New microorganism isolation techniques with emphasis on laser printing

Cheptsov, V. S.; Tsypina, S. I.; Минаев, Н. В.; Yusupov, V. I.; Chichkov, Boris N.

doi:10.18063/ijb.v5i1.165

Cited by 29 publications

(19 citation statements)

References 72 publications

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“…Subsequently, the parameters of the experiment, namely, Energy (E), Distance (D), Thickness (T), and Spot size (SS) are set and a trial transfer is performed. Based on the results of our works[ 13 , 32 , 44 , 63 , 64 ] and the results of other authors[ 14 , 15 , 54 ], for a wide variety of hydrogels[ 24 , 65 , 66 ], the threshold fluence is 2 – 3 J/cm 2 (nanosecond laser and Au absorbing layer). In practice, the starting values for the experiments can be: E = 15 – 20 μJ and SS = 30 μm for a laser with a wavelength of 1064 nm and a pulse duration of ~ 10 ns.…”

Section: Decision Tree For Controlled Hydrogel Transfermentioning

confidence: 57%

“…Laser-induced forward transfer (LIFT) is a digital printing technique that allows for the nozzle-free direct transfer of matter. This technology has been used (1) to print a wide range of biological materials, such as proteins[ 1 , 2 ] and DNA[ 3 , 4 ]; (2) to separate microorganisms[ 5 ]; (3) to print various types of cells to create three-dimensional (3D) bioequivalents of natural tissues[ 6 - 10 ]; and (4) to transfer stimulatory factors for cell differentiation[ 6 - 10 ].…”

Section: Introductionmentioning

confidence: 99%

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Laser-induced Forward Transfer Hydrogel Printing: A Defined Route for Highly Controlled Process

Yusupov¹,

Churbanov²,

Churbanova³

et al. 2020

ijb

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Laser-induced forward transfer is a versatile, non-contact, and nozzle-free printing technique which has demonstrated high potential for different printing applications with high resolution. In this article, three most widely used hydrogels in bioprinting (2% hyaluronic acid sodium salt, 1% methylcellulose, and 1% sodium alginate) were used to study laser printing processes. For this purpose, the authors applied a laser system based on a pulsed infrared laser (1064 nm wavelength, 8 ns pulse duration, 1 – 5 J/cm2 laser fluence, and 30 μm laser spot size). A high-speed shooting showed that the increase in fluence caused a sequential change in the transfer regimes: No transfer regime, optimal jetting regime with a single droplet transfer, high speed regime, turbulent regime, and plume regime. It was demonstrated that in the optimal jetting regime, which led to printing with single droplets, the size and volume of droplets transferred to the acceptor slide increased almost linearly with the increase of laser fluence. It was also shown that the maintenance of a stable temperature (±2°C) allowed for neglecting the temperature-induced viscosity change of hydrogels. It was determined that under room conditions (20°C, humidity 50%), the hydrogel layer, due to drying processes, decreased with a speed of about 8 μm/min, which could lead to a temporal variation of the transfer process parameters. The authors developed a practical algorithm that allowed quick configuration of the laser printing process on an applied experimental setup. The configuration is provided by the change of the easily tunable parameters: Laser pulse energy, laser spot size, the distance between the donor ribbon and acceptor plate, as well as the thickness of the hydrogel layer on the donor ribbon slide.

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Section: Decision Tree For Controlled Hydrogel Transfermentioning

confidence: 57%

Section: Introductionmentioning

confidence: 99%

Laser-induced Forward Transfer Hydrogel Printing: A Defined Route for Highly Controlled Process

Yusupov¹,

Churbanov²,

Churbanova³

et al. 2020

ijb

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“…Single cell technology is a powerful tool for studying the growth, physiology, function and biodiversity of microorganisms, especially for the as-yet-unculturable microorganisms in nature (1)(2)(3)(4)(5). As the basis and most important step of a single cell study, single cell isolation has been applied in many research fields such as single cell genomics (6), neurobiology (7) and analysis of disease processes (8).…”

Section: Introductionmentioning

confidence: 99%

Laser induced isolation and cultivation of single microbial cells

Peng

Wang

Wang³

et al. 2020

Preprint

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Single cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. Laser Induced Forward Transfer Technique (LIFT) has been applied to isolate single cells but the cell viability after sorting is unclear. We demonstrate that a three-layer LIFT system could be applied to isolate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae) and the sorted single cells were able to be cultured. The experiment results showed that the average cultivation recovery rate of the ejected single cells were 58% for Saccharomyces cerevisiae, 22% for E. coli, and 74% for Lactobacillus rhamnosus GG (LGG). The identities of the cultured cells from single cell sorting were confirmed by using colony PCR with 16S-rRNA for bacteria and large unit rRNA for yeast and subsequent sequencing. This precise sorting and cultivation technique of live single microbial cells can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture single microorganisms with specific functions, revealing their roles in the natural community.ImportanceSingle cell isolation and cultivation are crucial to recover microorganisms for the study of physiology, gene expression and functions. We developed a laser induced cell sorting technology to precisely isolate single microbial cells from a microscopic slide. More importantly, the isolated single microbial cells are still viable for cultivation. We demonstrate to apply the live sorting method to isolate and cultivate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae). This precise sorting and cultivation technique can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture specifically targeted single microorganisms from microbial community.Abstract Graphic

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“…Figure 5 presents the dependences of the diameter of the cavitation bubble in water on time for three different energies of the laser pulse. Red curves modeling the dynamics of cavitation bubbles obtained using relation (5) are plotted in the same figures. The initial pressure in bubble was the fitting parameter (until the calculated maximal radius will be equal to the obtained in the experiment).…”

Section: Figurementioning

confidence: 99%

“…A promising modification of the laser printing method is laser engineering of microbial systems (LEMS) technology. It has already been proven that LEMS will make it possible to separate symbionts [4] and isolate individual microorganisms that are difficult to cultivate or not cultivated at all by standard methods [5][6][7]. It is important that this process is necessary not only for solving the urgent problem of creating the demanded "Noah's Ark"-the World Bank of Microorganisms [8]-but also for the production of biologically active substances [9] and the synthesis of new antibiotics [10].…”

Section: Introductionmentioning

confidence: 99%

Evolution of Shock-Induced Pressure in Laser Bioprinting

et al. 2021

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Laser bioprinting with gel microdroplets that contain living cells is a promising method for use in microbiology, biotechnology, and medicine. Laser engineering of microbial systems (LEMS) technology by laser-induced forward transfer (LIFT) is highly effective in isolating difficult-to-cultivate and uncultured microorganisms, which are essential for modern bioscience. In LEMS the transfer of a microdroplet of a gel substrate containing living cell occurs due to the rapid heating under the tight focusing of a nanosecond infrared laser pulse onto thin metal film with the substrate layer. During laser transfer, living organisms are affected by temperature and pressure jumps, high dynamic loads, and several others. The study of these factors’ role is important both for improving laser printing technology itself and from a purely theoretical point of view in relation to understanding the mechanisms of LEMS action. This article presents the results of an experimental study of bubbles, gel jets, and shock waves arising in liquid media during nanosecond laser heating of a Ti film obtained using time-resolving shadow microscopy. Estimates of the pressure jumps experienced by microorganisms in the process of laser transfer are performed: in the operating range of laser energies for bioprinting LEMS technology, pressure jumps near the absorbing film of the donor plate is about 30 MPa. The efficiency of laser pulse energy conversion to mechanical post-effects is about 10%. The estimates obtained are of great importance for microbiology, biotechnology, and medicine, particularly for improving the technologies related to laser bioprinting and the laser engineering of microbial systems.

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New microorganism isolation techniques with emphasis on laser printing

Cited by 29 publications

References 72 publications

Laser-induced Forward Transfer Hydrogel Printing: A Defined Route for Highly Controlled Process

Laser-induced Forward Transfer Hydrogel Printing: A Defined Route for Highly Controlled Process

Laser induced isolation and cultivation of single microbial cells

Evolution of Shock-Induced Pressure in Laser Bioprinting

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