3. Imaging deep inside neuronal tissue by compensating spherical aber-
3.2.4. Optimizing experimental parameters
Understanding the optical consequences of the aberrations, we could explore the effects of easily variable experimental parameters, such as coverslip thickness, immersion media, heating and coverslip tilt, on the correction and then select the ideal settings for the appropriate situation. To do this we again used gold nanobeads, as they are by nature impervious to bleaching and can reveal more nuances of the occurring aberrations (especially non-spherical).
3.2.4.1. Coverslip thickness and immersion medium
Varying the coverslip thickness or the refractive index of the immersion medium had a severe impact on the spherical aberrations of the entire system. The glycerol objective lens was pre-corrected to compensate fixed aberrations incurred by specific values of both coverslip thickness and refractive index. So altering either parameter could be regarded as introducing (or removing) an additional aberrating layer, thereby shifting the dynamic correction range up or down by a fixed amount.92 Thinner coverslips would additionally increase the effective working distance of the objective lens, which constituted a hard limit for the maximal penetration depth inside a sample.
We measured these potential shifts to the dynamic correction range by observing the beam PSFs using gold nanobeads for different coverslip thicknesses (70µm, 140µm and 170µm) as well as for different immersion media. We tested glycerol with varying water content (1.44 ≤ n ≤ 1.46), because added water reduces the refractive index, as well as ’refractive index liquids’ with fixed refractive indices
positive aberrations
negative aberrations increase depth
thicker coverslip heating
higher refractive index
x/y z
x/y z
increase correction
Figure 3.5. | Shifting the correction range by altering experimental parameters.
Additional spherical aberrations can be introduced by altering some of the experimental parameters, such as the coverslip thickness and the refractive index of the immersion medium. This shifts the range in which the correction collar can compensate aberrations, potentially offsetting the increasing aberrations incurred by imaging deeper and deeper inside dense neuronal tissue.
(1.43 ≤ n≤ 1.47). Obviously, if the additionally induced aberrations were greater than the correction capabilities of the objective lens, then we were not able to precisely quantify the ideal correction value. By varying two parameters with opposite effects simultaneously, however, it was possible to keep the optimum within the quantifiable correction range of the objective lens. Together, this allowed a quantification of the individual effects.
We observed increasing shifts toward lower optimal correction values for thicker coverslips as well as for immersion media with a higher refractive index than n=1.45.
Changing the refractive index by∆n = 0.01shifted the correction value by more than a full rotation of the correction ring (∆cv < −10). Changes in coverslip thickness induced lesser aberrations, with ∆d = 15µm shifting the correction range by ∆cv ≈ −1. Heating the setup from room temperature (21◦C) to more physiological temperatures (35◦C) also caused the correction range to shift towards lower values by∆cv ≈ −2(Figure 3.5). Shifts to lower correction values imply the introduction of positive aberrations, necessitating additional negative aberrations to be introduced by the correction collar in order to compensate.
3.2. Measuring and compensating spherical aberrations
Finally, we tested the viability of using two ’sandwiched’ 70µm coverslips, with a thin layer of air, water or TDE (2,2’-thiodiethanol93) in between. This kind of configuration might be of interest for experimental situations in which the brain slices need to be replaced rapidly and/or frequently, as is commonly the case in patch-clamping experiments. Such experiments would therefore be slowed down considerably by the necessity of glueing each individual slice into the chamber (and to remove the previous slice and clean the chamber afterwards). Overall, the results were quite encouraging. The additional aberrating layer in between both coverslips introduced only small shifts in correction (∆cv ≈ +1 for ACSF and∆cv ≈ −1for TDE). Furthermore, the length of the PSFs along the optical axis matched the results from single (140µm) coverslip samples, indicating a comparable quality of the correction. What was evident, however, was a reduced intensity and a worse signal to noise ratio. An explanation for this might be the Fresnel reflection occurring at the two additional refractive index discontinuities at the coverslip–medium interfaces.
3.2.4.2. Coverslip tilt and unstable immersion media
An additional cause of PSF distortions when using a non-oil immersion objective lens arises when the coverslip is tilted in relation to the optical plane. In the case of a glycerol immersion objective the tilt needs to be kept within0.5◦in order to limit the intensity losses of the STED beam to less than 10%.94We could easily observe these distortions using gold beads when viewing xz- and yz- sections of the otherwise rotationally symmetrical PSF of the STED donut. Depending on the direction of the tilt, one of the donut lobes would be distorted and drawn out in a crescent-shape along the direction of the side lobes. We could avoid this to a surprising degree by meticulously eliminating any unevenness and tilt of the microscope stage and the sample chamber. Even though the coverslips with the brain slices were all glued in by hand, almost no tilt towards the optical plane was visible. This was more difficult when observing two ’sandwiched’ coverslips, as discussed earlier. Here we observed slightly more pronounced tilts more often, yet still not severe enough to cause a noticeable loss of image quality.
We identified an additional source of aberrations when working with self-mixed glycerol as immersion medium. Over the course of several hours during an experiment we observed a gradual increase of negative spherical aberrations, as
the optimal correction value shifted upwards. We traced this effect back to a gradual decrease of the refractive index, as the hygroscopic glycerol drew water from the air, thus self-diluting. This caused severe problems over the course of long experiments, requiring regular re-alignment procedures to be undertaken. We could easily prevent this problem by either using a refractive index liquid or by using commercial immersion glycerol, which had an added agent, preventing the glycerol from drawing water from the air.