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Single Cell Isolation Using Optical Tweezers.

ABSTRACT: Optical tweezers offer a non-contact method for selecting single cells and translocating them from one microenvironment to another. We have characterized the optical tweezing of yeast S. cerevisiae and can manipulate single cells at 0.41 ± 0.06 mm/s using a 26.8 ± 0.1 mW from a 785 nm diode laser. We have fabricated and tested three cell isolation devices; a micropipette, a PDMS chip and a laser machined fused silica chip and we have isolated yeast, single bacteria and cyanobacteria cells. The most effective isolation was achieved in PDMS chips, where single yeast cells were grown and observed for 18 h without contamination. The duration of budding in S. cerevisiae was not affected by the laser parameters used, but the time from tweezing until the first budding event began increased with increasing laser energy (laser power × time). Yeast cells tweezed using 25.0 ± 0.1 mW for 1 min were viable after isolation. We have constructed a micro-consortium of yeast cells, and a co-culture of yeast and bacteria, using optical tweezers in combination with the PDMS network of channels and isolation chambers, which may impact on both industrial biotechnology and understanding pathogen dynamics.

SUBMITTER: Keloth A

PROVIDER: S-EPMC6187562 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Single Cell Isolation Using Optical Tweezers.

Keloth Anusha A Anderson Owen O Risbridger Donald D Paterson Lynn L

Micromachines 20180829 9

Optical tweezers offer a non-contact method for selecting single cells and translocating them from one microenvironment to another. We have characterized the optical tweezing of yeast <i>S. cerevisiae</i> and can manipulate single cells at 0.41 ± 0.06 mm/s using a 26.8 ± 0.1 mW from a 785 nm diode laser. We have fabricated and tested three cell isolation devices; a micropipette, a PDMS chip and a laser machined fused silica chip and we have isolated yeast, single bacteria and cyanobacteria cells ...[more]

PMID: 30424367

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Project description:IntroductionIn response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an 'ultrasound tweezers'-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed "actinin-sstFRET," to monitor in situ single-cell allostasis in response to transient stimulation in real time.MethodsThe ultrasound tweezers utilize 1 Hz, 10-s transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca2+ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis.ResultsBy subjecting a 1 Hz, 10-s physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30 min that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca2+ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation.ConclusionsBy integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging.

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Single Cell Isolation Using Optical Tweezers.

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Single Cell Isolation Using Optical Tweezers.

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