Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Summary

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 10⁶ measurements of individual cell morphology and 10584 cell cycle tracks.

Abstract

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 10⁶ cell morphological measurements.

Introduction

Monitoring cultured mammalian cells throughout several cell cycles and measuring accurately cell size and cell dry mass is a challenging task. Several label-free optical techniques are able to perform this task^1,2: phase-shifting interferometry³, digital holographic microscopy (DHM)^4,5,6,7, quadriwave lateral shearing interferometry^8,9 and quantitative phase tomography^10,

Protocol

1. Cell Culture Monitoring Acquisition

1. Grow HeLa cells in DMEM + glutamine (e.g., GlutaMAX) medium supplemented with 10% (v/v) heat-inactivated fetal calf serum and 1% penicillin and streptomycin.
2. Coat 6-well glass bottom culture plates with fibronectin (25 µg/mL) for 1 h. Then seed 2 x 10⁴ cells per well.
3. During the acquisition, change the medium every 3 days.
4. For the time-lapse acquisition, use the video lens-free microscope (commercially available).
   NOTE: This is based on the lens-free computational imaging technique as described by Ozcan et al.¹⁶ which was modified ....

Results

For the holographic reconstruction process, the light field is described by a scalar field A (where is the complex value of A on the plane at distance z from the sample, and lateral position

Discussion

In this paper, we show that lens-free video microscopy can be used inside an incubator to capture the kinetics of thousands of cells. In order to describe the overall methodology we explained how a 2.7 day time-lapse acquisition of HeLa cells in culture can be analyzed with standard cell-tracking algorithms. The result is a dataset featuring 2.2 x 10⁶ cell measurements and 10,584 cell cycle tracks. The acquisitions were performed on a culture of Hela cells with a relatively large cell-to-cell distance (cell de.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cytonote lens-free video microscope	Iprasense
Horus acquisition software	Iprasense
6-well glass bottom culture plates	MatTek corporation	Part No: P06G-0-14-F
DMEM + GlutaMAX medium	Gibco
heat-inactivated fetal calf serum	Eurobio
penicillin and streptomycin	Gibco
Fibronectin	Sigma Aldrich
Matlab, image processing toolbox	Mathworks