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Autor/a
Moral Blázquez, Nuria
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Abstract
Super-resolution light microscopy allows us to visualize the cell interior with a resolution that exceeds by ten times the resolution previously achieved with conventional microscopes. Invented more than 25 years ago, Stimulated emission depletion (STED) microscopy has become a standard and widely used imaging method in the life sciences. Thanks to continuous technological progress, STED microscopy can provide effective resolution while preserving the most valuable aspects of fluorescence microscopy, such as optical sectioning, molecular specificity and sensitivity, and compatibility of studying living cells.
Early in the development of STED microscopy, the number of fluorophores used in the process was minimal. Rhodamine B was named in the first theoretical description of STED. As a result, the first markers used were laser emitters in the red spectrum. To enable STED analysis of biological systems, fluorophores and laser sources must be tailored to the system. Consequently, not all fluorophores are suitable for use as markers for STED microscopy. These must have particular characteristics, high photostability, high fluorescence intensity, and extinction coefficients of approximately 105 cm-1 M-1. This desire for better analysis of these systems has led to live-cell STED and multi-color STED. Still, it has also required increasingly advanced markers and excitation systems to fit with greater functionality.
One of those advances was the usage of immunolabelled cells. These cells are labeled by antibodies conjugated with fluorophores suitable for STED microscopy. Although these can be purchased, the researchers can also perform the conjugation, thus having the possibility of varying the number of fluorophores conjugated per antibody. In this project, the optimization of fluorescent labeling by immunostaining of chemically fixed cells for STED microscopy has been studied. The first approach for optimization was to compare commercial fluorescent conjugated antibodies and fluorescently conjugated antibodies developed in the laboratory (In-House). Parameters such as photostability, and
Signal-to-noise ratio were compared. On the other hand, fluorescent labeling in living cells has also been studied. Different specific commercial markers suggested for the STED technique have been evaluated to optimize the concentration and incubation times in different cell lines. Finally, the possibility of using other existing markers for living cells not previously tested for the STED technique has also been studied. With which the concentration, incubation times, and photostability were studied.
Furthermore, an attempt was made to optimize the image analysis parameters of each fluorophore for STED microscopy, and codes have been generated to automatize this analysis.
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