Biomedical Engineering Reference
In-Depth Information
Fig. 10.6 Resolution enhancement with STED microscopy. Synaptic vesicles in GABAergic
terminals of hippocampal neurons labeled with VGAT observed in standard confocal mode (a).
In contrast, STED (b) reveals details of single vesicles which are unobservable in the confocal
image. Both images show raw data. (c) Profile along the line indicated by arrows in the STED
image reveals a resolution around 40 nm (
red
), corresponding to the average size of individual
synaptic vesicles. Excitation 572/15 nm. Detection 641/75 nm. Depletion 720/20 nm. Pixel size
15 nm. Excitation average power
4
μ
W; STED average power
4.2 mW. Scale bar of 0.5
μ
m.
After Galiani et al. (
2012
) (Credit: P. Bianchini and S.Galiani)
et al.
2012
; Cella Zanacchi et al.
2013
; Takasaki et al.
2013
). In fact, STED
microscopy was initially used to image cortical spines in brain slices in depth, and
combining STED with two-photon excitation represents an optimal opportunity for
multicolor imaging in living brain tissue (Bethge et al.
2013
). Two-photon excitation
can also be used to improve localization-based techniques by confining the
photoactivation process to increase imaging depth. More recently, 2PE and temporal
focusing have been also used to confine the activation process, to perform 3D super-
resolution at the whole cell level (York et al.
2011
).
Combining STED with two-photon microscopy was realized with continuous-
wave STED beams (Ding et al.
2009
; Moneron and Hell
2009
), a solution that
simplifies the combination of 2PE and STED. A new approach that allows
performing 2PE-STED imaging using a single wavelength (SW) and, consequently,
the very same laser source for 2PE and depletion (Bianchini et al.
2012
), SW
2PE-STED, simplifies the image-formation scheme, especially for thick samples
and deep-penetration imaging. It is important to note that even if stimulated
emission is a one-photon process, scattering of stimulating photons will not
increase background, because in most cases, their wavelength is far away from
the absorption spectral window of the dye. However, even in the worst scenario, the
fluorescence signal induced by the STED beam can be subtracted by lock-in
technique (Vicidomini et al.
2013
; Ronzitti et al.
2013
) to get super-resolved
images. Gould et al. (
2012
) showed that using spatial light modulators in both the
excitation and STED beam can compensate sample-induced aberration in a three-
dimensional STED implementation. Super-resolution techniques are still in their
infancy, yet improvements are rapidly being implemented as demand for possible
applications increase, especially in neuroscience.
Search WWH ::
Custom Search