Optimizing Multi-Color 3D-STED Fluorescence Nanoscopy to Unveil Transcriptional Cluster Nano- Architecture in Embryonic Cells

Over the past decades, quantitative far-field optical fluorescence microscopy techniques have
contributed significantly to the investigation of biomolecular interactions in living systems at
the highest possible spatial resolution. As one of these methods, Stimulated Emission Depletion
(STED) fluorescence nanoscopy has become a cornerstone in cell research since it provides a
unique window to directly observe complicated dynamics that underlie life. Among its benefits
over conventional light microscopy, STED's ability to surpass the diffraction limit allows for
visualization of nanoscale features crucial for knowledge of cellular architecture. By depleting
excited fluorophores within the periphery region of excitation volume, STED nanoscopy
performs excitation focal volume engineering to effectively decrease the volume of excitation
so that the diffraction barrier can be broken. Also, with the extension to three-dimensional
imaging, 3D-STED allows one to get high-resolution volumetric reconstructions of complex
biological specimens, further extending its functionality in the study of complicated biological
systems.
... mehrDespite the increased applications due to superior spatial resolution, there are challenges in
improving data quality while conducting multi-color 3D imaging deep inside the cells. For
instance, in densely labeled biological samples, the strong fluorescence background signal
superimposed on the true signal impairs data quality significantly; hence, strategies to reduce
background contribution effectively are required for correct data interpretation. Besides, the
nature of optical imaging systems induces wavefront aberrations that could further degrade the
achievable imaging resolution. The deterioration caused by aberrations might even be more
severe for super-resolution techniques relying on focal volume engineering, like STED, which
are extremely sensitive to deviations in focal volume from the ideal distribution of intensity. In
such cases, effective deep super-resolution imaging in cells, for instance in zebrafish embryos,
requires not only system but also sample aberration minimization, primarily due to refractive
index mismatch between the sample and the medium, as well as structural heterogeneities in
the specimen itself.

This PhD thesis studies different optimization strategies to improve the performance of multi-
color 3D-STED fluorescence microscopy and its application to investigate the nano-
architecture at transcriptional clusters in embryonic cells. Chapter 2 describes the theoretical
background of this nanoscopy technique, introducing the basics of fluorescence and optical
principles. In Chapter 3, the structure and functioning of the custom-built STED microscope
setup enhanced in this work are described. Moreover, this chapter discusses one of the existing
potent methods to address the fluorescence background while imaging sparsely labeled
samples. Chapter 4 examines a novel method developed in this research project for correcting
fluorescence background, not only in sparsely labeled specimens but, more importantly, in the
highly challenging environment of densely labeled samples. Chapter 5 focuses on the
application of background-free 3D multi-color super-resolution microscopy to study
morphological characteristics and spatial organization of transcriptional clusters and DNA in
zebrafish embryonic cells. Chapter 6 examines the impact of wavefront aberrations in STED
microscopy and the implementation of a photon-efficient adaptive optics strategy as a solution.
Lastly, Chapter 7 provides a summary of the findings and discusses potential future directions
for this work.