Integration of Droplet Microfluidic Tools for Single-cell Functional Metagenomics: An Engineering Head Start

<p>Droplet microfluidics techniques have shown promising results to study single-cells at high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant because of the field’s complex and multidisciplinary nature. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single-cells in droplets at a rate of ~ 250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescent activated droplet sorting (FADS) systems to integrate the use of 4 independent fluorescence-exciting lasers (e.g., 405, 488, 561, 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger was also integrated into our method to enable adding new reagents to already made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput (> 50,000 cells/day) capabilities to mining and bioprospecting metagenomic data.</p>

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Integration of Droplet Microfluidic Tools for Single-cell Functional Metagenomics: An Engineering Head Start

10.36227/techrxiv.14760726.v1 ◽

2021 ◽

Author(s):

David Conchouso ◽

Amani Al-Ma’abadi ◽

Hayedeh Behzad ◽

Mohammed Alarawi ◽

Masahito Hosokawa ◽

...

Keyword(s):

Head Start ◽

Single Cell ◽

High Throughput ◽

Single Cells ◽

Droplet Microfluidics ◽

Metagenomic Data ◽

Functional Screening ◽

Functional Metagenomics ◽

Technical Details ◽

Droplet Sorting

<p>Droplet microfluidics techniques have shown promising results to study single-cells at high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant because of the field’s complex and multidisciplinary nature. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single-cells in droplets at a rate of ~ 250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescent activated droplet sorting (FADS) systems to integrate the use of 4 independent fluorescence-exciting lasers (e.g., 405, 488, 561, 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger was also integrated into our method to enable adding new reagents to already made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput (> 50,000 cells/day) capabilities to mining and bioprospecting metagenomic data.</p>

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Ultrahigh-Throughput Screening of Single-Cell Lysates for Directed Evolution and Functional Metagenomics

Methods in Molecular Biology - Protein Engineering ◽

10.1007/978-1-4939-7366-8_18 ◽

2017 ◽

pp. 297-309 ◽

Cited By ~ 2

Author(s):

Fabrice Gielen ◽

Pierre-Yves Colin ◽

Philip Mair ◽

Florian Hollfelder

Keyword(s):

Single Cell ◽

Directed Evolution ◽

Functional Metagenomics ◽

Cell Lysates

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Electron microscopy of keratinocytes cultured on a collagen substrate used as an allograft for the treatment of chronic wounds

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100130158 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1104-1105

Author(s):

Debby A. Jennings ◽

Michael J. Morykwas ◽

Louis C. Argenta

Keyword(s):

Electron Microscopy ◽

Cell Suspension ◽

Single Cell ◽

Chronic Wounds ◽

Mechanical Stability ◽

Uv Light ◽

Type I ◽

Single Cell Suspension ◽

Collagen Substrate ◽

Dermal Collagen

Grafts of cultured allogenic or autogenic keratlnocytes have proven to be an effective treatment of chronic wounds and burns. This study utilized a collagen substrate for keratinocyte and fibroblast attachment. The substrate provided mechanical stability and augmented graft manipulation onto the wound bed. Graft integrity was confirmed by light and transmission electron microscopy.Bovine Type I dermal collagen sheets (100 μm thick) were crosslinked with 254 nm UV light (13.5 Joules/cm2) to improve mechanical properties and reduce degradation. A single cell suspension of third passage neonatal foreskin fibroblasts were plated onto the collagen. Five days later, a single cell suspension of first passage neonatal foreskin keratinocytes were plated on the opposite side of the collagen. The grafts were cultured for one month.The grafts were fixed in phosphate buffered 4% formaldehyde/1% glutaraldehyde for 24 hours. Graft pieces were then washed in 0.13 M phosphate buffer, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Polybed 812.

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The IBM-PC in electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100165525 ◽

1996 ◽

Vol 54 ◽

pp. 612-613

Author(s):

James F. Mancuso

Keyword(s):

Head Start ◽

Image Acquisition ◽

Cost Effective ◽

Financial Applications ◽

Number Of Factors ◽

Instrument Control ◽

The Cost ◽

Training And Support ◽

Control Image ◽

Ibm Pc

IBM PC compatible computers are widely used in microscopy for applications ranging from control to image acquisition and analysis. The choice of IBM-PC based systems over competing computer platforms can be based on technical merit alone or on a number of factors relating to economics, availability of peripherals, management dictum, or simple personal preference.IBM-PC got a strong “head start” by first dominating clerical, document processing and financial applications. The use of these computers spilled into the laboratory where the DOS based IBM-PC replaced mini-computers. Compared to minicomputer, the PC provided a more for cost-effective platform for applications in numerical analysis, engineering and design, instrument control, image acquisition and image processing. In addition, the sitewide use of a common PC platform could reduce the cost of training and support services relative to cases where many different computer platforms were used. This could be especially true for the microscopists who must use computers in both the laboratory and the office.

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Unravelling Single Cell Genomics

10.1039/9781849732284 ◽

2010 ◽

Keyword(s):

Single Cell ◽

Single Cell Genomics

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Dynamic Assessment Language Tasks and the Prediction of Performance on Year-End Language Skills in Preschool Dual Language Learners

American Journal of Speech-Language Pathology ◽

10.1044/2019_ajslp-19-00120 ◽

2020 ◽

Vol 29 (3) ◽

pp. 1226-1240

Author(s):

Janet L. Patterson ◽

Barbara L. Rodríguez ◽

Philip S. Dale

Keyword(s):

Head Start ◽

Language Learners ◽

Language Impairment ◽

Dual Language ◽

Dynamic Assessment ◽

Dual Language Learners ◽

Achievement Testing ◽

Potential Applications ◽

Language Screening ◽

Relationship Of

Purpose Early identification is a key element for accessing appropriate services for preschool children with language impairment. However, there is a high risk of misidentifying typically developing dual language learners as having language impairment if inappropriate tools designed for monolingual children are used. In this study of children with bilingual exposure, we explored performance on brief dynamic assessment (DA) language tasks using graduated prompting because this approach has potential applications for screening. We asked if children's performance on DA language tasks earlier in the year was related to their performance on a year-end language achievement measure. Method Twenty 4-year-old children from Spanish-speaking homes attending Head Start preschools in the southwestern United States completed three DA graduated prompting language tasks 3–6 months prior to the Head Start preschools' year-end achievement testing. The DA tasks, Novel Adjective Learning, Similarities in Function, and Prediction, were administered in Spanish, but correct responses in English or Spanish were accepted. The year-end achievement measure, the Learning Accomplishment Profile–Third Edition (LAP3), was administered by the children's Head Start teachers, who also credited correct responses in either language. Results Children's performance on two of the three DA language tasks was significantly and positively related to year-end LAP3 language scores, and there was a moderate and significant relationship for one of the DA tasks, even when controlling for age and initial LAP3 scores. Conclusions Although the relationship of performance on DA with year-end performance varies across tasks, the findings indicate potential for using a graduated prompting approach to language screening with young dual language learners. Further research is needed to select the best tasks for administration in a graduated prompting framework and determine accuracy of identification of language impairment.

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