Fluorescence-lifetime image scanning microscopy
by Niels Johann Radmacher
Date of Examination:2025-02-03
Date of issue:2025-03-13
Advisor:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Silvio O. Rizzoli
Persistent Address:
http://dx.doi.org/10.53846/goediss-11145
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Abstract
English
Improving optical resolution and image quality has always been an important goal in the field of microscopy. One technique that histori- cally played a major role in the optimization of image quality has been confocal microscopy. With the ability to remove out of focus contribu- tion to the final image it has revolutionized the field. Especially for dense and highly inhomogeneous samples, it is highly important to separate foreground from background light. An additional benefit of Confocal Laser Scanning Microscopy (CLSM) is the possibility to combine it with Fluorescence-Lifetime Imaging Microscopy (FLIM). Gaining access to a whole new dimen- sion of information. Because fluorescence lifetime can be influenced by the surrounding of the specimen. In addition, it can also be used to differentiate between different fluorescent labels. By integrating an array detector in a confocal laser scanning mi- croscope, it is possible to double the spatial resolution with Image Scanning Microscopy (ISM). This requires no further adjustments to the microscope, the measurement procedure or the sample prepara- tion. On the contrary, this technique does no longer require a confocal pinhole. Thus increasing the light detection efficiency dramatically. Hence, with the introduction of Single Photon Avalanche Diode-arrays the full potential of FLIM could also be applied to ISM. In this thesis, I will introduce a novel and unique combination of Image Scanning Microscopy and Single-Molecule Localization. That brings this resolution doubling to the world of single-molecule mi- croscopy. We achieved a localization precision in the single digit nanometre range. And with the addition of a multichannel high-speed Time-Correlated Single Photon Counting (TCSPC) electronics we im- plemented chromatic aberration free fluorescence-lifetime multiplex- ing. I also present a new and straightforward method to determine the optimal Optical Transfer Function (OTF) for any bandwidth limited imaging method. This is relevant in all areas of microscopy to correct aberration or other imaging artefacts. And due to its fundamental nature, this method can be applied to all forms of imaging processes and not only to light microscopy. In addition, I will discuss many of my unpublished contributions. Such as the development of an open-source focus hold system that works for a wide verity of microscopy methods and implementa- tions. And the upgrade to an already existing single partial tracking framework to include Image Scanning Microscopy with single photon sensitive array detectors.
Keywords: Super-resolution; Microscopy; Fluorescence lifetime
