Biolistic transfection of Physcomitrella filaments was performed as described (Jaedicke et al., 2012 (link)). For subcellular protein localization studies, transfected filaments were incubated for 2 days in D and observed via fluorescence microscopy without pre-treatment and following 1 h R pre-treatment (660 nm LEDs, 3 μmol m-2 s-1). For split-YFP studies, plant material was incubated for 3 days in D and then subjected to confocal microscopy with and without analogous pre-treatments.
Transformed cells were identified by R fluorescence of the co-transfected nuclear marker mCherry:VirD2NLS using a Leica Z16 apo fluorescence macroscope. For localization and split-YFP studies, an automated DM6000b fluorescence microscope and a TCS SP2 AOBS confocal laser scanning microscope (both Leica) were used, respectively. For filter cube and look-up table details, see Jaedicke et al. (2012) (link). Single scans of YFP fluorescence were recorded with 4x line and 6x frame average. In order to distinguish YFP signals from possible mCherry bleed-through, YFP emission detection was narrowed to 525–535 nm. The red mCherry- and chlorophyll autofluorescence and the transmission images were taken with 4x line and 2x frame average. Adobe Photoshop CS5 was used for image processing (enhancement of brightness and contrast).
Transformed cells were identified by R fluorescence of the co-transfected nuclear marker mCherry:VirD2NLS using a Leica Z16 apo fluorescence macroscope. For localization and split-YFP studies, an automated DM6000b fluorescence microscope and a TCS SP2 AOBS confocal laser scanning microscope (both Leica) were used, respectively. For filter cube and look-up table details, see Jaedicke et al. (2012) (link). Single scans of YFP fluorescence were recorded with 4x line and 6x frame average. In order to distinguish YFP signals from possible mCherry bleed-through, YFP emission detection was narrowed to 525–535 nm. The red mCherry- and chlorophyll autofluorescence and the transmission images were taken with 4x line and 2x frame average. Adobe Photoshop CS5 was used for image processing (enhancement of brightness and contrast).