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Archivio digitale delle tesi discusse presso l’Università di Pisa

Tesi etd-06222023-222254


Tipo di tesi
Tesi di laurea magistrale
Autore
MAGNI, LORENZO
URN
etd-06222023-222254
Titolo
Advancing robustness and sensitivity in DNA-based super-resolution microscopy
Dipartimento
BIOLOGIA
Corso di studi
BIOLOGIA MOLECOLARE E CELLULARE
Relatori
relatore Prof. Marangoni, Roberto
relatore Dott. Stein, Johannes
Parole chiave
  • adapters
  • cryo-fixation
  • DNA-origami
  • DNA-PAINT
  • gel-embedding
  • multiplexing
  • non-specific binding
  • probe screening
  • super-resolution microscopy
  • ultracryotomy
Data inizio appello
11/07/2023
Consultabilità
Non consultabile
Data di rilascio
11/07/2026
Riassunto
DNA-points accumulation for imaging in nanoscale topography (PAINT) is a Single-Molecule Localization Microscopy (SMLM) technique exploiting transiently binding fluorescent DNA probes to visualize target DNA, RNA, or DNA-labelled molecules. Despite significant developments over the past decade, DNA-PAINT remains an advanced imaging method requiring specialist expertise with respect to optics, microscopy, biochemistry and cellular biology. In this thesis, some of the limitations of DNA-PAINT – first and foremost non-specific binding (NSB) and impaired diffusion of probes – will be assessed and tackled with the objective of 1) improving the user-friendliness and reproducibility of DNA-PAINT and 2) expanding its multiplexing and multi-omics potential. Different sample preparation protocols will be explored and optimized, from cell fixation to the combination of DNA-PAINT with ultracryotomy and gel embedding. Adapter secondary probes will be proposed to decrease the costs of DNA-PAINT, and to expand its versatility and multiplexing capabilities. In vitro hybridization assays combined with automated postprocessing pipelines will be designed to quantitatively screen adapter probe sequences, tested on proof of principle tasks, and proposed as the first step of an in vitro to in situ optimization pipeline.
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