Analysis and conclusions

Can we be sure that the measured spine neck widths (Figure 3.7) accurately reflect the true distribution? If so, this can be true only for the lower end of the spine neck spectrum, because we selectively measured thin spines and excluded ones that looked too large. The apparent skew of the distribution towards thinner neck diameters and the tailing off towards thicker neck values reflected this. What about the lower boundary of the measured distribution, which we used to judge the spatial resolution? Whereas the neck widths were evenly distributed between130nm and 70nm, the frequency dropped off quickly from 70nm to 50nm. This corresponds well to electron microscopy measurements on fixated samples that found no spine necks smaller than40nm.^95,96 If the spatial resolution of our microscope in tissue, however, had been insufficient to resolve these small structures, we would have

expected to observe peaking values at the actual resolution, followed by a sharp drop-off at thinner values; all structures smaller than the resolution would have appeared to be larger – of similar size than the true resolution. This peak would be far less obvious, however, if coinciding with a (sharp) decline instead of a flat distribution. The spatial resolution of our microscope was measured in Section 3.2.3 to be better than60nm to 70nm for fluorescent beads on a coverslip. A detailed comparison of the spine neck widths measured using STED or electron microscopy would be interesting, as a method to evaluate the impact of fixation artifacts on the neuronal morphology.

The measured neck widths cannot be exact, however, considering that the spine neck widths were not constant but could vary over the entire length of the spine. Because we measured the thinnest section of each spine, the distribution can be considered to reflect a lower boundary of spine neck widths. These local fluctuations may have benefited the resolution measurement by increasing the chance to find a structure thin enough to be used as a measure. Furthermore, because our fluorescent label attached to actin, it was not possible to distinguish between the true morphology of the spine neck and the actin distribution. But because dendritic spines are highly enriched in actin,⁹⁷ in thin spine necks this label should be similar in appearance to a volume label. ^VII To measure the lower boundary of the spine neck diameters more precisely, a microscope with a resolution better than40nm would be needed, as well as an unambiguous labeling, such as a membrane-bound fluorophore.

Given that the correction collar optimally corrects the image by inducing aberrations that are equal but opposite to the aberrations in the sample, we can deduce some information about these aberrations and their nature. In Section 3.2.2 we determined that higher correction values induce increasing positive aberrations, and in Section 3.3.2 that we need ever higher correction values to offset the aberrations in increasing depths beneath the sample surface. Consequently, the spherical aberrations induced by the refractive index mismatch sample were negative. This implies that the average refractive index of the brain tissue was lower than that of the glycerol immersion (n=1.451).⁸⁸ These observations are in good agreement both with our direct measurements of the refractive index of hippocampal tissue, as well

VIIRegardless, this had no ramifications for the estimation of the spatial resolution of our STED nanoscope.

3.4. Discussion

as comparative tests of correction values with other refractive index liquids, thus further supporting our approximation of the average refractive index of hippocampal tissue.

We observed a clear loss of image contrast as the imaging depth increased beyond 80µm; this was both due to increased background fluorescence and declining signal intensities. The reduced signal could not be fully offset by increasing excitation intensities. Whereas uncompensated aberrations could account for the reduced signal, they cannot explain the increasing, diffuse background that occurred even in very sparsely labeled areas. The most probable explanation is increased scattering, which should intensify deeper inside the dense brain tissue. The average scattering length inside grey brain matter is95µm,^17,98thus close to this observed depth. The loss in image contrast was still tolerable at our maximum depths of 120µm, but would surely constitute a serious problem at even greater depths.

The maximum depth to which neurons could be imaged in this setup was not inherently limited by insufficient aberration compensation. More basic restrictions were the cause, such as the maximum thickness of the brain slices and the limited working distance of the objective lens. The working distance of the glycerol immersion objective lens was280µm, but this could have been extended to a certain degree by using thinner coverslips and compensating the additional aberrations with a higher refractive index immersion medium. This, however, would have incurred further complications, such as severe alignment difficulty (as discussed in Section 3.3.2). The maximal thickness of the organotypic brain slices was limited by the technique: if brain slices were cut thicker than 400µm, then the bottommost cells near the coverslip would not receive adequate nourishment from the medium and consequently die off. This could have been alleviated by using alternative protocols, such as cultivating the brain slices on porous membranes instead of glass coverslips,^52,99 thus allowing nutrients to be taken in from both sides of the brain slice. In general, such cultured slices are thicker than slices used in our method and could possibly be grown even thicker. But these methods would have caused additional imaging problems, such as imaging through the membrane or necessitating an upright microscope. Other options include using acute brain slices or conductingin vivoexperiments in live animal brains. Increased scattering can be

expected in these cases. In acute slices there is a thick layer of dead and damaged cells at the interface, forming a dense, turbid layer with increased scattering and autofluorescence. In the living brain, scattering, motion artifacts and different tissue layers would need to be considered. But even with greatly enhanced depth penetration, this approach would still not be able to examine the hippocampusin vivo, which is locked deep inside the mammalian brain.