STED microscopy reveals sub-cellular details in living mice

06 Apr 2021

Yale project achieves super-resolution 3D imaging in native tissue environment.

Spot the diference: 3D STED

As published in Optica, the breakthrough was based on the use of stimulated emission depletion (STED) microscopy, one of the family of techniques developed to allow imaging beyond the normal diffraction limit of optical microscopy.

The principles behind STED, for which Stefan Hell shared the 2014 Nobel prize for chemistry, involve the addition of a quenching laser pulse to conventional fluorescence excitation, one delivered in a ring-shaped profile. Resolution at the central hole of this pulse can be driven down below the diffraction limit under certain conditions.

Various improvements to the optics of STED have been developed since then, such as a mirror-enhanced variant designed to improve axial and lateral resolution.

It has remained challenging to use the technique to image thick tissues or apply it to living animals, however, due to the inherent optical aberrations in biological specimens that prevent STED from focusing properly.

The Yale team tackled the problem through a platform combining several individual enhancements: two-photon emission (2PE), specific red-emitting organic dyes, wavefront-sensing adaptive optics for aberration correction, and a water-immersion objective lens with a long 2-millimeter working distance.

"Our microscope is the first instrument in the world to achieve 3D STED super-resolution deep inside a living animal," said Joerg Bewersdorf from Yale School of Medicine.

"Such advances in deep-tissue imaging technology will allow researchers to directly visualize subcellular structures and dynamics in their native tissue environment."

Where the brain's interesting connections happen

Yale first tested its platform by imaging samples of fixed ex vivo mouse skin, and then by visualizing the internal 3D structures of keratinocyte skin cells. According to the project, the 3D-2PE-STED operation "resolved volumes more than 10 times smaller compared to using 2PE alone," and revealed the distribution of DNA in the cell nucleus.

Trials using a 300-micron-thick mouse brain tissue section also showed that the technique could successfully image 164-microns below the tissue surface. "Two astrocyte branches that appear as one in the 2PE image are distinguishable in the corrected 2PE-STED image, elucidating the STED effect," commented the project in its paper.

To demonstrate the technique's full potential, the team then carried out aberration-corrected 3D-2PE-STED imaging of neurons in the intact brain of a living mouse, achieving an imaging depth of 76 microns.

The team took care to assess whether the in vivo STED operation itself caused any structural changes through phototoxicity; the results to date are encouraging, although further investigations will be carried out.

"Dendritic spines are so small that without super-resolution it is difficult to visualize their exact 3D shape, let alone any changes to this shape over time," said Yale's Mary Velasco. "3D-2PE-STED now provides the means to observe these changes and to do so not only in the superficial layers of the brain, but also deeper inside, where more of the interesting connections happen."