Single cell isolation process with laser induced forward transfer
© The Author(s). 2017
Received: 22 September 2016
Accepted: 26 December 2016
Published: 13 January 2017
A viable single cell is crucial for studies of single cell biology. In this paper, laser-induced forward transfer (LIFT) was used to isolate individual cell with a closed chamber designed to avoid contamination and maintain humidity. Hela cells were used to study the impact of laser pulse energy, laser spot size, sacrificed layer thickness and working distance. The size distribution, number and proliferation ratio of separated cells were statistically evaluated. Glycerol was used to increase the viscosity of the medium and alginate were introduced to soften the landing process.
The role of laser pulse energy, the spot size and the thickness of titanium in energy absorption in LIFT process was theoretically analyzed with Lambert-Beer and a thermal conductive model. After comprehensive analysis, mechanical damage was found to be the dominant factor affecting the size and proliferation ratio of the isolated cells. An orthogonal experiment was conducted, and the optimal conditions were determined as: laser pulse energy, 9 μJ; spot size, 60 μm; thickness of titanium, 12 nm; working distance, 700 μm;, glycerol, 2% and alginate depth, greater than 1 μm. With these conditions, along with continuous incubation, a single cell could be transferred by the LIFT with one shot, with limited effect on cell size and viability.
LIFT conducted in a closed chamber under optimized condition is a promising method for reliably isolating single cells.
KeywordsSingle cell isolation Laser induced forward transfer Proliferation Mechanical stress Cell damage
Single cell technology is used to study the growth, metabolism and apoptosis of individual cells . Recently, it has been widely applied to studies of personalized medicine , oncology , cardiovascular disease , fertility  and AIDS . The most crucial step for the implementation of single cell technology is isolation of a single cell.
Currently, there are several methods developed to separate an individual cell, including fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), limiting dilution, micro-chips, laser capture micro-dissection (LCM) and optical tweezers. In FACS or MACS, the cells are labeled with fluorescence activated antigen or protein. The cells are then separated by either fluidic or magnetic force, respectively. However, these methods cannot be used to obtain single cells of rare sample or those lacking a known antigen [7–9]. Limiting dilution is the most used method of single cell separation. The cell suspension is repeatedly dilute into new medium untill the density of the cells is below 10 cells/mL. Although it is simple and easy to use, but the target cell cannot be traced since there is no signal feedback , and its randomness limits its applications [11, 12]. With the development of micro-fluidics, various chips have been prototyped to capture single cells basing on the certain properties of the cells, such as size [13, 14], rigidity  and metabolic function [16, 17]. However, these chips cannot be used to isolate cells that lack clearly distinguishing characteristics . In LCM, cell samples are fixed on a glass slide with either ethanol or formalin. The laser burns out the neighbor cells but keeps the target ones for further research . Because the cells are fixed, the isolated cells with LCM are not viable and cannot be cloned . Optical tweezers, which were originally developed to manipulate micro particles, have recently been introduced to manipulate single cells from channel to a culture well , but the manual process is inefficient and complicated .
Laser-induced forward transfer (LIFT) utilizes the laser with high energy to partially evaporate the materials coated on the glass, resulting in high pressure to reject the remaining material to a work piece, namely the receptor . Recently, LIFT has been developed for biological materials [24, 25], and has been successfully used to transfer stem cells in tissue engineering , to obtain a vein with fully function , and to deposit DNA or protein on bio-chips . In these applications, a random number of cells were transferred within each laser shot since there is no specific requirement for the number of deposited cells.
In this paper, we use LIFT to isolate single Hela cell. To prevent lossing of viability from contamination and drying, a chamber was developed with polydimethylsiloxane (PDMS) to seal LIFT environment and regulate humidity. The impact of critical parameters of the process, including laser pulse energy, spot size, working distance and the thickness of the scarified layer was investigated comprehensive. In addition, glycerol was added to the medium and a layer of alginate is deposited on the receptor to optimize the LIFT process. The modifications successfully protected the cells from damage.
Laser induced forward transfer setup
HELA cell (CCL-2™, ATCC, USA) was chosen as the object for its easy-culture, uniformly growth and fast proliferation. Accordingly the cell culture medium is made of 89 vol% DMEM (Dulbecco’s Modified Eagle’s Medium, D5796-Sigma Aldrich, Switzerland), 10 vol% FBS (Fetal Bovine Serum, F6765- Sigma Aldrich, Switzerland) and 1 vol% P/S (Penicillin-Streptomycin, P4333-Sigma Aldrich, Switzerland). The cells were grown at 5% CO2, 100% humidity and 37 °C for 5 days untill cells covered over 90% of the flask (3151-Corning, USA). The culture medium was replaced with 4 mL of PBS (Phosphate buffered saline, P5368- Sigma Aldrich, Switzerland) to wash away the dead cells. After dispersing the PBS, 2 mL of trypsin (T4049- Sigma Aldrich) was added to the flask for 3 min to detach the cells. The cells were transferred to a 15 mL tube filled with 8 mL of culture medium and centrifuged. After removing the medium from the tube, fresh medium was added to resuspend cells to a concentration of 103 cells/mL.
The base of the donor was quartz glass that was carefully cleaned with Piranha cleaning process. After drying overnight at 80 °C, the sacrificed layer, a layer of Titanium with defined thickness, was deposited on quartz by sputtering coating. The coated quartz was cleaned with ethanol and, rinsed with sterilized water, and then was sterilized with UV light. The prepared cell suspension with cell density of 103 cells/mL was coated on the surface of the Titanium by spin coating process. The depth of the suspension was no more than 30 μm, ensuring a single layer of cells.
Quart was also used as receptor for its great transparency that makes it suitable for microscope. It was sterilized as for the donor, and then Alginate solution (2 wt% in culture medium) was stacked on the quartz by spin coating process.
Viability analysis of cell after LIFT
The cells were stained with trypanblue (T8154-Sigma Aldrich) to distinguish the dead cells from live ones. The trypan blue was diluted 10 times in PBS, then transferred to the flask of cultured cells. After 30 min, the tryan blue solution was removed and the cell samples were observed under the microscope immediately. The transferred cells were stained after culturing for 1 to 5 days to calculate the proliferation ratio each day.
The experimental arrangement of single factor experiments
Laser pulse energy (E)
1 μJ, 3 μJ, 5 μJ, 7 μJ, 9 μJ, 11 μJ, 13 μJ, 15 μJ
Laser spot sizea(Diameter, D)
20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm,50 μm, 55 μm, 60 μm, 65 μm
Thickness of Titanium (T -t )
20 nm, 40 nm, 60 nm, 80 nm, 100 nm, 120 nm, 140 nm
Working distance (l)
0 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm
Concentration of Glycerol (d)
0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%
Thickness of Alginate (T -A )
0 μm, 2 μm, 4 μm, 6 μm, 8 μm, 10 μm
The damage to isolated cells in LIFT process, and their ability to recover were analyzed by comparing their size and proliferation ratio to those of untreated cells (Fig. 2).
Effect of laser pulse energy
Effect of laser spot size
Effect of the thickness of titanium
Effect of working distance
Effect of concentration of glycerol
Effect of alginate
The result of orthogonal experiment
T -t /nm
No. of Cells
No. of Cells
Mechanism of single cell isolation
When the laser energy was raised from 1 μJ to 15 μJ, the laser fluency increased from 79.58 mJ/cm2 to 1193.69 mJ/cm2. And the jet formation changed through four types, a bump with titanium partially ablated, a bump with titanium completely melted, a narrow jet, and a less controlled jet. Accordingly, the number of cell rose up from 11 to 47.
Conversely, in Fig. 4, the spot size was enlarged from 20 to 65 μm, the laser fluency decreased from 557.06 mJ/cm2 to 52.74 mJ/cm2, causing the jet to narrow down and even disappear, so there were fewer cells isolated successfully. The spot size also changed the Oh number and We number by shifting the value of R. With a larger spot, a less control jet could form, with which multiple cells were transferred. Generally, increasing the radius of spot size decreased the number of cells separated with 25 laser shots but eventually the number increased again.
The function of the titanium was to convert laser energy to drive the cell separation. But in LIFT process, the thickness of titanium should be carefully selected. On one hand, Titanium functionally absorbs the laser energy to induce cavitation. On the other hand, if not completely evaporated by laser, the remained titanium constrains the expansion of the cavitation and consums the energy to deform. When the titanium is thin (20 nm), The LIFT process is not efficient enough to to isolate cell successfully. In contrast, when the titanium is thick (140 nm), the laser fluency was fully transformed into the process which was 139.26 mJ/cm2, but some of the energy was wasted in deformation as shown in Fig. 10a and b).
The critical distance within which the jet was stably formed was 500 μm, so the working distance should be set at least 500 μm to ensure that only a target cell is isolated with each laser pulse.
Modifying the culture medium with glycerol mainly varied the properties of the medium, especially the viscosity. As the glycerol concentration increased, more energy were consumed by viscous dissipation during jetting. As a result, adding glycerol increased the chance that a cell was not transferred with each shot.
The cell damage
The cells suffered from three types of damage during the LIFT isolation process : mechanical, thermal and UV light. The damage can be recognized by a reduced cell radius or a reduced the proliferation ratio.
The mechanical damage results from three processes. During cavitation bubble expansion, the isolated cell was subject to the high pressure. Duringthe jetting process, the cell is rapidly accelerated to a high velocityThen the cell decelerates when it lands on the receptor. Young reported that the velocity can be high as 1000 m/s and the acceleration/deceleration can be 105-109 g [31, 32]. The pressure, acceleration and deceleration lead to high shear stress on the cell, which shrinks the cell and slows the proliferation. Since the pressure is generated from energy absorbed by titanium, the laser fluency, the thickness of titanium and the working distance are the main factors affecting the shear stress on the cell.
In addition, the interaction between the laser and the titanium produces heat. The heat injures cells by deactivating the enzymes, denaturing protein and carbonization . In fact, the thermal damage is dependent on both temperature and exposure time. Within several nanoseconds, the thermal effect zone was found to be dominated by Fourier heat conduction meaning that the zone was confined to within several micrometers depth. With a 30 μm thick suspension, the thermal injury of separated cell was negligible in this study.
UV lights can kill cells effectively, but in LIFT with a sacrificed layer, the UV light was constrained to the interface between the glass and titanium. Even with a 20 nm thick layer of titanium will absorbed, only 40% of laser passed through the sacrificed layer. Using Lambert-Beer law, with less than 4 μJ of pulse energy and 45 μm of spot size, 60% of laser radiation was absorbed by titanium, even with only a 20 nm thick layer. For the other 40% of laser fluency, the damage threshold depth in culture medium was estimated to be about 2 μm meaning that only 7% of the suspension was affected by UV light, resulting in minimal injury. So in this paper, UV light damage was negligible also.
Among mechanical, thermal and UV damage, under the conditions used in our experiments, mechanical stress was the dominant factor contributing to cell damage. Since the pressure resulted from energy absorbed by titanium, the laser fluency and the thickness of titanium were the main factors affecting the shear stress acting on the cell. As shown in Figs. 3 and 4, increasing the laser pulse energy, reducing the laser spot diameter helped to reduce the cell size, as well as to slow down the proliferation of the transferred target cell. Increasing Titanium thickness from 20 nm to 80 nm decreased the cell size and the proliferation because the titanium contributes on the conversion of energy to cavitation. When it was greater 80 nm, the cells shrank less and the proliferation ratio increased. As previously mentioned, the working distance had a limited impact on jetting, but decreasing the length of path that the isolated cell had to pass through decreased the mechanical damage to the cell, leading to larger cells with higher viability., Glycerol is used to increase the viscosity to form a better droplet but itchanged the osmotic pressure of the medium, making the cell smaller, and reducing the viability. Coating alginate on the receptor was a method to provide enough cushion to reduce the deceleration during the landing process.
We have analyzed the effects of laser pulse energy, laser spot size, thickness of Titanium, working distance, glycerol and alginate on the number, size and proliferation ratio of cells isolated with LIFT. It revealed that the laser fluency and the thickness of titanium were the main factors affecting the viability of isolated cell because they influence the energy introduced into the process. Providing a sufficient work distances and increasing the viscosity with glycerol helped to control the cell transfer and coating with alginate was employed to soften the cell landing.
The optimal settings for obtaining a viable cellare: laser pulse energy, 9 μJ; spot size, 60 μm; thickness of titanium, 12 nm; working distance, 700 μm, 2–4% glycerol in culture medium and alginate thickness greater than 1 μm.
Dulbecco’s modified eagle’s medium
Fluorescence activate cell sorting
Fetal bovine serum
Laser capture micro-dissection
Laser induced forward transfer
Magnetic activate cell sorting
Phosphate buffered saline
The discussion with Arnaud Bertsch and Yufei Ren of École Polytechnique Fédérale de Lausanne, and clean room technologies support from Joffrey Pernollet of CMI inÉcole Polytechnique Fédérale de Lausanne.
The study was partially supported by National Natural Science Foundation of China (51175091) and the project of young innovative talents in university of Guangdong (2015KQNCX027).
Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information files].
YD took charge of lab work, data analysis and participated in the design of the study and drafted the manuscript; PR participated in the design of the study; ZG participated on statistical analyses, coordinated the study and helped to draft the manuscript; ZH took part in lab work, sequence alignment and revised the manuscript. YC conducted the data analysis and partially involved in design of the study. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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- Hoppe PS, Coutu DL, Schroeder T. Single-cell technologies sharpen up mammalian stem cell research. Nat Cell Biol. 2014;16(10):919–27.View ArticleGoogle Scholar
- Bhagat AAS, Bow H, Hou HW, Tan SJ, Han J, Lim CT. Microfluidics for cell separation. Med Biol Eng Comput. 2010;48(10):999–1014.View ArticleGoogle Scholar
- Leslie M. The power of one. Science. 2011;331(6013):24–6.Google Scholar
- Altelaar AM, Heck AJ. Trends in ultrasensitive proteomics. Curr Opin Chem Biol. 2012;16(1):206–13. doi:10.1016/j.cbpa.2011.12.011.View ArticleGoogle Scholar
- Giorgetti A, Montserrat N, Aasen T, Gonzalez F, Rodríguez-Pizà I, Vassena R, Raya A, Boué S, Barrero MJ, Corbella BA, Torrabadella M. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell. 2009;5(4):353.View ArticleGoogle Scholar
- Levine JH, Lin Y, Elowitz MB. Functional roles of pulsing in genetic circuits. Science. 2013;342(6163):1193–200.View ArticleGoogle Scholar
- Sanchez A, Golding I. Genetic determinants and cellular constraints in noisy gene expression. Science. 2013;342(6163):1188–93.View ArticleGoogle Scholar
- Toli D, Buttigieg D, Blanchard S, Lemonnier T, d’Incamps BL, Bellouze S, Baillat G, Bohl D, Haase G. Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique. Neurobiol Dis. 2015;82:269–80.View ArticleGoogle Scholar
- Plouffe BD, Murthy SK, Lewis LH. Fundamentals and application of magnetic particles in cell isolation and enrichment: a review. Rep Prog Phys. 2014;78(1):016601.View ArticleGoogle Scholar
- Wilson A, Chen WF, Scollay R, Shortman K. Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes. J Immunol Methods. 1982;52(3):283–306.View ArticleGoogle Scholar
- Cheshier SH, Morrison SJ, Liao X, Weissman IL. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci. 1999;96(6):3120–5.View ArticleGoogle Scholar
- Chen WF, Wilson A, Scollay R, Shortman K. Limit-dilution assay and clonal expansion of all T cells capable of proliferation. J Immunol Methods. 1982;52(3):307–22.View ArticleGoogle Scholar
- Kim H, Lee S, Lee JH, Kim J. Integration of a microfluidic chip with a size-based cell bandpass filter for reliable isolation of single cells. Lab Chip. 2015;15(21):4128–32.View ArticleGoogle Scholar
- Khamenehfar A, Beischlag TV, Russell PJ, Ling MTP, Nelson C, Li PCH. Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip. Biomicrofluidics. 2015;9(6):064104.View ArticleGoogle Scholar
- Huang WC, Burnouf PA, Su YC, Chen BM, Chuang KH, Lee CW, Wei PK, Cheng TL, Roffler SR. Engineering chimeric receptors to investigate the size-and rigidity-dependent interaction of PEGylated nanoparticles with cells. ACS Nano. 2016;10(1):648–62.View ArticleGoogle Scholar
- Meister A, Gabi M, Behr P, Studer P, Vörös J, Niedermann P, Bitterli J, Polesel-Maris J, Liley M, Heinzelmann H, Zambelli T. FluidFM: combining atomic force microscopy and nanofluidics in a universal liquid delivery system for single cell applications and beyond. Nano Lett. 2009;9(6):2501–7.View ArticleGoogle Scholar
- Guillaume-Gentil O, Zambelli T, Vorholt JA. Isolation of single mammalian cells from adherent cultures by fluidic force microscopy. Lab Chip. 2014;14(2):402–14.View ArticleGoogle Scholar
- Palima D, Aabo T, Bañas A and Glückstad J. Cell handling, sorting, and viability. PHOTONICS: Scientific Foundations, Technology and Applications, IV,2015; 197–237.
- Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, Zhou W, Coukos G, Geho DH, Petricoin EF, Liotta LA. Laser-capture microdissection. Nat Protoc. 2006;1(2):586–603.View ArticleGoogle Scholar
- Kolijn K, Leenders GJ. Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue. BMC Res Notes. 2016;9(1):1.View ArticleGoogle Scholar
- Probst C, Grünberger A, Wiechert W, Kohlheyer D. Microfluidic growth chambers with optical tweezers for full spatial single-cell control and analysis of evolving microbes. J Microbiol Methods. 2013;95(3):470–6.View ArticleGoogle Scholar
- Liberale C, Cojoc G, Bragheri F, Minzioni P, Perozziello G, La Rocca R, Ferrara L, Rajamanickam V, Di Fabrizio E, Cristiani I. Integrated microfluidic device for single-cell trapping and spectroscopy. Sci Rep. 2013;3:1258.Google Scholar
- Nagel M, et al. Aryltriazene photopolymers for UV‐laser applications: improved synthesis and photodecomposition study. Macromol Chem Phys. 2007;208(3):277–86.View ArticleGoogle Scholar
- Ma H, Mismar W, Wang Y, Small DW, Ras M, Allbritton NL, Sims CE and Venugopalan V. Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells. J Royal Soc Interface. 2011; doi:rsif20110691.
- Chatzipetrou M, Tsekenis G, Tsouti V, Chatzandroulis S, Zergioti I. Biosensors by means of the laser induced forward transfer technique. Appl Surf Sci. 2013;278:250–4.View ArticleGoogle Scholar
- Nguyen AK, Narayan RJ. Liquid-Phase Laser Induced Forward Transfer for Complex Organic Inks and Tissue Engineering. Ann Biomed Eng. 2017;45(1):84–99.
- Ovsianikov A, Gruene M, Pflaum M, Koch L, Maiorana F, Wilhelmi M, Haverich A, Chichkov B. Laser printing of cells into 3D scaffolds. Biofabrication. 2010;2(1):014104.View ArticleGoogle Scholar
- Serra P, Colina M, Fernández-Pradas JM, Sevilla L, Morenza JL. Preparation of functional DNA microarrays through laser-induced forward transfer. Appl Phys Lett. 2004;85(9):1639–41.View ArticleGoogle Scholar
- Zhigilei LV, Garrison BJ. Mechanisms of laser ablation from molecular dynamics simulations: dependence on the initial temperature and pulse duration. Appl Phys A. 1999;69(1):S75–80.View ArticleGoogle Scholar
- Zhang Z, Xiong R, Mei R, Huang Y, Chrisey DB. Time-resolved imaging study of jetting dynamics during laser printing of viscoelastic alginate solutions. Langmuir. 2015;31(23):6447–56.View ArticleGoogle Scholar
- Barron JA, Young HD, Dlott DD, Darfler MM, Krizman DB, Ringeisen BR. Printing of protein microarrays via a capillary‐free fluid jetting mechanism. Proteomics. 2005;5(16):4138–44.View ArticleGoogle Scholar
- Lin Y, Huang Y, Wang G, Tzeng TRJ, Chrisey DB. Effect of laser fluence on yeast cell viability in laser-assisted cell transfer. J Appl Phys. 2009;106(4):043106.View ArticleGoogle Scholar
- Åkerfelt M, Morimoto RI, Sistonen L. Heat shock factors: integrators of cell stress, development and lifespan. Nat Rev Mol Cell Biol. 2010;11(8):545–55.View ArticleGoogle Scholar