Jun 02, 2022

Public workspaceYeast cells live fluorescence imaging

DOI
dx.doi.org/10.17504/protocols.io.n92ldzqjnv5b/v1
  • 1Max Planck Institute of Biophysics, Frankfurt;
  • 2Max Planck Institute of Biochemistry
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DOI: dx.doi.org/10.17504/protocols.io.n92ldzqjnv5b/v1
Protocol Citation: Florian Wilfling, Fabian Fiedler, Anna Bieber, Cristina Capitanio 2022. Yeast cells live fluorescence imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldzqjnv5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 24, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63135
Keywords: #yeast, #fluorescence, #glass slides coating, ASAPCRN
Abstract
Protocol for sample preparation and live fluorescence imaging of yeast cells.
Materials
MEDIA
- LoFlo medium (Yeast Nitrogen Base w/o Amino Acids and w/o Folic Acid and Riboflavin. Formedium)

REAGENTS
- Concanavalin A solution: 1 mg ml−1 Concanavalin A dissolved in purified water. (sterile filtered 0.2 µm)

INSTRUMENTS
- Nikon Ti2 Eclipse microscope, with Olympus Apo TIRF 100x 1.49 oil objective and Hamamatsu ORCA-Flash 4.0 LT+ Digital CMOS camera

SOFTWARE
- CellCounter plugin, ImageJ


Yeast cell culture in low fluorescence medium
Yeast cell culture in low fluorescence medium
12h
12h
Grow yeast cells overnight in Low Fluorescence (LoFlo) medium.
12h
In the morning dilute cells in LoFlo to OD 0.1.
Grow until mid-log phase (0.5–0.8 OD).
Pre-treat microscope slides with Concanavalin A solution
Pre-treat microscope slides with Concanavalin A solution
1h
1h
Add 10 µl Concanavalin A to the glass surface.
Let the slides dry completely before use.
Prepare the sample for imaging
Prepare the sample for imaging
20m
20m
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.
Imaging procedure:
Imaging procedure:
1h
1h
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.
Image analysis
Image analysis
Count cells and analyse signal with Fiji Cell counter Plug-in.