Super-resolved imaging by STED nanoscopy
(Ensayo defensa de tesis)
Since the studies made by Ernst Abbe, by the end of the XIXth century, it was accepted that far field optical microscopy had a limit in its spatial resolution: objects found closer together than half the wavelength of the light used would not be distinguished apart. However, in the last two decades, a number of techniques based on fluorescence have been developed, that keep all the advantages of far-field optical microscopy and provide spatial resolution only limited by the size of the fluorescent marker. These techniques are called super-resolution fluorescence microscopies or fluorescence nanoscopies and they involve the transition between molecular states of the fluorophores that allow or not fluorescence emission. Particularly, in STED (Stimulated Emission Depletion) nanoscopy, this transition is produced by stimulated emission depletion, in order to disable fluorophores in controlled regions of space and thereby reduce the effective volume of observation.
In the framework of this thesis, and supported by a formal and active collaboration with Stefan W. Hell (Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany), awarded in 2014 with the Nobel Prize in Chemistry for his pioneering work in fluorescence nanoscopies, the first STED nanoscope of Argentina was built (Grupo de Nanofísica Aplicada, Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires (BA), Argentina).
The STED nanoscope was applied to investigate and elucidate biophysical questions with nanometric resolution. In interdisciplinary collaborations it was studied: (a) the organization of proteins in the membrane of Trypanosoma cruzi, in collaboration with the Group of Molecular Immunology, Instituto de Investigaciones Biotecnológicas (IIB), Universidad Nacional de San Martín (UNSAM), CONICET, BA, Argentina; (b) processes of cytoskeleton restructuring in growth cones during neuronal polarization, in collaboration with the Laboratory of Neurobiology, Instituto de Investigación Médica de Córdoba (INIMEC), CONICET, Córdoba, Argentina; and (c) the formation and stability of periodic structures of actin in the process of axonal degeneration, in collaboration with the Laboratory of Neurobiology, INIMEC, CONICET, Córdoba, Argentina and the Group of Molecular Neurobiology, Instituto de Investigación en Biomedicina de Buenos Aires (IBIOBA), CONICET, BA, Argentina.
Also, a new implementation scheme of STED nanoscopy was developed, which expands considerably its field of application. Generally, light of a wavelength located in the far-red zone of the emission spectrum of the fluorophore is used to induce stimulated emission. In this thesis, it is shown that continuous wave STED (CW), is also feasible using STED wavelengths located at the maximum of emission, where the stimulated emission cross section can be up to 10 times higher. As a result, super-resolved STED images are obtained using proportionally less power. Also, fluorophores considered unsuitable for certain spectral configurations of STED nanoscopy, may become usable with this scheme.