Plan apochromat 63 1.4 n a objective lens

Yeast cells were analyzed with an Axiovert 200M microscope (Carl Zeiss) equipped with an Exfo X-cite 120 excitation light source, band pass filters (Carl Zeiss and Chroma Technology), a Plan-Apochromat 63×1.4 NA objective lens (Carl Zeiss) and a digital camera (Orca ER; Hamamatsu Photonics). Image acquisition was performed using Volocity software (PerkinElmer). Fluorescence images were collected as 0.5 μm z-stacks, merged into 1 plane as maximum intensity projections using Openlab software (PerkinElmer) and processed further in Photoshop (Adobe). Bright-field images were collected in 1 focal plane and processed to highlight the circumference of the cells in blue. Each imaging experiment was performed at least 3 times, and representative images are shown. For quantitation, 1 experiment was used.

Live cell imaging of D. hansenii and S. cerevisiae cells was performed as described previously [20 (link)]. Essentially, cells were imaged with an inverted motorized microscope (Axiovert 200 M; Carl Zeiss, Oberkochen, Germany) equipped with an Exfo X-cite 120 excitation light source containing band pass filters (Carl Zeiss, Oberkochen, Germany and Chroma Technology, Bellows Falls, VT, USA), a Plan-Apochromat 63 × 1.4 NA objective lens (Carl Zeiss, Oberkochen, Germany) and a digital camera (Orca ER; Hamamatsu Photonics, Shizuoka, Japan). Image acquisition was performed using Volocity software (PerkinElmer, Waltham, MA, USA). Fluorescence images were collected as 0.5 μm z-stacks and presented as maximum intensity projections into one plane using Openlab software (openlab 5.5.2 demo; PerkinElmer, Waltham, MA, USA) and processed further in Photoshop (Adobe). Bright-field images were collected in one plane and processed where necessary to highlight the circumference of the cells in blue and overlaid on fluorescent images.