Lsm5 microscope

Confocal images of in situ hybridizations (~30 μM z-stacks) were captured on a Zeiss LSM5 microscope using ZEN software. Time-lapse imaging of doubly transgenic fli1a:EGFP; sox10:DsRed and col2a1a_BAC:GFP; sox10:DsRed larvae followed [117 (link)], with ~130 μM of z-stacks collected every 10 or 12 minutes starting at 48 hpf. Skeletal preparations were photographed using a Leica DM2500 microscope. Image levels were adjusted in Adobe Photoshop CS6, with care taken to apply identical adjustments to images from the same data set and to avoid removing information from the image.

Light and fluorescence microscopic pictures were captured with a 63x C-apochromatic objectives on an inverted LSM 5 microscope equipped with a laser scanning-disk confocal system (Zeiss). For life imaging of fungal hyphae, conidia were pre-grown on MM agar plate at 30 °C for 1 day. An agar piece containing mycelium was cut out and placed, upside down, onto an objective glass. To prevent drying-out of the agar/mycelium piece, 50 μl of MM was applied between the colony and the objective glass. After cells resumed growth (around one hour after the transfer) images were captured.

Phase contrast and epifluorescence imaging were performed using a Delta Vision system (Applied Precision Inc.) on an Olympus IX inverted microscope equipped with a cooled CCD camera. For high magnification images a 60×/1.3 NA (Olympus) oil-immersion objective was used. Epifluorescence microscopy was additionally performed using a Leica DM6000B upright microscope equipped with a CCD camera. A water immersion objective 40×/0.8 NA (Leica) was used. Laser scanning confocal microscopy (LCSM) was performed on a Zeiss LSM 5 microscope using a 40×/1.2 NA (Zeiss) water immersion objective.
Cells were fixed with 4% paraformaldehyde in PBS (15 minutes in PBS) and stained with fluorescent agents. Wheat germ agglutinin, AlexaFluo 488 conjugate (WGA; 10 µg/ml) was used to stain plasma membrane, phalloidin-tetramethyl rhodamine B isothiocyanate (Phalloidin-TRITC; 2.5 µg/ml) stained filamentous actin and 4′,6-diamidino-2-phenylindole (DAPI; 1.0 µg/ml) labeled cell nuclei. Projected cell area was calculated using the Cell Outliner plugin of ImageJ software (NIH) from fluorescence microscopy images of WGA-stained cells.