Imaging of microtubules in fixed Vero cells

The beforehand characterized depletion pattern is subsequently employed for imaging biological structures. Microtubules in Methanol-fixed Vero cells are stained with Abberior STAR 635P as fluorescent dye via antibody-staining. A confocal image is shown in figure 4.4(a) (lower right corner) with the scale bar indicating a length of 2µm. For a tomoSTED image, a decision on the number of pattern orientations for the acquisition needs to be taken based on the resolution enhancement as described in section 2.2. For a chosen STED laser power of 38 mW, a resolu-tion enhancement of k≈7 is expected, necessitating at least six different pattern orientations for an artifact-free Richardson-Lucy reconstruction (see figure 2.5 and the description thereof).

These six equally spaced pattern orientations are switched on a single-pixel level with a pixel dwell time of 5µs per direction, amounting to a total pixel dwell time of 30µs. By scaling the pixel dwell times of the sub-images, a comparable pixel dwell time as in the 2D case is ensured

[Krü17], enabling imaging at half of the light dose compared to conventional 2D STED mi-croscopy. Reordering the signal yields a sub-image per direction, as displayed in figure 4.4(c-h) on a subset of the whole field of view. A two-dimensionally super-resolved tomoSTED image (figure 4.4(a), upper left corner) is reconstructed from these sub-images by a Richardson-Lucy algorithm with 30 iteration steps and regularization parameter α_Rch = 0.0001. A zoom-in is shown in figure 4.4(b). The analysis of small fluorophore aggregations yields a width of less than 40 nm, which confirms the expected 7-fold resolution enhancement (data not shown).

This resolution is sufficient to resolve immunolabeled microtubule filaments. Their expected diameter amounts to approximately 60 nm, consisting of the size of the unlabeled microtubule of around 25 nm and the size of the antibodies [WRO78]. Indeed, an exemplary line profile perpendicular to a filament (figure 4.4(i)) shows a measured structure size of 58 nm.

In order to further reduce the light dose, either less pattern orientations or a shorter pixel dwell time can be employed. The number of pattern orientations is critical for an artifact-free reconstruction and is already reduced to the minimum possible value for Richardson-Lucy deconvolution. On the other hand, the collected signal is still relatively high, allowing a

re-(b)

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Figure 4.4: (a) Confocal image (lower right corner) and reconstructed two-dimensionally resolved STED image (upper left corner) ofα-tubulin in fixed Vero cells (fixed in MeOH, Abberior STAR 635P as fluorescent dye, embedded in Mowiol), based on measurements with six pixel-wise switched pattern orientations. The scale bar indicates a length of 2µm. Here, PSTED = 38 mW, Pexc = 3.2µW, the pixel dwell-time is set to pT = 5µs per pattern orientation and the pixel size to px = 20 nm. The reconstruction was performed by employing the Richardson-Lucy deconvolution with 30 iteration steps andα_Rch = 0.0001. (b) shows a close-up of the reconstructed STED image, with (c-h) showing the six sub-images for the six pattern orientations for the same area (scale bar 1µm). An exemplary line profile (averaged over 5 neighboring lines) of a filament for excitation and STED image as visualized in (b) is shown in (i), exhibiting a FWHM of 58 nm in the STED image.

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0 100 200 300 400 500 600

0.0 0.2 0.4 0.6 0.8 1.0

intensity (a.u.)

distance (nm)

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(a) 0 max (b) (c)

FWHM = 58nm y

Figure 4.5: (a) Confocal and (b) reconstructed tomoSTED image (Richardson-Lucy deconvolution with 30 iterations,αRch = 0.0001) ofα-tubulin in fixed Vero cells, based on 6 sub-images for different pattern orientations. The pixel size is set topx = 20 nm, the excitation power to Pexc = 2.5µW and the STED laser power toP_STED= 32 mW. The pixel dwell time is pT = 2µs per sub-image, resulting in a total effective pixel dwell time of only 12µs. The scale bar indicates a length of 2µm. (c) shows a line profile of a filament (averaged over 5 neighboring lines), yielding the same resolution enhancement as in figure 4.4.

duction of the pixel dwell time to 2µs per sub-image. In this way, a total pixel dwell time of 12µs can be realized, which is considerably less than typically employed under similar imaging conditions (cf. subsection 4.2.2). Thus, this yields a tomoSTED acquisition at a significantly reduced light dose compared to the 2D STED acquisition. Exemplary results on microtubules are displayed in figure 4.5. Figure 4.5(a) shows a confocal scan and figure 4.5(b) the respec-tive tomoSTED image. Six different pattern orientations at a pixel dwell time of 2µs each are switched on a pixel level and reconstructed employing the Richardson-Lucy deconvolution. The color bar is chosen individually for each image to cover the full dynamic range, while the scale bar is the same for both images and indicates a length of 2µm. A line profile, averaged over 5 neighboring lines, is displayed in figure 4.5(c) and indicates the same image FWHM as for the results presented in figure 4.4. Consequently, tomoSTED microscopy enables the acquisition of super-resolved images of uncompromised quality at a significantly reduced depletion light dose for a pixel-wise switching of the depletion pattern orientation.

4.2. FastRESCue

TomoSTED microscopy has successfully proven its ability to reduce the light dose imposed on the sample by a factor of 4 [Krü17]. As discussed in section 2.3, several other techniques have been developed in the recent years in order to minimize photodamage, with RESCue [SER⁺11]

being among them. This section is dedicated to the implementation of the novel FastRESCue

technique (see section 3.5), which enhances the performance of RESCue by not only lowering the light dose, but also reducing the acquisition time necessary to obtain a high-resolution image. First, the performance of the scan system introduced in section 3.5 is compared to the one of the conventional scan via the Imspector software in order to guarantee comparability of the results. Second, FastRESCue applications on both fluorescent microspheres as well as various cellular structures are presented.

Being an evolution of RESCue, FastRESCue can obviously be combined with any imaging technique for which RESCue is applicable. Thus, both tomoSTED and 2D STED microscopy can take advantage by the novel features introduced by this technique. A 1D depletion pattern is the ideal candidate for the characterization of the scan system, since potential artifacts or jit-ters are better visible for the elongated 1D STED PSF. However, to ensure a direct comparison with the RESCue applications demonstrated in literature during the past years, conventional 2D STED microscopy is employed for most of the imaging part.

Please note that all further measurements within this thesis utilizing the novel scan system are performed with self-written LabVIEW routines and the resulting images are acquired without any pulse gating.