πSPIM: high NA high resolution isotropic light-sheet imaging in cell culture dishes.
Light-sheet fluorescence microscopy (LSFM), also termed single plane illumination microscopy (SPIM), enables live cell fluorescence imaging with optical sectioning capabilities superior to confocal microscopy and without any out-of-focus exposure of the specimen. However, the need of two objective lenses, one for light-sheet illumination and one for imaging, imposes geometrical constraints that require LSFM setups to be adapted to the specific needs of different types of specimen in order to obtain optimal imaging conditions. Here we demonstrate the use of an oblique light-sheet configuration adapted to provide the highest possible Gaussian beam enabled resolution in LSFM. The oblique light-sheet configuration furthermore enables LSFM imaging at the surface of a cover slip, without the need of specific sample mounting. In addition, the system is compatible with simultaneous high NA wide-field epi-fluorescence imaging of the specimen contained in a glass-bottom cell culture dish. This prevents cumbersome sample mounting and enables rapid screening of large areas of the specimen followed by high-resolution LSFM imaging of selected cells. We demonstrate the application of this microscope for in toto imaging of endocytosis in yeast, showing for the first time imaging of all endocytic events of a given cell over a period of >5 minutes with sub-second resolution.
Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.|Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Patrick
P
Theer
Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Denitsa
D
Dragneva
Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.|Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Michael
M
Knop
D045744:Cell Line, Tumor; D004705:Endocytosis / Q000502:physiology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology