Simulated light dose reduction for different samples

As described in section 5.4, three basic structure geometries have been simulated with a 3-step DyMIN scan (Figure 5.3). Those were dots (left), lines (middle) and rings (right), representing basic structure geometries of biological samples. Dots:

central channel of nuclear pores, single fluorophores etc.; Rings: clathrin, virus particles, synapses, gephyrin etc.; Lines: vimentin, tubulin etc.

The lines and rings were rendered by single points with a spatial distance much closer than the resolution of the DyMIN step N −1 . Since individual points are not resolved by that step, the structure appears quasi-continuous. For all samples the structure was generated randomly. For the simulations in this section, similar imaging parameters were used. The resolution at the final step N was 25 nm, the dwell time was 100 µs, and the pixel size was 15 nm (slightly to large, but does not affect the results). For RESCue a decision timet_dec of 30 µs was selected.

5.4. Assessment of DyMIN

Figure 5.3.: Simulated DyMIN scan for different structure geometries. 3-step DyMIN, the final resolution was 25 nm, dwell time 100 µs. Left column: single spots (density of 20 points / µm²). Middle column: assembly of linear structures.

Right column: assembly of ring structures. (A) The specific random fluorophore distribution in the sample. White: pixels including a fluorophore, black: pixels without fluorophore. (B) Illumination map of the STED powers applied throughout the scan. White: illumination stopped after the confocal probing; green: stopped after the intermediate probing; pink: final STED power applied. (C) The spatial STED light dose which originates from the particular DyMIN scan. The dose is highly heterogeneous and much lower than needed for conventional scanning (dose of 1). (D) Shown is the mean STED light dose on the structures in dependence of the intermediate resolution. For the structures shown here, a global minimum exists, demonstrating an optimal intermediate resolution. The optimum for the dots

In a 3-step DyMIN process mainly the resolution of the intermediate step (second step) has to be optimized which strongly influences the total light dose on the structure (Figure 5.3 (D)). The first step has a confocal resolution. Here, the intermediate resolution was changed in 1 nm increments starting at the confocal resolution (230 nm) up to the final resolution (25 nm). For all three structures the influence of the intermediate step on the total light dose is shown.

In figure 5.3 (C) the spatial distribution of the total light dose is shown which occurs for the optimal resolution in step 2, as calculated by the minimization. For the structures simulated, a global optimum of the light dose exists with respect to the intermediate resolution. This optimum is relatively broad, which allows a coarse adjustment of the step 2 resolution without a significant performance loss. The optimum appears roughly at half the resolution of the final step for all three structures. The simulations were carried out with a set of scan parameters which are close to the practical application (realistic values). Parameter sets or samples exist which feature no optimum (e.g. where DyMIN has a negative effect), for instance a very short dwell time or very dense samples. However, a useful DyMIN implementation allows to use a much longer dwell time (since bleaching is reduced) which will always reduce the light dose minimum when compared with the conventional scan. In fact, the optimum depth in respect to the conventional scan is the bleaching reduction which is convertible into a higher dwell time (by the same factor) and signal. An appropriate choice of the step 2 resolution should be near the optimum but a little bit lower (further right on the curve in figure 5.3 (D)). Thereby, a kind of clearance area is generated around the sample structure (without much higher bleaching), which assures that the structure will be completely contained in the illumination mask N−1.

In figure 5.4 (A) the simulated light dose is plotted for different densities of a dot structure, using the same sample type as in figure 5.3, left column. The sample density was simulated in the range between 0 and 200 points / µm². The STED light dose is shown for conventional scanning, RESCue, 2-step DyMIN, 3-step DyMIN and 4-step DyMIN scanning. Here, the same imaging parameters were used as for figure 5.3 (25 nm final resolution). The probing resolution sets for the different DyMIN methods which were used in this density study are listed in table 5.3.

The resolutions for probing were optimized for a sample density of 20 / µm²and vary if the sample density is different. A different sample density (or type) will most of the time require a different probing resolution set (compared to the one used here) to achieve the lowest light dose possible. In this study it is shown that a 3-step DyMIN scan gives a lower light dose compared to RESCue at sample densities which are in a realistic range, compare figure 5.4 (B). At low densities, RESCue features a high light exposure base line which originates from the fixed (relatively long) decision time while using the highest OFF-switching light power. However, at high densities,

5.4. Assessment of DyMIN

Figure 5.4.: Light dose for different scan schemata in dependence on the sample density. (A) The simulated relative STED light dose on the structure vs. the sample density (dot structure). Final resolution 25 nm, dwell time 100 µs.

Simulated are conventional scanning, RESCue and DyMIN with 2/3/4 steps for typical sample densities (e.g. ∼5−20 nuclear pore complexes / µm²). With DyMIN scanning the light dose is more than a magnitude lower compared to conventional scanning. DyMIN parameters were optimized for a density of 20 / µm². (B) DyMIN recording of NPCs (immunofluorescence), featuring a density of ∼7/µm² (image is smoothed with a Gaussian of 1.5 pixels). The scale bars (x, y) are 500 nm (B). Figure reproduced from ref. [2].

the intermediate DyMIN steps cannot resolve individual dots, in this case RESCue is the better choice due to the higher probing resolution. When comparing 4-step DyMIN and 3-step DyMIN, the 4-step method conceptually provides a lower light dose and is especially well-fitting for very small structures, due to the good chance for empty probing at higher resolutions.