Superresolution fluorescence microscopy with structured illumination

• Autores: Awoke Atena Negash
• Directores de la Tesis: Pablo Loza Álvarez (dir. tes.), Hugues Giovannini (codir. tes.)
• Lectura: En la Universitat Politècnica de Catalunya (UPC) ( España ) en 2017
• Idioma: español
• Tribunal Calificador de la Tesis: Rainer Heintzmann (presid.), Chrysanthe Preza (secret.), Stefan Wieser (voc.)
• Programa de doctorado: Programa de Doctorado Erasmus Mundus en Ingeniería Fotónica, Nanofotónica y Biofotónica / Photonics Engineering, Nanophotonics and Biophotonics por la Universidad Politécnica de Catalunya; Aix-Marseille Université(Francia); Karlsruhe Institute of Technology (KIT)(Alemania) y Università degli Studi di Firenze(Italia)
• Materias:
  • Física
    • Óptica
• Enlaces
  • Tesis en acceso abierto en: TDX
• Resumen

  • The resolution of a conventional fluorescence microscope image is diffraction limited which achieves a spatial resolution of 200nm lateral and 500nm axial. Recently, many superresolution fluorescence microscopy techniques have been developed which allow the observation of many biological structures beyond the diffraction limit. Structured illumination microscopy (SIM) is one of them. The principle of SIM is based on using a harmonic light grid which down modulates the high spatial frequencies of the sample into the observable region of the microscope. The resolution enhancement is highly dependent on the reconstruction technique, which restores the high spatial frequencies of the sample to their original position. Common SIM reconstructions require the perfect knowledge of the illumination pattern. However, to perfectly control the harmonic illumination patterns on the sample plane is not easy in experimental implementations and this makes the experimental setup very technical. Reconstructing SIM images assuming the perfect knowledge of the illumination intensity patterns may, therefore, introduce artifacts on the estimated sample due to the misalignment of the grid that can occur during experimental acquisitions. To tackle this drawback of SIM, in this these, we have developed blind-SIM reconstruction strategies which are independent of the illumination patterns. Using the 3D blind-SIM reconstruction strategies we extended the harmonic SIM to speckle illumination microscopy which uses random unknown speckle patterns that need no control, unlike the harmonic grid patterns. For harmonic-SIM images, since incorporating some information about illumination patterns is valuable, we have developed a 3D positive filtered blind-SIM reconstruction which confines the iterative estimation of the illuminations in the vicinity of the Fourier peaks (using carefully designed Fourier filter masks) in the Fourier space. Using blind-SIM reconstruction techniques a lateral resolution of about 100nm and axial resolution of about 200nm is obtained in both speckle and harmonic SIM. In addition, to reduce the out-of-focus problem in widefield images, a simple computational technique which is based on reconstructing 2D data with 3D PSF is developed based on blind-SIM reconstruction. Moreover, to combine the functionalities of SIM and light sheet microscopy, as a proof of concept, we have developed a simple microscope setup which produces a structured light sheet illumination pattern.