Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy.
Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.
Keller
Philipp J
PJ
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany. kellerp@janelia.hhmi.org
Schmidt
Annette D
AD
Santella
Anthony
A
Khairy
Khaled
K
Bao
Zhirong
Z
Wittbrodt
Joachim
J
Stelzer
Ernst H K
EH
D000818:Animals; D004331:Drosophila melanogaster / Q000254:growth & development; D004625:Embryo, Nonmammalian; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D015027:Zebrafish / Q000254:growth & development