Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Summary

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Abstract

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

Introduction

Single-cell analysis of gene expression has furthered our understanding of many aspects of gene regulation. Static snapshots of fluorescent reporter expression using flow cytometry or microscopy provide useful information on the distribution of single-cell expression, but lack the history and evolution of time series data required to directly inform gene expression dynamics. Fluorescence time-lapse microscopy presents a means to obtain both single-cell measurements and their history. Various experimental and analytical techniques have been developed to obtain and quantify movies of fluorescent reporter expression, thus imparting insights into gene regulation features ....

Protocol

1. Obtain Fluorescent Microscopy Movies of Single Yeast Cells Growing in a Microfluidic Chamber

1. Inoculate 1 ml SC media (synthetic defined media with 2% glucose and complete complement of amino acids) with cells from a freshly growing plate, and incubate the culture overnight ~16 hours on a roller drum at 30 °C. Prepare the culture such that the final OD_600nm is ~0.1 for early log-phase growth.
2. Dilute the starter culture as needed and regrow 1 ml in a fresh test tube an additional 6-8 hours to OD_600nm = 0.1. This provides cells growing at a nutrient-rich steady state at a density suitable for loading into the microfluidi....

Results

A successfully performed and analyzed experiment will yield mostly continuous time series for single whole cells with realistically assigned bud emergence and division times. As an example, we performed the above protocol with a haploid yeast strain expressing an integrated copy of Cerulean fluorescent protein (CFP) driven by the constitutive ADH1 promoter to observe how growth and global expression may vary over time in single cells (Table 4, Y47). We ran the time series analysis to ob.......

Discussion

The above protocol describes a simple method to obtain and analyze fluorescence time series data with limited experience in microfluidics or in software development. It allows one to obtain time-lapse fluorescence movies of single yeast cells; extract relevant cell size and expression measurements; curate tracking and lineage assignments; and analyze the behavior of whole cells over time using a commercially available microfluidic culture device and a versatile graphical user interface (GUI). While the experimental, segm.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Y04C Yeast Perfusion Plate	CellAsic	Y04C-02
ONIX Microfluidic Perfusion Platform	CellAsic	EV-262
Axio Observer.Z1 Microscope	Zeiss
Plan-Apochromat 63x/1.40 Oil DIC objective	Zeiss	440762-9904-000
Cascade II EMCCD camera	Photometrics
Lumen 200 metal-halide arc lamp	PRIOR Scientific
Triple-bandpass dichroic filter cube and excitation and emission filter set	Chroma Technology Corp	set #89006	Used for YFP (Venus/Citrine), CFP (Cerulean), RFP (mCherry/tdTomato)
MAC 5000 controller and filter wheels	Ludl Electronic Products
MATLAB R2011a	Mathworks		64-bit version handles large data files better than 32-bit