Preliminary applications of aberration-corrected microscopy and lithography are conducted using poke mode aberration correction. In this section, multiple examples of aberration correction are discussed. For adaptive optics in mi-croscopy, aberrations are corrected through fixed mouse brain tissue sample sections. Aberration correction is conducted on a⇠50µm thick mouse brain tissue followed by a⇠85µm mouse brain tissue sample. For adaptive optics in lithography, aberrations caused by the refractive index mismatch between the immersion medium, the coverglass and the photoresist are discussed.
correction using poke modes in two fixed mouse brain tissue samples with different thicknesses are illustrated.
5.1.1. Sample Preparation
A sketch of the sample structure is shown in Figure5.1. First, a microscope slide and a coverslip with200nm diameter Crimson Beads are prepared. Then the harvested mouse brain is sliced coronally to the required thicknesses and mounted in a PBS/FITC solution on the slide with the coverslip on top. The sample is then sealed with transparent nail polish. A detailed description of the sample preparation can be found in A.2
Figure5.1.: Illustration of the microscopy sample. A microscopy slide and a coverslip with200 nm Crimson Beads are prepared. Then the fixed brain tissue, which is immersed in a PBS/FITC solution, is sandwiched between the two glasses.
5.1.2. System Calibration
Before imaging the Crimson Beads without and with aberration correction, the built-in sensor-less AO is used to compensate for system aberrations. Then the reference spot diagram with the system flattened is recorded with the
guide star created by excited FITC solution. Afterwards the control matrix is generated.
5.1.3. Imaging through 50 µm and 85 µm Mouse Brain Tissue
50 µm Mouse Brain Tissue
Image5.2shows 200nm diameter Crimson Beads imaged via TPE through a 50µm thick mouse brain tissue section without aberration correction (see Figure5.2B) and after one iteration of aberration correction, see Figure5.2C.
It has to be emphasized that both images are scaled to the same color table.
Figure5.2A illustrates the averaged aberration in the FOV. By decomposition of the averaged aberration in Zernike polynomials, defocus and spherical aberration could be identified as the main contributing aberrations.
As can be seen from the example, after one iteration of aberration correction with the illustrated wavefront, a strong improvement of the bead image is visible. First, the intensity of the beads after aberration correction is approx-imately3.5 times higher. Thus, more beads are visible in that color regime.
Second, the beads gain in shape-uniformity after aberration correction and the resolution improves. Consequently, the aberration correction after one iteration of poke mode correction improves the image quality when imaging through⇠50µm fixed mouse brain tissue.
The same experiment was then conducted for a⇠85µm thick fixed mouse
brain tissue section.
Figure5.2.: Illustration of beads imaged through a⇠50µm mouse brain tissue. A illustrates the averaged aberration in the FOV. The color bar is in unit of radians. B shows 200nm Crimson Beads without aberration correction. C depicts the same FOV after one iteration of aberration correction using poke modes. Both bead images are scaled to the same color table.
85 µm Mouse Brain Tissue
Figure5.3shows200nm diameter Crimson Beads imaged via TPE through a
⇠85µm thick fixed mouse brain tissue section without aberration correction (B) and after one iteration of aberration correction (C). Figure5.3A illustrates the averaged aberration in the FOV. By decomposition of the averaged aberra-tion in Zernike polynomials, it illustrates that the aberraaberra-tion is a mixture of
multiple low and high order Zerinke polynomials with defocus and spherical aberration being to approximately the same extend the main contributing aberrations.
Again, Figure5.3shows for the given example an improvement of the bead images after one aberration correction iteration with the illustrated wavefront.
Both, intensity increase (⇠ 1.45times) and bead shape uniformity enhance-ment after aberration correction indicate an improved imaging condition after aberration correction. Consequently, the image quality improves when the aberration correction with poke modes is conducted.
Figure5.3.: Illustration of beads imaged through a⇠85µm mouse brain tissue. A illustrates the averaged aberration in the FOV. The colorbar is in units of radians. B shows 200nm Crimson Beads without aberration correction. C depicts the same FOV after one iteration of aberration correction using poke modes. Both bead images are scaled to the same color table.
5.1.4. Discussion
In the given examples, aberration correction with the implemented SHWFS and DM using poke modes leads to improved image quality. However, mul-tiple problems may influence the reliability of the implemented method, including pseudo inversion, mechanical drift and spot quality in the SHWFS.
A possible solution for the first challenge, the pseudo inversion, might be to use regularization methods for ill-posed problems [70]. One promising direct regularization method, called Tikhonov regularization, might improve the calculated compensation mode coefficients leading to enhanced image quality [71]. Another reason that might degrade aberration compensation is mechanical drift, especially during matrix calibration. The mechanical drift is most likely introduced by the manually driven stage. The grease between the screw and the thread in the stage have to settle before calibration, for example, and also before taking the spot diagram for compensation. A further reason could be the spot quality of the SHWFS spot diagram, especially regarding the SNR and the spot aberrations. Due to challenges in the sample preparation process, some areas have less NL-GS signal. These areas with less SNR show more difficult compensation in experimental observations. This challenge, however, can be addressed by improving the sample preparation protocol or neglecting dim spots. Although, the latter has to be handled with care since the system has140degrees of freedoms and only145sampling points.