Name: live-cell imaging of single-cell arrays (lisca) - a versatile technique to quantify cellular kinetics
Uploaded: 2021-03-18T14:30:00
Description: Watch this Scientific Journal Video about Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics at JoVE.com

This work was supported by grants from the German Science Foundation (DFG) to Collaborative Research Center (SFB) 1032. Support by the German Federal Ministry of Education, Research and Technology (BMBF) under the cooperative project 05K2018-2017-06716 Medisoft as well as a grant from the Bayerische Forschungsstiftung are gratefully acknowledged. Anita Reiser was supported by a DFG Fellowship through the Graduate School of Quantitative Biosciences Munich (QBM).

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62025/62025fig02.jpg" /> 
Figure 2: Data acquisition combining single-cell microarrays (A) with scanning time-lapse microscopy (B).&#160;As preparation of the time-lapse experiment, a single-cell array with a 2D micropattern of adhesion squares is prepared (1), followed by cell seeding and the alignment of the cells on the micropattern (2) as well as the connection of a perfusion system to the six-channel slide,...

<ol>
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MIAAB, 29262 Available from: <a target="_blank" href="https://github.com/SoftmatterLMU-RaedlerGroup/pyama">https://github.com/SoftmatterLMU-RaedlerGroup/pyama</a> (2020)</li><li>Reiser, A., 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=Correlation+of+mRNA+delivery+timing+and+protein+expression+in+lipid-based+transfection.">Correlation of mRNA delivery timing and protein expression in lipid-based transfection.</a> Integrative Biology. 11 (9), 362-371 (2019).</li><li>Reiser, A., Wosch&#233;e, D., Mehrotra, N., Krzyszto&#324;, R. S., Strey, H. H., R&#228;dler, J. 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Here we described LISCA as a versatile technique to follow cellular kinetics of intracellular fluorescent labels at the single-cell level. In order to perform a successful LISCA experiment, each of the described steps of the protocol section must be established individually and then all steps must be combined. Each of the three major aspects of LISCA feature crucial steps.
Single-cell microarray fabrication 
The quality of the microarray is crucial as the cellular alignment on th...

In recent years, the importance of single-cell experiments has become apparent. Data from single cells allow the investigation of cell-to-cell variability, the resolution of intracellular parameter correlations and the detection of cellular kinetics that remain hidden in ensemble measurements1,2,3. In order to investigate cellular kinetics of thousands of single cells in parallel, new approaches are needed that enable monitoring the cells under standardized conditions over a time period of several hours up to several days followed by a quantitative data analysis 4. Here, we present Live-cell Imaging of Single-Cell Arrays (LISCA), which combines the use of microstructured arrays with time-lapse microscopy and automated image analysis.
Several methods for generating microstructured single-cell arrays have been established and published in literature5,6. Here, we briefly describe Microscale Plasma-Initiated Protein Patterning (&#181;PIPP). A detailed protocol of the single-cell array fabrication using &#181;PIPP is also found in reference7. The use of single-cell arrays enables alignment of thousands of cells on standardized adhesion spots presenting defined microenvironments for each cell and thus reduces a source of experimental variability (Figure 1A). Single-cell arrays are used to monitor the time courses of fluorescent markers purposed to indicate a variety of cellular processes. Long-term microscopy in scanning time-lapse mode allows for monitoring a large area of the single-cell arrays and hence sampling single-cell data in high-throughput over an observation time of several hours or even days. This generates time-line stacks of images from each position of the array (Figure 1B). In order to reduce the large amount of image data and to extract the desired single-cell fluorescence time courses in an efficient way, automated image processing is required that takes advantage of the positioning of cells (Figure 1C).
The challenge of LISCA is to adapt the experimental protocols and computational tools to form a high-throughput assay that generates quantitative and reproducible data of cellular kinetics. In this article we provide a step-by-step description of the individual methods and how they are combined in a LISCA assay. As an example, we discuss the time course of enhanced green fluorescent protein (eGFP) expression after artificial mRNA delivery. eGFP expression following mRNA delivery is described by reaction rate equations modeling translation and degradation of mRNA. Fitting the model function for the time course of eGFP concentration to the LISCA readout of the fluorescence intensity for each individual cell over time yields best estimates of model parameters such as the mRNA degradation rate. As a representative result we discuss the mRNA delivery efficiency of two different lipid-based transfection agents and how their parameter distributions differ.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62025/62025fig01.jpg" /> 
Figure 1: Representation of the LISCA workflow combining (A) micro-patterned single-cell arrays (B) scanning time-lapse microscopy and (C) automated image analysis of recorded image series.&#160;The single-cell arrays consist of a two-dimensional pattern of cell-adhesive squares with a cell-repellent interspace leading to an arrangement of the cells on the micropattern, as can be seen in the phase-contrast image as well as the fluorescence image of eGFP expressing cells (A). The entire microstructured area is imaged in a scanning time-lapse mode repeatedly taking images at a sequence of positions (B). Recorded image series are processed to read out the fluorescence intensity per cell over time (C). Scale bars: 500 &#181;m (A), 200 &#181;m (C).&#160;<a href="https://www.jove.com/files/ftp_upload/62025/62025fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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			<td>Fisher Scientific</td>
			<td>10178071</td>
			<td></td>
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		<tr>
			<td>baking oven</td>
			<td>Binder</td>
			<td>9010-0190</td>
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			<td>CFI Plan Fluor DL 10x</td>
			<td>Nikon</td>
			<td>MRH20100</td>
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			<td>Desiccator</td>
			<td>Roth</td>
			<td>NX07.1</td>
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			<td>Eclipse Ti-E</td>
			<td>Nikon</td>
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			<td>eGFP mRNA</td>
			<td>Trilink</td>
			<td>L-7601</td>
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			<td>Female Luer to Tube Connector</td>
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			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>10270106</td>
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			<td>Fibronectin</td>
			<td>Yo Proteins</td>
			<td>663</td>
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			<td>Filter set eGFP</td>
			<td>AHF</td>
			<td>F46-002</td>
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			<td>Fisherbrand&#160;Translucent Platinum-Cured Silicone Tubing</td>
			<td>Fisher Scientific</td>
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			<td>Thermo Fisher</td>
			<td>15630080</td>
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			<td>Incubation Box</td>
			<td>Okolab</td>
			<td>OKO-H201</td>
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			<td>incubator</td>
			<td>Binder</td>
			<td>9040-0012</td>
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			<td>L-15 without phenol red</td>
			<td>Thermo Fisher</td>
			<td>21083027</td>
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			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher</td>
			<td>11668027</td>
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			<td>Male Luer</td>
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			<td>in-house fabricated consisting of teflon</td>
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			<td>Male Luer to Tube Connector</td>
			<td>MEDNET</td>
			<td>MTLS210-6005</td>
			<td>alternative to in-house fabricated male luers</td>
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			<td>NaCl (5 M)</td>
			<td>Thermo Fisher</td>
			<td>AM9760G</td>
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			<td>Needleless Valve to Male Luer Connector</td>
			<td>MEDNET</td>
			<td>NVFMLLPC</td>
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			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Imaging software Version 5.02.00</td>
		</tr>
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			<td>NOA81</td>
			<td>Thorlabs</td>
			<td>NOA81</td>
			<td>Fast Curing Optical Adhesive for tube system assembly</td>
		</tr>
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			<td>Opti-MEM</td>
			<td>Thermo Fisher</td>
			<td>31985062</td>
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			<td>PCO edge 4.2 M-USB-HQ-PCO</td>
			<td>pco</td>
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			<td>Phosphate buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td>in-house prepared</td>
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			<td>Plasma Cleaner</td>
			<td>Diener Femto</td>
			<td>Pico-BRS</td>
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			<td>PLL(20 kDa)-g[3.5]-PEG(2 kDa)</td>
			<td>SuSoS AG</td>
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			<td>silicon wafer mit mircorstructures</td>
			<td></td>
			<td></td>
			<td>in-house fabricated</td>
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			<td>Sola Light Engine</td>
			<td>Lumencor</td>
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			<td>sticky slide VI 0.4&#160;</td>
			<td>ibidi</td>
			<td>80608</td>
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			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>1673921</td>
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			<td>Tango 2</td>
			<td>M&#228;rzh&#228;user</td>
			<td>00-24-626-0000</td>
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			<td>Ultrapure water</td>
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			<td>in-house prepared</td>
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			<td>uncoated coverslips</td>
			<td>ibidi</td>
			<td>10813</td>
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			<td>Injekt-F Solo, 1 mL</td>
			<td>Omilab</td>
			<td>9166017V</td>
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</table>

live-cell imaging of single-cell arrays (lisca) - a versatile technique to quantify cellular kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

The LISCA approach enables to efficiently collect fluorescence time courses from single cells. As a representative example we outline how the LISCA method is applied to measure single-cell eGFP expression after transfection. The data of the LISCA experiment is used to assess mRNA delivery kinetics, which is important for the development of efficient mRNA drugs.
In particular we demonstrate the different impact of two lipid-based mRNA delivery systems with respect to the time point of translati...

Watch this Scientific Journal Video about Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics at JoVE.com

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

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