Multiple-view microscopy with light sheet based fluorescence microscope [Elektronische Ressource] / put forward by Uroš Kržič

Uros

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• Naturwissenschaften

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 39
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

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
thOral examination: 8 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 - v i e w m i c r o s c o p y w i t h L S F M

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
1 INTRODUCTION

The human mind prefers something, which it can
recognize to something for which it has no name, and,
whereas thousands of persons carry field glasses to
bring horses, ships, or steeples close to them, only a few
carry even the simplest pocket microscope. Yet a small
microscope will reveal wonders a thousand times more
thrilling than anything, which Alice saw behind the
looking-glass.

David Fairchild, American botanist
The World Was My Garden (1938)


Sight is regarded as the single most important channel through which the human mind perceives
its surrounding world. It see