3D STED Microscopy with Pulsed and Continuous Wave Lasers
3D STED Mikroskopie mit gepulsten und Dauerstrichlasern
Benjamin Harke
Doctoral thesis
Date of Examination:
2008-04-02
Date of issue:
2008-04-16
Advisor:
Hell, Stefan Prof. Dr.
Referee:
Hell, Stefan Prof. Dr.
Referee:
Münzenberg, Markus Prof. Dr.
Persistent Address: http://hdl.handle.net/11858/00-1735-0000-000D-F146-5
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Abstract
English
STED microscopy improves the resolution of fluorescence microscope by effectively quenching the population of the excited state in the outer regions of the focus. The resolution capability in STED microscopy has already been shown in optical sections particularly in the lateral directions. Biological samples have been prepared in a manner, that the resolution enhancement in the lateral plane is sufficient to tackle the biological problem. However, a cell for example is a three dimensional object and though has to be recorded in all spatial directions in order to get information about structural details. The way to three dimensional imaging with an enhanced resolution in all directions will be presented in this work. The influence of different depletion patterns on the resolution of the STED microscope is thereby a very important parameter. A quantitative investigation of the resolution enhancement in a STED microscope will be discussed in the following chapter. The capability of performing three dimensional imaging will be presented in chapter 3. Thereby for the first time the incoherently combination of two depletion patterns - one for the resolution enhancement in the lateral plane and one for the axial direction - will be presented. The resulting minimized extent of the focal spot enables the three dimensional imaging of colloidal crystals with an enhanced resolution in all three directions. Currently the implementation of STED microscopy as a standard imaging tool is mainly inhibited by the complex experimental platform including sophisticated and expensive laser sources as well as electrical equipment. In chapter 4, this thesis shows the first ever use of continuous wave (CW) lasers for the excitation and STED light sources. The simplicity of the setup can facilitate the way to a wide use of STED microscopy allowing in principle every existing scanning fluorescence microscope to perform high resolution.
Keywords: Microscopy; Fluorescence; High resolution; STED
Other Languages
STED Mikroskopie erhöht das
Auflösungsvermögen eines Fluoreszenzmikroskops, indem die
Bevölkerung des angeregten Zustandes in den äußeren Regionen des
Fokus effektiv reduziert wird.
Die Auflösungserhöhung in der lateralen Ebene wurde bereits in
Publikationen validiert. Viele Proben können in der Weise
präpariert werden, dass diese Auflösungserhöhung ausreichend ist.
Allerdings gibt es Proben, beispielsweise biologische Strukturen,
die eine Auflösungserhöhung in alle 3 Raumrichtungen benötigen, um
den Aufbau der Zelle voll aufzulösen. Die Problematik der
dreidimensionalen Auflösungserhöhung wird in dieser Arbeit
diskutiert.
Die Komplexität des daraus resultierenden experimentellen Aufbaus
kann durch den Einsatz von Dauerstrichlasern anstatt gepulster
Strahlquellen deutlich reduziert werden. Der Einsatz von
Dauerstrichlasern in einem STED Mikroskop wird im letzten Kapitel
dieser Arbeit präsentiert.
Schlagwörter: Mikroskop; Fluoreszenz; Hochauflösung; STED
