Name: lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture
Uploaded: 2018-02-23T14:00:00
Description: Watch this Scientific Journal Video about Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture at JoVE.com

The authors have nothing to acknowledge.

1. Cell Culture Monitoring Acquisition
<ol>
	<li>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.</li>
	<li>Coat 6-well glass bottom culture plates with fibronectin (25 &#181;g/mL) for 1 h. Then seed 2 x 104 cells per well.</li>
	<li>During the acquisition, change the medium every 3 days.</li>
	<li>For the time-lapse acquisition, use the video lens-free microscope (commercially available).<br...

<ol>
	<li>Zangle, T. A., Teitell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+mass+profiling:+an+emerging+approach+in+quantitative+biophysics.">Live-cell mass profiling: an emerging approach in quantitative biophysics.</a> Nat Methods. 11 (12), 1221-1228 (2014).</li><li>Popescu, G., Park, K., Mir, M., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+technologies+for+measuring+single+cell+mass.">New technologies for measuring single cell mass.</a> Lab Chip. 14 (4), 646-652 (2014).</li><li>Reed, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+massively+parallel+single-cell+drug+response+measurements+via+live+cell+interferometry.">Rapid, massively parallel single-cell drug response measurements via live cell interferometry.</a> Biophys J. 101 (5), 1025-1031 (2011).</li><li>Mir, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurement+of+cycle-dependent+cell+growth.">Optical measurement of cycle-dependent cell growth.</a> Proc Natl Acad Sci. USA. 108 (32), 13124-13129 (2011).</li><li>Girshovitz, P., Shaked, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+cell+morphological+parameters+based+on+interferometric+phase+microscopy+and+their+application+to+cell+life+cycle+characterization.">Generalized cell morphological parameters based on interferometric phase microscopy and their application to cell life cycle characterization.</a> Biomed Opt Express. 3 (8), 1757-1773 (2012).</li><li>Kemper, B., Bauwens, A., Vollmer, A., Ketelhut, S., Langehanenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+quantitative+cell+division+monitoring+of+endothelial+cells+by+digital+holographic+microscopy.">Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy.</a> J Biomed Opt. 15 (3), (2010).</li><li>Mir, M., Bergamaschi, A., Katzenellenbogen, B. S., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+quantitative+imaging+for+monitoring+single+cancer+cell+growth+kinetics+and+drug+response.">Highly sensitive quantitative imaging for monitoring single cancer cell growth kinetics and drug response.</a> PLoS One. 9 (2), 1-8 (2014).</li><li>Bon, P., Savatier, J., Merlin, M., Wattellier, B., Monneret, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+detection+and+measurement+of+living+cell+morphometric+features+with+single-shot+quantitative+phase+microscopy.">Optical detection and measurement of living cell morphometric features with single-shot quantitative phase microscopy.</a> J Biomed Opt. 17 (7), (2012).</li><li>Aknoun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+cell+dry+mass+measurement+usinq+quantitative+phase+imaging+with+quadriwave+lateral+shearing+interferometry:+an+accuracy+and+sensitivity+discussion.">Living cell dry mass measurement usinq quantitative phase imaging with quadriwave lateral shearing interferometry: an accuracy and sensitivity discussion.</a> J Biomed Opt. 20 (1), 1-4 (2015).</li><li>Cotte, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marker-free+phase+nanoscopy.">Marker-free phase nanoscopy.</a> Nature Photonics. 7 (2), 113-117 (2013).</li><li>Choi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+phase+microscopy.">Tomographic phase microscopy.</a> Nat Methods. 4 (9), 717-719 (2007).</li><li>Kesavan, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+monitoring+of+major+cell+functions+by+means+of+lensfree+video+microscopy.">High-throughput monitoring of major cell functions by means of lensfree video microscopy.</a> Sci Rep. 4, 1-11 (2014).</li><li>Zheng, G., Lee, S. A., Antebi, Y., Elowitz, M. B., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ePetri+dish,+an+on-chip+cell+imaging+platform+based+on+subpixel+perspective+sweeping+microscopy+(SPSM).">The ePetri dish, an on-chip cell imaging platform based on subpixel perspective sweeping microscopy (SPSM).</a> Proc Natl Acad Sci USA. 108 (41), 16889-16894 (2011).</li><li>Pushkarsky, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+single-cell+motility+analysis+on+a+chip+using+lensfree+microscopy.">Automated single-cell motility analysis on a chip using lensfree microscopy.</a> Sci Rep. 4, 4717 (2014).</li><li>Allier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+dense+cell+cultures+by+multiwavelength+lens-free+video+microscopy.">Imaging of dense cell cultures by multiwavelength lens-free video microscopy.</a> Cytom Part A. 91 (5), 1-10 (2017).</li><li>Su, T. -. W., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+lensfree+imaging+and+characterization+of+a+heterogeneous+cell+solution+on+a+chip.">High-throughput lensfree imaging and characterization of a heterogeneous cell solution on a chip.</a> Biotechnol Bioeng. 102 (3), 856-868 (2009).</li><li>Delacroix, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+lens-free+microscopy:+a+new+tool+for+the+laboratory+diagnosis+of+meningitis.">Cerebrospinal fluid lens-free microscopy: a new tool for the laboratory diagnosis of meningitis.</a> Sci Rep. 7, 39893 (2017).</li><li>Tinevez, J. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrackMate:+an+open+and+extensible+platform+for+single-particle+tracking.">TrackMate: an open and extensible platform for single-particle tracking.</a> Methods. 115, 80-90 (2016).</li><li>Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+cell+mass+and+growth+dynamics.">Optical imaging of cell mass and growth dynamics.</a> Am J Physiol Physiol. 295 (2), 538-544 (2008).</li><li>Liu, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+refractive+index+for+cell+biology+and+disease+diagnosis:+past,+present+and+future.">Cell refractive index for cell biology and disease diagnosis: past, present and future.</a> Lab Chip. 16, 634-644 (2016).</li><li>Rapoport, D. H., Becker, T., Mamlouk, A. M., Schicktanz, S., Kruse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+validation+algorithm+allows+for+automated+cell+tracking+and+the+extraction+of+biologically+meaningful+parameters.">A novel validation algorithm allows for automated cell tracking and the extraction of biologically meaningful parameters.</a> PLoS One. 6 (11), e27315 (2011).</li><li>Al-Kofahi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+cell+lineage+construction:+A+rapid+method+to+analyze+clonal+development+established+with+murine+neural+progenitor+cells.">Automated cell lineage construction: A rapid method to analyze clonal development established with murine neural progenitor cells.</a> Cell Cycle. 5 (3), 327-335 (2006).</li><li>Meijering, E., Dzyubachyk, O., Smal, I., van Cappellen, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+in+cell+and+developmental+biology.">Tracking in cell and developmental biology.</a> Semin Cell Dev Biol. 20 (8), 894-902 (2009).</li><li>Posakony, J. W., England, J. M., Attardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+growth+and+division+during+the+cell+cycle+in+HeLa+cells.">Mitochondrial growth and division during the cell cycle in HeLa cells.</a> J Cell Biol. 74 (2), 468-491 (1977).</li><li>Zocchi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD38+Increases+Intracellular+Calcium+Concentration+and+Reduces+Doubling+Time+in+HeLa+and+3T3+Cells.">Expression of CD38 Increases Intracellular Calcium Concentration and Reduces Doubling Time in HeLa and 3T3 Cells.</a> J Biol Chem. 273 (14), 8017-8024 (1979).</li><li>Reitzer, L. J., Wice, B. M., Kennell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+glutamine,+not+sugar,+is+the+major+energy+source+for+cultured+HeLa+cells.">Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells.</a> J Biol Chem. 254 (8), 2669-2676 (1979).</li><li>Benedetti, A. D. E., Joshi-barve, S., Rinker-Schaeffer, C., Rhoads, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Antisense+RNA+against+Initiation+Factor+eIF-4E+mRNA+in+HeLa+Cells+Results+in+Lengthened+Cell+Division+Times,+Diminished+Translation+Rates,+and+Reduced+Levels+of+Both+eIF-4E+and+the+p220+Component+of+eIF-4F.">Expression of Antisense RNA against Initiation Factor eIF-4E mRNA in HeLa Cells Results in Lengthened Cell Division Times, Diminished Translation Rates, and Reduced Levels of Both eIF-4E and the p220 Component of eIF-4F.</a> Mol Cell Biol. 11 (11), 5435-5445 (1991).</li><li>Kumei, Y., Nakajima, T., Sato, A., Kamata, N., Enomoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+G1+phase+duration+and+enhancement+of+c-myc+gene+expression+in+HeLa+cells+at+hypergravity.">Reduction of G1 phase duration and enhancement of c-myc gene expression in HeLa cells at hypergravity.</a> J Cell Sci. 93 (2), 221-226 (1989).</li><li>Ginzberg, M. B., Kafri, R., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+being+the+right+(cell)+size.">On being the right (cell) size.</a> Science. 348 (6236), 1245075 (2015).</li><li>Mathieu, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+lens-free+imaging+of+cell+migration+in+diverse+physical+microenvironments.">Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.</a> Lab Chip. 16 (17), 3304-3316 (2016).</li></ol>

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...

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 task1,2: phase-shifting interferometry3, digital holographic microscopy (DHM)4,5,6,7, quadriwave lateral shearing interferometry8,9 and quantitative phase tomography10,11. These methods have led to many new insights into the understanding of the cell cycle of mammalian cells. However they are seldom coupled with automatic cell tracking algorithms and their throughput remains limited when measuring cell mass trajectories1 (N&#60;20 in respectively3,4,5,6). Hence a novel optical method is needed to measure cell mass trajectories with large statistics (N&#62;1000).
In this paper, we demonstrate the capability of lens-free video microscopy to simultaneously image thousands of cells directly inside the incubator, and then quantify single cell metrics along thousands of individual cell cycle tracks. Lens-free microscopy is a quantitative phase imaging technique which allows the acquisition of phase image of densely packed cells over a very large field of view (typically several tens of mm2, here 29.4 mm2)12,13,14,15. Several metrics at the single cell level are determined, e.g., cell area, cell dry mass, cell thickness, cell major axis length and cell aspect ratio12,15, from each image. Then, by applying a cell-tracking algorithm, these features can be plotted for every single cell as a function of the experiment time14,15. Furthermore, by detecting the occurrence of cell divisions in the cell tracks, it is possible to extract other important information such as the initial cell dry mass (just after the cell division), the final cell dry mass (just before the cell division) and the cell cycle duration, i.e., the time between two consecutive divisions15. All these measurements can be computed with very good statistics (N&#62;1000) since the large field of view would typically allow the analysis of 200 to 10,000 cells in a single lens-free acquisition.
In order to explain this methodology based on lens-free video microscopy, we describe the protocol to monitor and quantify a HeLa cell culture for 2.7 days. Data analysis is a four-step process based on multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division algorithms. Here it is shown that the spatial resolution and the relatively fast frame rate (one acquisition every 10 minutes) obtained with this lens-free video microscopy setup is compatible with standard cell-tracking algorithms. The full analysis of this dataset results in the measurement of 10,584 cell tracks over complete cell cycles.
To summarize, lens-free video microscopy is a powerful tool to automatically monitor thousands of unlabeled, unsynchronized, and unmodified cells per experiment; each cell being tracked over several cell cycles. Our measurements thus provide the mean value of several cell parameters, but more importantly, the inter-cell variability over a large population of cells.

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Cytonote lens-free video microscope&#160;</td>
			<td style="width:208px;">Iprasense</td>
			<td style="width:156px;"></td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horus acquisition software</td>
			<td>Iprasense</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well glass bottom culture plates</td>
			<td>MatTek corporation</td>
			<td>Part No: P06G-0-14-F&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM + GlutaMAX medium&#160;</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">heat-inactivated fetal calf serum&#160;</td>
			<td>Eurobio</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin and streptomycin&#160;</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin&#160;</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matlab, image processing toolbox&#160;</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture

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.

For the holographic reconstruction process, the light field is described by a scalar field A (where <img alt="Equation 4" src="/files/ftp_upload/56580/56580eq4v3.jpg" /> is the complex value of A on the plane at distance z from the sample, and lateral position <img alt="Equation 5" src="/files/ftp_upload/56580/56580eq5v3.jpg" /> and at wavelength &#955;). Light propagation is modeled by the Huygens-Fresnel theory which provides a propagator kernel <img alt="Equa...

Watch this Scientific Journal Video about Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture at JoVE.com

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

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.

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Research

JoVE Journal

Biology

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...