Hyperspectral light sheet microscopy.
To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos.
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany.|DIGS-BB, Technische Universität Dresden, Pfotenhauer Str. 108, 01307 Dresden, Germany.
Wiebke
http://orcid.org/0000-0003-0201-2315
W
Jahr
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany.
Benjamin
B
Schmid
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany.
Christopher
C
Schmied
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany.
Florian O
FO
Fahrbach
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany.
Jan
http://orcid.org/0000-0001-7250-3756
J
Huisken
D000818:Animals; D004331:Drosophila melanogaster; D004625:Embryo, Nonmammalian; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence / Q000379:methods*; D030781:Organisms, Genetically Modified; D015027:Zebrafish
D005456:Fluorescent Dyes