Yeast cells live fluorescence imaging

Grow yeast cells overnight in Low Fluorescence (LoFlo) medium.

In the morning dilute cells in LoFlo to OD 0.1.

Grow until mid-log phase (0.5–0.8 OD).

Add 10 µl Concanavalin A to the glass surface.

Let the slides dry completely before use.

Once the cell culture has reached 0.5–0.8 OD, spin down 2 ml of the original culture. Resuspend the pellet in 30 µl LoFlo to obtain a concentrated sample.

Apply 3.5 µl of the concentrated yeast culture on the Concanavalin A-treated glass slide.

Cover the sample with a glass coverslip.

First, choose a group of cells to use to set up adequate imaging settings. Use lasers corresponding to the right fluorophores and regulate exposure time based on the brightness of the features of interest.

Set up stack acquisition. For yeast cells set a total stack size of 6-8 µm. We used a step size of 300 nm.

In order to have an unbiased approach, use Transmitted Light to select a new a field of view for data acquisition.

The field of view should contain 25-100 cells in the same focal plane.

Acquire stacks.

Count cells and analyse signal with Fiji Cell counter Plug-in.