scan of the same filament in reverse. After returning to the crossing point, the tracing can continue along any other filament. In this way, also ending filaments can be correctly detected and scanned.

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Figure 4.21: Tracing of microtubules in fixed fibroblasts (dye: Abberior STAR 635P). (a) Confocal overview, acquired with Pexc = 1.19µW, a pixel size of 200 nm and pixel dwell time of 10µs, and (b) confocal closeup of the highlighted area, imaged with Pexc = 3.4µW, a pixel size of 20 nm and pixel dwell time of 30µs. The tracing algorithm is performed twice on this field of view for different starting positions, indicated in (b). The tracing is performed for 15 pixels per line for blocks of 7 lines each (starting position 1) and 19 pixels per line for blocks of 5 lines each (starting position 2), respectively. The corresponding results are displayed via the (c) maximum-value reconstruction and the (d) Richardson-Lucy deconvolution (10 iteration steps,α_Rch = 0.0001). The acquisition parameters are set asP_exc= 3.2µW,P_STED= 48 mW, a pixel size of 20 nm and pixel dwell time of 10µs per direction.

(e) Confocal overview of a different cell, acquired with the same parameters as for (a), and (f) confocal closeup of the highlighted area, imaged with the same acquisition parameters as chosen for (b). Tracing is performed for the starting position indicated in (b) for 19 pixels per line for blocks of 5 lines each, with the (g) maximum-value reconstruction and (h) Richardson-Lucy deconvolution of the raw data.

The acquisition parameters are set asPexc= 7.9µW,PSTED= 48 mW, a pixel size of 20 nm and pixel dwell time of 10µs per direction. The scale bar in (a,e) is set to 10µm, whereas in all other images it indicates a length of 2µm.