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Dissertation Submitted to the Combined Dissertation submitted to the Combined faculties for the natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Put forward by Dipl.-Phys. Uroš Kržič Born in Slovenj Gradec, Slovenia Oral examination: 8th July 2009 Multiple-view microscopy with light-sheet based fluorescence microscope Referees: Prof. Dr. Jörg Schmiedmayer, Vienna University of Technology Prof. Dr. Bernd Jähne, Heidelberg University Zusammenfassung: Die axiale Auflösung jedes auf einem einzelnen Objektiv basierenden Lichtmikroskops ist schlechter als seine laterale Auflösung. Daher ist auch die Auflösung in einem konfokalen oder einem zweiphotonenabsorbierenden Fluoreszenzmikroskop entlang der optischen Achse schlechter als in der Fokalebene. Das Verhältnis der Auflösungen ist 3 bis 4 bei hohen numerischen Aperturen (NA 1,2 – 1,4) und kann für niedrige numerische Aperturen (NA < 0,2) sogar Werte von 10 bis 15 erreichen. Damit ist der Einsatz der konventionellen Lichtmikroskopie gerade für große Objekte eventuell sehr stark eingeschränkt. Die schlechte axiale Auflösung ist nicht ausreichend, um kleine Objekte innerhalb einer Zelle zu lokalisieren. Dazu kommt, dass große Objekte nicht komplett erfasst werden können. Die Beobachtung entlang mehrerer Raumrichtungen stellt sich dieser Aufgabe, indem sie Bildstapel desselben Objekts entlang verschiedener Winkel aufzeichnet. Diese unabhängig voneinander aufgezeichneten Bildstapel werden in einem nachfolgenden Prozess zu einem neuen Datensatz zusammengefasst. Die Datenaufzeichnung entlang unterschiedlicher Raumrichtungen wurde am EMBL im Rahmen der Fluoreszenz-Lichtscheibenmikroskopie entwickelt (LSFM). Das LSFM ist bislang das einzige bekannte Mikroskop, an dem ein solches Konzept zur Datenfusion erfolgreich demonstriert werden konnte. In dieser Arbeit werden die Aspekte eines LSFM, die für die Aufnahmen entlang unterschiedlicher Raumrichtungen wichtig sind, charakterisiert. Außerdem wird die Implementierung eines LSFM ausführlich beschrieben. Wesentliche Aspekte werden sorgfältig diskutiert und in den allgemeinen Kontext bereits publizierter Arbeiten gestellt. Die Bilderfassung unterschiedlicher Objekte (u.a. Medaka Fisch, Fruchtfliege, Bäckerhefe) illustriert zwar die Grenzen aber vor allem die Möglichkeiten. Abstract: The axial resolution of any standard single-lens light microscope is lower than its lateral resolution. The ratio is approximately 3-4 when high numerical aperture objective lenses are used (NA 1.2 -1.4) and more than 10 with low numerical apertures (NA 0.2 and below). In biological imaging, the axial resolution is normally insufficient to resolve subcellular phenomena. Furthermore, parts of the images of opaque specimens are often highly degraded or obscured. Multiple-view fluorescence microscopy overcomes both problems simultaneously by recording multiple images of the same specimen along different directions. The images are digitally fused into a single high-quality image. Multiple-view imaging was developed as an extension to the light-sheet based fluorescence microscope (LSFM), a novel technique that seems to be better suited for multiple-view imaging than any other fluorescence microscopy method to date. In this contribution, the LSFM properties, which are important for multiple-view imaging, are characterized and the implementation of LSFM based multiple-view microscopy is described. The important aspects of multiple-view image alignment and fusion are discussed, the published algorithms are reviewed and original solutions are proposed. The advantages and limitations of multiple-view imaging with LSFM are demonstrated using a number of specimens, which range in size from a single yeast cell to an adult fruit fly and to Medaka fish. TABLE OF CONTENTS 1 Introduction .............................................................................................................................. 5 1.1 Optical microscopy ........................................................................................................... 6 1.2 Trends in biological imaging ............................................................................................. 7 1.3 Fluorescence microscopy ................................................................................................. 7 1.4 Fluorophores .................................................................................................................... 8 1.5 Fluorescent dyes ............................................................................................................. 11 1.5.1 Fluorescent proteins ............................................................................................... 12 1.5.2 Quantum dots ......................................................................................................... 13 1.6 Common fluorescence microscopy techniques ............................................................. 13 1.6.1 Wide-field fluorescence microscope ...................................................................... 14 1.6.2 Confocal microscope .............................................................................................. 19 1.6.3 Two-photon microscope ........................................................................................ 22 1.6.4 Other optical sectioning microscopes .................................................................... 24 1.6.5 Lateral vs. axial resolution ...................................................................................... 25 1.6.6 Super-resolution methods ...................................................................................... 27 2 Light-sheet based fluorescence microscope (LSFM) .............................................................. 31 2.1 Use of light-sheets in light microscopy .......................................................................... 33 2.2 Basic principles ............................................................................................................... 35 2.2.1 Detection unit ......................................................................................................... 35 2.2.2 Illumination unit ..................................................................................................... 39 2.2.3 Single plane illumination microscope (SPIM) ......................................................... 41 2.2.4 Digital scanned laser light sheet microscope (DSLM)............................................. 51 2.2.5 Specimen translation and rotation ......................................................................... 52 2.3 Specimen preparation and mounting ............................................................................ 53 4 | M u l t i p l e - view microscopy with LSFM 2.4 LSFM application: imaging of hemocyte migration in a Drosophila melanogaster embryo ....................................................................................................................................... 57 2.4.1 Drosophila m. hemocytes ...................................................................................... 57 2.4.2 Drosophila transgenes ........................................................................................... 59 2.4.3 Automated hemocyte tracking .............................................................................. 60 2.4.4 Laser induced wounding and wound induced hemocyte migration ..................... 62 3 Multiple-view microscopy with LSFM .................................................................................... 65 3.1 Motivation ...................................................................................................................... 66 3.2 Multiple-view imaging in microscopy ............................................................................ 69 3.3 Multiple-view microscopy with LSFM ............................................................................ 71 3.4 Multiple-view image acquisition .................................................................................... 72 3.5 Multiple-views image fusion .......................................................................................... 76 3.5.1 Image formation and sampling .............................................................................. 78 3.5.2 Preprocessing ......................................................................................................... 79 3.5.3 Image registration .................................................................................................. 80 3.5.4 Final image fusion .................................................................................................. 96 3.6 Examples of multiple-view microscopy on biological specimens ................................ 124 4 Summary and outlook .......................................................................................................... 133 Bibliography ................................................................................................................................. 135 Abbrevations ................................................................................................................................ 145 Table of figures ............................................................................................................................ 147 Acknowledgements ...................................................................................................................... 151
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