우리는 도움이 토론과 편집을위한 프로토콜, 키 에런 스위니 테스트에 도움이 몰리 라자 베로니카 델가도를 인정, 그리고 원고의 중요한 읽기 테일러 스콧, 내 한 - adirekkun, 그리고 스테파니 겔러 것이다. 메건 니콜 소매점을, 박사 버로우즈 웰컴 기금에서 과학 인터페이스에서 경력 상을 보유하고있다.

<ol>
	<li>Motta-Mena, L. B., Reade, 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=An+optogenetic+gene+expression+system+with+rapid+activation+and+deactivation+kinetics.">An optogenetic gene expression system with rapid activation and deactivation kinetics.</a> Nature chemical biology. 10 (3), 196-202 (2014).</li><li>Hughes, R. M., Bolger, S., Tapadia, H., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-mediated+control+of+DNA+transcription+in+yeast.">Light-mediated control of DNA transcription in yeast.</a> Methods. 58 (4), 385-391 (2012).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nature biotechnology. 20 (10), 1041-1044 (2002).</li><li>Polstein, L. R., Gersbach, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Light-inducible+spatiotemporal+control+of+gene+activation+by+customizable+zinc+finger+transcription+factors.">a Light-inducible spatiotemporal control of gene activation by customizable zinc finger transcription factors.</a> J Am Chem Soc. 134 (40), 16480-16483 (2012).</li><li>Polstein, L. R., Gersbach, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+A+light-inducible+CRISPR-Cas9+system+for+control+of+endogenous+gene+activation.">a A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.</a> Nature Chemical Biology. 11 (3), 198-200 (2015).</li><li>Yang, X., Jost, A. P. T., Weiner, O. D., Tang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-inducible+organelle-targeting+system+for+dynamically+activating+and+inactivating+signaling+in+budding+yeast.">A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast.</a> Molecular biology of the cell. 24 (15), 2419-2430 (2013).</li><li>Lee, S., Park, H., 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=Reversible+protein+inactivation+by+optogenetic+trapping+in+cells.">Reversible protein inactivation by optogenetic trapping in cells.</a> Nature methods. 11 (6), 633-636 (2014).</li><li>Kennedy, M. J., Hughes, R. M., Peteya, L. A., Schwartz, J. W., Ehlers, M. D., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+blue-light-mediated+induction+of+protein+interactions+in+living+cells.">Rapid blue-light-mediated induction of protein interactions in living cells.</a> Nature Methods. 7 (12), 973-975 (2010).</li><li>Yazawa, M., Sadaghiani, A. M., Hsueh, B., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+protein-protein+interactions+in+live+cells+using+light.">Induction of protein-protein interactions in live cells using light.</a> Nat Biotechnol. 27 (10), 941-945 (2009).</li><li>Zoltowski, B. D., Motta-Mena, L. B., Gardner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light-induced+dimerization+of+a+bacterial+LOV-HTH+DNA-binding+protein.">Blue light-induced dimerization of a bacterial LOV-HTH DNA-binding protein.</a> Biochemistry. 52 (38), 6653-6661 (2013).</li><li>Renicke, C., Schuster, D., Usherenko, S., Essen, L. O., Taxis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+LOV2+Domain-Based+Optogenetic+Tool+to+Control+Protein+Degradation+and+Cellular+Function.">A LOV2 Domain-Based Optogenetic Tool to Control Protein Degradation and Cellular Function.</a> Chemistry &amp; Biology. 20 (4), 619-626 (2013).</li><li>Novick, A., Szilard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+the+chemostat.">Description of the chemostat.</a> Science (New York, N.Y.). 112 (2920), 715-716 (1950).</li><li>Monod, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=La+technique+de+culture+continue.+Th&#233;orie+et+applications.">La technique de culture continue. Th&#233;orie et applications.</a> Ann. Inst. Pasteur. 79 (4), 390-410 (1950).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nature Biotechnology. 20, 1041-1044 (2002).</li><li>Kennedy, M. J., Hughes, R. M., Peteya, L. A., Schwartz, J. W., Ehlers, M. D., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+blue-light-mediated+induction+of+protein+interactions+in+living+cells.">Rapid blue-light-mediated induction of protein interactions in living cells.</a> Nature Methods. 7, 973-975 (2010).</li><li>Olson, E. J., Tabor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+characterization+methods+overcome+key+challenges+in+synthetic+and+systems+biology.">Optogenetic characterization methods overcome key challenges in synthetic and systems biology.</a> Nature chemical biology. 10 (7), 502-511 (2014).</li><li>Botstein, D., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast:+An+experimental+organism+for+21st+century+biology.">Yeast: An experimental organism for 21st century biology.</a> Genetics. 189 (3), 695-704 (2011).</li><li>Galanie, S., Smolke, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+yeast-based+production+of+medicinal+protoberberine+alkaloids.">Optimization of yeast-based production of medicinal protoberberine alkaloids.</a> Microbial cell factories. 14 (1), 144 (2015).</li><li>Gerngross, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+production+of+human+therapeutic+proteins+in+yeasts+and+filamentous+fungi.">Advances in the production of human therapeutic proteins in yeasts and filamentous fungi.</a> Nature biotechnology. 22 (11), 1409-1414 (2004).</li><li>van Maris, A. J. A., Abbott, D. 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=Alcoholic+fermentation+of+carbon+sources+in+biomass+hydrolysates+by+Saccharomyces+cerevisiae:+current+status.">Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status.</a> Antonie van Leeuwenhoek. 90 (4), 391-418 (2006).</li><li>Edelstein, A., Amodaj, N., Hoover, K., Vale, R., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+control+of+microscopes+using+&#181;Manager.">Computer control of microscopes using &#181;Manager.</a> Curr Protoc Mol Biol. , (2010).</li><li>Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+methods+of+microscope+control+using+&#181;Manager+software.">Advanced methods of microscope control using &#181;Manager software.</a> Journal of biological methods. 1 (2), (2014).</li><li>Schindelin, J., Arganda-Carreras, I., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature methods. 9 (7), 676-682 (2012).</li><li>Bhattacharya, S., Datta, A., Berg, J. M., Gangopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+surface+wettability+of+poly(dimethyl)+siloxane+(PDMS)+and+glass+under+oxygen-plasma+treatment+and+correlation+with+bond+strength.">Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength.</a> Journal of Microelectromechanical Systems. 14 (3), 590-597 (2005).</li><li>Saldanha, A. J., Brauer, M. J., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+homeostasis+in+batch+and+steady-state+culture+of+yeast.">Nutritional homeostasis in batch and steady-state culture of yeast.</a> Molecular Biology of the Cell. 15, 4089-4104 (2004).</li><li>Boer, V. M., Tai, S. L., 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=Transcriptional+responses+of+Saccharomyces+cerevisaie+to+preferred+and+nonpreferred+nitrogen+sources+in+glucose-limited+chemostat+cultures.">Transcriptional responses of Saccharomyces cerevisaie to preferred and nonpreferred nitrogen sources in glucose-limited chemostat cultures.</a> FEMS Yeast Research. 7, 604-620 (2007).</li><li>Boer, V. M., Amini, S., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+genotype+and+nutrition+on+survival+and+metabolism+of+starving+yeast.">Influence of genotype and nutrition on survival and metabolism of starving yeast.</a> Proceedings of the National Academy of Sciences. 105, 6930-6935 (2008).</li><li>Brauer, M. J., Huttenhower, C., 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=Coordination+of+growth+rate,+cell+cycle,+stress+response+and+metabolic+activity+in+yeast.">Coordination of growth rate, cell cycle, stress response and metabolic activity in yeast.</a> Molecular Biology of the Cell. 19, 352-367 (2008).</li><li>Gresham, D., Dunham, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enduring+utility+of+continuous+culturing+in+experimental+evolution.">The enduring utility of continuous culturing in experimental evolution.</a> Genomics. 104 (6), 399-405 (2014).</li><li>Liu, H., Yu, X., 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=Photoexcited+CRY2+interacts+with+CIB1+to+regulate+transcription+and+floral+initiation+in+Arabidopsis.">Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis.</a> Science (New York, N.Y.). 322 (5907), 1535-1539 (2008).</li><li>McIsaac, R. S., Oakes, B. L., Wang, X., Dummit, K. A., Botstein, D., Noyes, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+gene+expression+perturbation+systems+with+rapid,+tunable,+single-gene+specificity+in+yeast.">Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast.</a> Nucleic Acids Research. 41 (4), 1-10 (2013).</li><li>McIsaac, R. S., Petti, A. A., Bussemaker, H. J., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation-based+analysis+and+modeling+of+combinatorial+regulation+in+the+yeast+sulfur+assimilation+pathway.">Perturbation-based analysis and modeling of combinatorial regulation in the yeast sulfur assimilation pathway.</a> Molecular biology of the cell. 23 (15), 2993-3007 (2012).</li><li>McIsaac, R. S., Silverman, S. J., 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=Fast-acting+and+nearly+gratuitous+induction+of+gene+expression+and+protein+depletion+in+Saccharomyces+cerevisiae.">Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae.</a> Molecular biology of the cell. 22 (22), 4447-4459 (2011).</li><li>Melendez, J., Patel, M., Oakes, B. L., Xu, P., Morton, P., McClean, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optogenetic+control+of+intracellular+protein+concentration+in+microbial+cell+cultures.">Real-time optogenetic control of intracellular protein concentration in microbial cell cultures.</a> Integrative biology quantitative biosciences from nano to macro. 6 (3), 366-372 (2014).</li><li>Brauer, M. J., Huttenhower, C., 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=Coordination+of+growth+rate,+cell+cycle,+stress+response,+and+metabolic+activity+in+yeast.">Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast.</a> Molecular biology of the cell. 19 (1), 352-367 (2008).</li><li>Boer, V. M., Crutchfield, C. A., Bradley, P. H., Botstein, D., Rabinowitz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-limiting+intracellular+metabolites+in+yeast+growing+under+diverse+nutrient+limitations.">Growth-limiting intracellular metabolites in yeast growing under diverse nutrient limitations.</a> Molecular biology of the cell. 21 (1), 198-211 (2010).</li><li>Markham, B. E., Byrne, W. J., Methods, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+storage+and+utilization+of+phosphate+by+yeast.">Uptake, storage and utilization of phosphate by yeast.</a> Journal of the Institute of Brewing. 73, 271-273 (1967).</li><li>Kazemi Seresht, A., Palmqvist, E. A., Olsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+phosphate+scarcity+on+pharmaceutical+protein+production+in+S.+cerevisiae:+linking+transcriptomic+insights+to+phenotypic+responses.">The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses.</a> Microbial cell factories. 10 (1), 104 (2011).</li><li>Shimizu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+Systems+of+Bacteria+such+as+Escherichia+coli+in+Response+to+Nutrient+Limitation+and+Environmental+Stresses.">Regulation Systems of Bacteria such as Escherichia coli in Response to Nutrient Limitation and Environmental Stresses.</a> Metabolites. 4 (1), 1-35 (2013).</li><li>Olson, E. J., Hartsough, L. A., Landry, B. P., Shroff, R., Tabor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+bacterial+gene+circuit+dynamics+with+optically+programmed+gene+expression+signals.">Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals.</a> Nature methods. 11 (4), 449-455 (2014).</li></ol>

저자는 공개 아무것도 없어.

우리는 마음의 유연성이 장치를 설계했습니다. 사용 된 모든 코드는 무료이며 오픈 소스입니다. 세그먼트 셀 기본 이미지 분석 과정은 간단하고 신속하게 실행. 사용자 분석, 피지 그래픽 유저 인터페이스로 대표 화상을 분석하는으로 Beanshell 스크립트 입력을 변환하고 스크립트를 호출하는 플러그인을 설정하는 동안 사용자 입력을 기록함으로써 구현 될 수있다. 이 호출 될 때,이 스크립트는 배경-감산 이미지의 가장 최근의 세트에 파일 경로를 포함하는 &quot;이미지&quot;라는 문자열 배열을 전송됩니다. 이미지 데이터는 실험이 갑자기 끝나는 경우가 손실되지 않도록 수집로 저장됩니다. 블루 LED 어레이 우리 optogenetic 시스템을 유도하는 선택되었다 있지만 다른 색의 LED로 교체 될 수있다. 쉽게 만드는 PCB에 추가적인 MOSFET 스위치 및 릴레이 스위치 용 마이크로 컨트롤러뿐만 아니라 관통 구멍을 사용 가능한 추가 입력 및 출력 핀이있다이 시스템에 대해 좀 더 복잡한 용도에 적합합니다. 예를 들면, LED에 전력을 제공하고, 배양 용기에 묶여 광 센서로부터의 값을 판독 한 후 탁도를 측정 할 수있는 소프트웨어를 수정하고 이에 따라 용기를 희석하기 위해 마이크로 컨트롤러에 추가 핀을 사용하는 turbidostat로 작동이 장치를 만드는 . 주의 성 현미경 장비를 보호하고, 상기 유체 유량이 일치되도록 배양 오염되지 않도록주의해야한다. 배양 용기 의도적 접종시에 앞서 오염은 용기 내부에 접종하고 성장이 없다는 것을 확인하기 전에 몇 일을 대기에 의해 검출 될 수있다. 현미경 목적에 세포 배양을 유출 방지하기 위해, 마이크로 유체 장치는 흡수성 천 위에 그것을 통해 처음 펌핑 세포 배양에 의해 누출되지 않습니다 있는지 확인하십시오. 배양 용기에 미디어의 흐름이 일치하지 않으면, 통상 튜브 주위 때문에연동 펌프의 롤러가 너무 느슨하게되고있다. 수족관 펌프로부터의 공기의 흐름이 일치하지 않는 경우, 액체 수영장 일관성 기류 레지스트있다 유출 관에는 U 자형 굴곡부가 없다는 것을 보장한다. 매체가 그 안에 건조 때문에 튜브는 실험 후 막히게되면, 방해물을 용해 온수 욕에서 막힌 튜브 소크. 실험을 계획 또는 결과를 분석 할 때 고려해야 할이 프로토콜에 고유 제한이 있습니다. 희석율 및 사용 튜브의 길이에 따라, 세포 배양, 현미경으로 배양 용기로부터 펌핑되는 동안의 약 10 분의 지연이있다. 따라서, 잘 짧은 시간 척도를 통해 발생하는 공부 이벤트에 적합하지 않습니다. 실험 매체의 용적에 의해서만 제한 약 10 일 동안 연속적으로 실행할 수있는 반면 또한, 세포 배양의 진화 실험 증가의 기간으로 고려되어야한다. LIMI미디어 팅 영양소 대사 유전자 발현을 35, 36, 37, 38, 39에 지대한 효과를 가지므로, 연구중인 생리학의 양태에 기초하여 결정되어야한다. 결과를 분석하면, 이미지 특히 외곽 데이터에서 이미지 포인트-한다 로아 이미지 분석 루틴에 의해 식별 된 방식에서 오류를 검토. 기포 아티팩트하여 측정 된 형광 비틀림 셀 착각하는 것이 가능하다. 이러한 측정 오류를 제거 할 수있는 간단한 방법은 자동 형광 강도 아래 형광 농도 모든 ROI를 폐기하는 것이다. 이 프로토콜은, 연속 배양에서 성장할 수있는 생물에 빛에 반응하여 형광 강도 및 / 또는 유출 세포 배양의 세포 형태를 측정하는 데 유용 할 설정합니다교반하지 일관된 초점면에 TLE, 자동 이미지 분석 알고리즘에 의해 식별 될 수있다. 여기에 사용되는 기본 셀 식별 알고리즘은 효모 신진과 유사한 대략 구형 미생물에 가장 직접적으로 적용 할 수 있습니다. 대안 40이 프로토콜을 하나 배치 배양의 세트에서 미생물을 성장하고, 각 시점에 대해 하나의 배치를 샘플링하고 유동 세포 계측법에 의해 시료의 특성이다. 그러나, 여기에 기술 된 프로토콜의 장점은 동일한 배양에서 가져온 샘플 많은 샘플을 취할 수 있고, 공정이 자동화되어있다. 이 프로토콜은 optogenetic 효모가 계속 배양 및 형광으로 ROI를 식별 바이어스 신속하고하지 여러 이미지를 획득하여 현미경 (34) 아래에 몇 군데 있었다있는 유사한 방법에 개선. 이 프로토콜의 큰 장점은 개별 셀의 많은 측정이 정기적으로 witho 며칠 동안 취득 할 수 있다는 것입니다유타 사용자 입력. 노광에 미생물 배양의 응답을 측정 한 후, 다음 단계는 반응 실리 폐쇄 루프 제어에 구현 될 수있다.

Optogenetic 시스템은 유전자 발현, 1, 2, 3, 4, 5 단백질 지역화 육 단백질 활성, 6, 7, 8, 단백질 결합, 8, 9, 10, 단백질 분해 등의 세포 과정의 성장 목록을 제어하는 광을 사용한다. 프로그래밍 광 자극으로 제어 된 환경에서 세포를 배양하고, 생물학적으로 중요한 시간 척도에 걸쳐 반응을 측정하기위한 방법 (11)은 세포 생물학 및 생명 공학 연구를 위해 이러한 도구의 전위를 이용하는 것이 필요하다. 우리의 방법은, AE, 잘 혼합에 일정한 세포의 성장 속도를 유지하기 chemostasis 활용평가 및 프로그램 조명에 노출되는 온도 조절 유리 배양 용기 (12, 13). 역 현미경 배양 유출 화상에서는 개별 셀이 프로그래밍 된 조명에 문화의 반응을 측정한다. 형광 강도 및 유출 세포 배양의 세포 형태는 사용자의 입력없이 여러 날에 걸쳐 측정 될 수 있도록 배양 샘플링, 이미지, 이미지 분석은 완전 자동화된다. 이 프로토콜은 성장하는 세포 배양과 현미경 익숙한 대부분 실험실에서 구현 될 수 있으며, 사용되는 장치는, 저렴하고 용이하게 가능한 구성 요소했다. 투명 배양 용기 광의 1 μW / cm 2 mW의 -10 / cm이 발광 가능한 발광 다이오드 (LED)의 행렬 위에 배치된다. 미생물 연속 배양 용기에서 성장된다; 하나의 연동 펌프는에 미디어를 추가하는 데 사용됩니다희석 비율은 다른들은 현미경 낮은 속도로 배양을 인출하는 데 사용되며, 그 차이는 오버 플로우 출구를 통해 빠져. 가열 패드 온도를 유지합니다. 공기는 연속적으로 양압을 유지할뿐만 아니라, 혼합 배양을 통기 배양 용기에 펌핑된다. 공기 펌프를 제외하고,이 장치의 전원은 온도계와 연결된 데스크탑 컴퓨터로부터 입력을 수신하는 마이크로 컴퓨터에 의해 조절된다. 유출 세포 배양은 거꾸로 현미경의 무대에서 미세 유체 장치로 펌핑된다. 비 형광 및 형광 이미지가 자동으로됩니다. 이미지 내의 셀은 ROI (region of interest)를 각 셀의 위치를 ​​각 ROI의 특성을 측정하는 알고리즘을 특징으로한다. 이 프로토콜의 응용 프로그램을 설명하기 위해, 우리는 푸른 빛 responsi으로 설계 사카로 마이 세스 세레 비지 세포의 변화하는 빛의 강도에 대한 응답을 측정형광 단백질의 전사를 제어하는 ​​시스템 optogenetic했습니다. 이 시스템에서 유전자 발현을 제어하기위한 여러 optogenetic 시스템이 이미 14, 15, 16가 존재하기 때문에 통상적으로 빵 효모 S. cerevisiae의 알려진이 선택되었다. 또한,이 모델 생물은 일반적으로 시스템 생물학 (17)과 생명 공학 응용 프로그램을 18, 19, 20 섀시로 연구에 사용됩니다. 우리의 대표적인 결과가이 프로토콜은 입력 광 강도를 변화 형광 리포터의 생산을 측정함으로써 복수 일 배양의 전사를 조절하는데 사용될 수 있다는 것을 보여준다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Extensive lab manual</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >An extensive, regularly updated lab manual is available in the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files). This also includes a description of the microfluidic mold used to generate the representative results.</td>
		</tr>
		<tr >
			<td >Fritzing Design Viewer</td>
			<td >Fritzing</td>
			<td >NA</td>
			<td >The free, open-sourced software to view and edit the .fzz type circuit board designs is available at &#34;http://fritzing.org/download/&#34;</td>
		</tr>
		<tr >
			<td >Arduino Uno R3 (Atmega328 - assembled)</td>
			<td >Adafruit</td>
			<td >50</td>
			<td >Microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >Arduino Stackable Header Kit</td>
			<td >SparkFun Electronics</td>
			<td >10007</td>
			<td >Female pin headers for connecting PCB to microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >Adjustable 30W 110V soldering iron - XY-258 110V</td>
			<td >Adafruit</td>
			<td >180</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >Soldering iron stand</td>
			<td >Adafruit</td>
			<td >150</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >Mini Solder spool - 60/40 lead rosin-core solder 0.031&#34; diameter - 100g</td>
			<td >Adafruit</td>
			<td >145</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >0.1 &#956;F capacitor</td>
			<td >SparkFun Electronics</td>
			<td >COM-08375</td>
			<td >Stabilizes voltage in PCB. 1 required.</td>
		</tr>
		<tr >
			<td >10 &#956;F capacitor</td>
			<td >SparkFun Electronics</td>
			<td >COM-00523</td>
			<td >Stabilizes voltage in PCB. 1 required.</td>
		</tr>
		<tr >
			<td >MAX7219CNG LED Matrix/Digit Display Driver - MAX7219</td>
			<td >Maxim</td>
			<td >MAX7219CNG</td>
			<td >LED driver. 1 required.</td>
		</tr>
		<tr >
			<td >8 pin IC Socket</td>
			<td >Mouser Electronics</td>
			<td >575-144308</td>
			<td >16 required. These will be stacked on top of each other to support the culture vessel above the LED matrix.</td>
		</tr>
		<tr >
			<td >24 Pin IC socket</td>
			<td >Mouser Electronics</td>
			<td >535-24-3518-10</td>
			<td >Optional. Use this to reversibly attach the MAXIM 7219CNG driver to the PCB.</td>
		</tr>
		<tr >
			<td >Digital multimeter</td>
			<td >Adafruit</td>
			<td >2034</td>
			<td >For troubleshooting electronics. 1 required.</td>
		</tr>
		<tr >
			<td >Break Away Headers - 40-pin Male (Long Centered, PTH, 0.1&#34;)</td>
			<td >SparkFun Electronics</td>
			<td >PRT-12693</td>
			<td >Male pin headers for connected LED matrix to printed circuit board. Ends can be trimmed with wire cutters. 1 set required.&#160;</td>
		</tr>
		<tr >
			<td >Flush diagonal wire cutters</td>
			<td >Adafruit</td>
			<td >152</td>
			<td >For trimming long pin headers and cutting power cables. 1 required.</td>
		</tr>
		<tr >
			<td >Premium Female/Female Jumper Wires - 40 x 12&#34; (300mm)</td>
			<td >Adafruit</td>
			<td >793</td>
			<td >Wire ribbon for connecting breadboard to LED matrix. Can be connected end-to-end with male pin-headers to be longer. 1 required.</td>
		</tr>
		<tr >
			<td >Half-size breadboard</td>
			<td >Adafruit</td>
			<td >64</td>
			<td >The LED matrix will connect to this and the culturing vessel will rest above it.</td>
		</tr>
		<tr >
			<td >Miniature 8x8 Blue LED Matrix</td>
			<td >Adafruit</td>
			<td >956</td>
			<td >Light source. Dominant wavelength is 470nm (blue). 1 required. Alternative miniature LED matrices from the same vendor are available with dominant wavelengths: 624 nm (red), 588 nm (yellow), 525 nm (green), 572 nm (yellow-green), and white.</td>
		</tr>
		<tr >
			<td >Stackable header-3 pin</td>
			<td >SparkFun Electronics</td>
			<td >13875</td>
			<td >8 required.</td>
		</tr>
		<tr >
			<td >Resistor Kit - 1/4W (500 total)</td>
			<td >SparkFun Electronics</td>
			<td >10969</td>
			<td >For electronics. 1 required.</td>
		</tr>
		<tr >
			<td >&#160;IRL520N MOSFET</td>
			<td >International Rectifier</td>
			<td >IRL520N</td>
			<td >Voltage regulating switch for controlling DC current. 4 required.</td>
		</tr>
		<tr >
			<td >Hook-Up Wire - Assortment (Solid Core, 22 AWG)</td>
			<td >SparkFun Electronics</td>
			<td >PRT 11367</td>
			<td >Wire for electronics. 1 required.</td>
		</tr>
		<tr >
			<td >5V 2A (2000mA) switching power supply - UL Listed</td>
			<td >Adafruit</td>
			<td >276</td>
			<td >Power supply for the heating pad and Arduino. 2 required.</td>
		</tr>
		<tr >
			<td >12 VDC 1000mA regulated switching power adapter - UL listed</td>
			<td >Adafruit</td>
			<td >798</td>
			<td >For peristaltic pumps. 2 required.</td>
		</tr>
		<tr >
			<td >Electric Heating Pad - 10cm x 5cm</td>
			<td >Adafruit</td>
			<td >1481</td>
			<td >For heating the bioreactor. 1 required.</td>
		</tr>
		<tr >
			<td >Low flow variable flow peristaltic pump</td>
			<td >Fisher Scientific</td>
			<td >13-876-1</td>
			<td >For pumping media. 1 required</td>
		</tr>
		<tr >
			<td >Medium flow variable flow peristaltic pump</td>
			<td >Fisher Scientific</td>
			<td >13-876-2</td>
			<td >For pumping culture. 1 required.</td>
		</tr>
		<tr >
			<td >9 VDC 1000mA regulated switching power adapter - UL listed</td>
			<td >Adafruit</td>
			<td >63</td>
			<td >For microcontroller power supply. Order 1.</td>
		</tr>
		<tr >
			<td >High Temp Waterproof DS18B20 Digital temperature sensor + extras</td>
			<td >Adafruit</td>
			<td >642</td>
			<td >Thermometer for the bioreactor. 1 required.</td>
		</tr>
		<tr >
			<td >Micromanager</td>
			<td >Micromanager</td>
			<td >NA</td>
			<td >The free, open-sourced microscope control software is available at &#34;https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release&#34;</td>
		</tr>
		<tr >
			<td >FIJI</td>
			<td >ImageJ</td>
			<td >NA</td>
			<td >The free, open-sourced image analysis software is available at &#34;http://fiji.sc/&#34;</td>
		</tr>
		<tr >
			<td >Arduino Integrated Development Environment</td>
			<td >Arduino</td>
			<td >NA</td>
			<td >The free, open-sourced IDE is available at &#34;https://www.arduino.cc/en/Main/Software&#34;</td>
		</tr>
		<tr >
			<td >Custom code</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >The custom microcontroller code and &#34;Bioreactor Controller&#34; plugin are available in the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td>
		</tr>
		<tr >
			<td >USB Cable A to B - 6 Foot</td>
			<td >SparkFun Electronics</td>
			<td >CAB-00512</td>
			<td >Used to download data to microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >bioreactorTimecourse_example.csv</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >The advantage of loading LED matrix values from a CSV file is that a program can be called by the plugin to update those values based on image analysis results, and those values can be reloaded to the microcontroller, enabling feed-back control. It is available from the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td>
		</tr>
		<tr >
			<td >Tota-frost gels (diffusion paper)</td>
			<td >B&#38;H</td>
			<td >B&#38;H # LOFSFTL 
			MFR # T1-72</td>
			<td >For LED matrix. 1 required.</td>
		</tr>
		<tr >
			<td >Kitting Sheet Crosslink 1/4x12x24in</td>
			<td >Grainger, inc</td>
			<td >20JL37</td>
			<td >Black foam for culturing vessel enclosure. 4 required.</td>
		</tr>
		<tr >
			<td >Standard Photodiode Power Sensor, Si, 200 - 1100 nm, 50 mW&#160;</td>
			<td >Thorlabs</td>
			<td >S120VC</td>
			<td >For measuring light intensity. 1 required.</td>
		</tr>
		<tr >
			<td >Labelling Tape</td>
			<td >Fisher Scientific</td>
			<td >159015N</td>
			<td >For labelling and securing loose components. 1 required.</td>
		</tr>
		<tr >
			<td >Compact Power and Energy Meter Console, Digital 4&#34; LCD</td>
			<td >Thorlabs</td>
			<td >PM100D</td>
			<td >For measuring light intensity. 1 required.</td>
		</tr>
		<tr >
			<td >100mL GL45 hybridization glass bottle</td>
			<td >Bellco Glass, Inc.</td>
			<td >(7910-40150)</td>
			<td >Bioreactor vessel. 1 required.</td>
		</tr>
		<tr >
			<td >Six port assembly</td>
			<td >Bellco Glass, Inc.</td>
			<td >Custom&#160;</td>
			<td >For the bioreactor vessel. Tubing Specs: .125&#34; OD x .055&#34;ID. Port A: 1.0&#34; long above cap slug and to bottom of tube. Ports B,C,E,F: 1.0&#34; long above cap slug, 33 mm long below. Port D: 1.0&#34; long above cap slug, 65 mm&#160; long below. 1 required. Includes 45 mm diameter polypropylene open top screw cap and a white silicone gasket to ensure a tight seal between the cap and the vessel.&#160;</td>
		</tr>
		<tr >
			<td >Scotch Magic Tape 3105, 3/4 x 300 Inches, Pack of 3</td>
			<td >Amazon</td>
			<td >B0009F3P3U</td>
			<td >Clear scotch tape. This is available from many other vendors. It is used to cover markings on the culturing vessel and to secure the coverglass with the PDMS channel to the aluminum support frame.</td>
		</tr>
		<tr >
			<td >1/16&#34; ID x 3/16&#34; OD x 1/16&#34; Wall Tygon Sanitary Silicone Tubing</td>
			<td >United States Plastic Corp.</td>
			<td >57288</td>
			<td >Tubing. ~25&#39; required.</td>
		</tr>
		<tr >
			<td >Cole-Parmer Twistit white rubber stopper, size 10</td>
			<td >Cole-Parmer</td>
			<td >EW-62992-32</td>
			<td >Media flask stopper and effluent flask stopper. 2 required.</td>
		</tr>
		<tr >
			<td >2L Laboratory Flask</td>
			<td >Pyrex</td>
			<td >4980</td>
			<td >Media flask and effluent flask. 2 required.</td>
		</tr>
		<tr >
			<td >Day pinchcock</td>
			<td >Fisher Scientific</td>
			<td >5867</td>
			<td >For pinching tubes shut. 3 required.</td>
		</tr>
		<tr >
			<td >Replacement tubing assembly 1/16&#34; ID</td>
			<td >Traceable Products</td>
			<td >3372</td>
			<td >The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.</td>
		</tr>
		<tr >
			<td >Replacement tubing assembly 1/50&#34; ID</td>
			<td >Traceable Products</td>
			<td >3371</td>
			<td >The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.</td>
		</tr>
		<tr >
			<td >Male luer with lock ring x 1/16&#34; hose barb, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-00</td>
			<td >Connectors. ~10 luers are required.</td>
		</tr>
		<tr >
			<td >Male luer with lock ring x 1/8&#34; hose barb, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-04</td>
			<td >Connectors. 5 required, one for each rubber stopper hole to fill with tubing.</td>
		</tr>
		<tr >
			<td >Female luer x 1/16&#34; hose barb adapter, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45502-00</td>
			<td >Connectors. ~10 luers required.</td>
		</tr>
		<tr >
			<td >Female luer x 3/16&#34; hose barb adapter</td>
			<td >Cole-Parmer</td>
			<td >EW-45502-08</td>
			<td >Connectors. ~10 luers required.</td>
		</tr>
		<tr >
			<td >Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk</td>
			<td >Cole-Parmer</td>
			<td >SC-45502-28</td>
			<td ></td>
		</tr>
		<tr >
			<td >Cole-Parmer Luer Accessory, Male Luer Lock Plug, Nylon, 25/Pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-56</td>
			<td ></td>
		</tr>
		<tr >
			<td >Microbore PTFE Tubing, 0.022&#34;ID x 0.042&#34;OD, 100 ft/roll</td>
			<td >Cole-Parmer</td>
			<td >EW-06417-21</td>
			<td >Tubing. 1 roll required.</td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone tubing, L/S 13, 25 ft</td>
			<td >Cole-Parmer</td>
			<td >EW-96410-13</td>
			<td >Tubing. ~25&#39; required.</td>
		</tr>
		<tr >
			<td >3/16&#34; ID x 1/4&#34; OD x 1/32&#34; Wall Tygon Sanitary Silicone Tubing</td>
			<td >United States Plastic Corp.</td>
			<td >57293</td>
			<td >Tubing. ~1&#39; required.</td>
		</tr>
		<tr >
			<td >Vacuum filter</td>
			<td >Fisher Scientific</td>
			<td >974107</td>
			<td >Nalgene vacuum filter for sterile filtering media.</td>
		</tr>
		<tr >
			<td >Aquel Oxy-Boost 200</td>
			<td >Rena Aquatic Supply</td>
			<td >AP200</td>
			<td >Dual diaphram adjustable flow air pump for aerating and mixing media. 1 required.&#160;</td>
		</tr>
		<tr >
			<td >0.2 &#956;m pore syringe filter</td>
			<td >Corning International</td>
			<td >431229</td>
			<td >This ensures that air from the aquarium pump does not contaminate the apparatus. 1 required.</td>
		</tr>
		<tr >
			<td >Slygard 184 Silicone Elastomer Kit</td>
			<td >Dow Corning</td>
			<td >Slygard 184</td>
			<td >For microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >American Safety Razor GEM Scientific Single-Edge Razor Blades</td>
			<td >Fisher Scientific</td>
			<td >17989000</td>
			<td >For cutting tubes and PDMS. 1 blade required.</td>
		</tr>
		<tr >
			<td >Harris Uni-Core hole puncher 1.2mm ID</td>
			<td >Sigma-Aldrich</td>
			<td >WHAWB100028 ALDRICH</td>
			<td >For punching inlet/outlet in microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >Microscope cover glass 22x60-1.5</td>
			<td >Fisher Scientific</td>
			<td >12-544-G</td>
			<td >For microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >Rectangular aluminum frame with a square window</td>
			<td >Custom</td>
			<td >Custom</td>
			<td >To support the microfluidic channel. Outer dimensions: 3 inches x 1.25 inches. 
			Inner dimmensions (cut out portion): 7/8 inches x 7/8 inches 
			Thickness: ~1/32 inches</td>
		</tr>
	</tbody>
</table>

design and implementation of an automated illuminating, culturing, and sampling system for microbial optogenetic applications

Optogenetic 시스템은 세포 과정을 변경하는 빛의 특정 파장에 대한 응답으로 형태를 변경 유전자 인코딩 된 단백질을 사용한다. optogenetic 조명 시스템의 프로그래밍 및 자극을 탑재 되 배양 시스템 및 측정이 필요하다. 우리 구축 광의 프로그래밍 용량과 균체를 조명하고, 자동으로 획득하고 유출 셀의 이미지를 분석하는 연속 배양 장치를 이용하기위한 프로토콜을 제시한다. chemostat 등이 장치의 동작은 성장 속도와 셀룰러 환경이 엄격하게 제어 될 수있다. 연속 세포 배양의 유출 물을 주기적으로 샘플링하고, 세포를 다 채널 현미경으로 이미지화된다. 형광 강도 배양 유출 물로부터 샘플링 된 세포의 세포 형태의 동적 응답이 여러 날에 걸쳐 측정되도록 배양 샘플링, 촬상 한 화상 분석이 완전히 자동화사용자 입력없이. 우리가 동적으로 전사를 활성화 optogenetic 시스템 설계 사카 세레 비지에 균주에서 단백질 생산을 유도하여 배양 장치의 유용성을 입증한다.

이 장치는 CRY2 / CIB1 단백질 쌍 (30)에 기초하여 유도 optogenetic 전사 시스템을 통해 푸른 빛의 질문에 답변 노란색 형광 단백질 (YFP)를 표현하는 S. cerevisiae의 문화를 자극하기 위해 사용되었다. 세포를 0.2 ± 0.008 평균 희석 속도 포스페이트 한정 배지에서 배양 하였다 chemostatically. 인산 제한은 일반적으로 성장 속도를 제어하는 S. 세레 비지 chemostat 실험에 사용되는 인산 제한의 효과는 잘 특징으로한다. 31 (32)는 연속적으로 희석 된 배양 용기 (33)로부터 유출 물은 거꾸로 현미경에서 미세 유체 소자로 샘플링 하였다. 도 8에 도시 된 바와 같이, 이미지는 자동으로 분석 하였다. 개별 셀 배경-감산 위상차 이미지와 자신의 YFP에 집중하고에서 확인되었다백그라운드 - 감산 형광 화상으로부터 측정 된 기들은 형광로부터 추정 하였다. 169,677 세포의 형광 실험 70 시간에 걸쳐 마이크로 유체 장치 (28)로부터 취득한 위치 유출 물의 33600 이미지에서 분석 하였다. 우리는 형광 조명 시스템 및 CMOS 카메라 장착 거꾸로 현미경을 사용 하였다. YFP 이미지는 20분의 500 nm의 여기 필터, 30분의 535 nm의 방출 필터와 T515lp 이색으로 획득 하였다. 이미지는 쾰러 조명 아래, 40X 위상 대비 목표로 촬영되었다. 마이크로 미터 제곱 당 이미지는 87 픽셀 16 비트 색 깊이에서 기록되었다. 문화는 6 시간 간격에 대한 완전한 어둠 다음, 6 시간 간격에 대한 푸른 빛의 강도 변화에 노출되었다. 배양은 그 형광 강도가 제 어두운 기간 동안 감소하는 이유는 첫번째 측정 전에 빛에 노출되어 있었다. &lt;/ P&gt; 도 9는 배양 용기의 조명에 응답 의한 optogenetic 시스템의 활성화시 YFP 생산의 형광을 나타낸다. 그것은 평균 단일 셀 측정 형광 강도의 인구 분포를 나타내 인구 위에 형광 단일 셀 측정의 유용성을 보여준다. 42 시간 26 분에서 특이를합니다. 해당 이미지를 검토 한 결과, 배경 화상 감산 된 세포 닮은 아티팩트의 결과로, 배경의 합성 이미지를 생성하는 데 사용되는 이미지의 대다수의 세포 덩어리가 있다고 명백했다. 또한, 배양 용기로부터 기포는 미세 유체 채널로 펌핑하고, 모서리 부분은 이미지 분석 알고리즘에 의해 세포와 같은 착각이었다. 기포 나 배경 차감으로부터 아티팩트도 실제 전지에 대응하기 때문에, 그 측정 된 형광 강도셀의 자동 형광보다 낮다. 이 수치는 자동으로 3 일 동안 획득 될 수있는 데이터의 품질을 보여준다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54894/54894fig9large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 8" src="/files/ftp_upload/54894/54894fig8.jpg" /> 그림 8 : 영상 분석 알고리즘의 시각적 묘사. 이러한 이미지는 잘립니다 및보기의 용이성을 위해 확장되었습니다. (A) 여섯 이미지는 세포 배양의 배경 - 감산 위상차 이미지를 생성하기 위해 취득된다. 메인 이미지가 획득 된 후, 5 개의 추가 이미지 간단히 세포 변위되도록 각각의 취득 사이에 온 유출 샘플링 펌프를 취득한다. 배경의 합성 화상은 다섯 요소 화상으로부터 생성된다. 각 화소의 값배경 화상의 5 성분 화상에 걸쳐 동일한 화소의 중간 값이다. (B)이 감산 된 배경 화상은 이진으로 변환된다. 이진은 다음 넓혀되고 이진의 연속 섹션 내에서 구멍이 채워집니다. 황색 윤곽선 크기 원형 기준에 따라 선택된 관심 영역 (ROI)에 대응한다. (C) 그 ROI 각 셀의 형광은 ROI 내에서 가장 밝은 화소의 값으로서 측정되는 형광 이미지로 매핑된다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54894/54894fig8large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 9" src="/files/ftp_upload/54894/54894fig9.jpg" /> 그림 9 : 시간이 지남에 따라 형광 강도의 인구 분포입니다. 측정 된 불소의 이러한 하위 도면에서 로그escence은 유동 세포 계측법의 표준 관행에 따라 표시됩니다. A 및 B의 광고들은 빛을 흡수하거나, 배양 용기 내에서 세포 배양을 통해 전송 멸균 매체 및 광 투과 한 빛의 강도 사이의 차이로서 측정된다 배양, 회절 강도를 나타낸다. 이것은 제 종축에 대해 도시된다. (A) 상자와 수염 플롯 시간에 따른 인구의 측정 된 형광의 대수 (5 번째 백분위 수, 25 번째 백분위, 중앙값, 75 번째 백분위 수, 95 번째 백분위 수). (B) 컬러 해당 빈의 범위에서 측정 된 형광과 세포의 규격화 주파수에 대응하는 동일한 데이터의 2 차원 히스토그램. 색상은 26 분 포함 42 시간의 특이없이 데이터의 범위를 확장했다. 가장 낮은 형광 빈에서 아웃 라이어의 정규화 된 주파수0.7. (C) 1 차원 광에 처음으로 기록 된 노광 전에 모집단의 측정 된 형광의 대수의 히스토그램 및 광 최대 노광 후. 그것은이 변형에 YFP의 빛에 의한 표현 바이 모달 있음을 보여줍니다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54894/54894fig9large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications at JoVE.com

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

우리는 거꾸로 현미경으로 유출 물에 미생물의 문화와 정기적으로 이미지 세포를 조명하기 optogenetic 시스템과 함께 사용 연속 배양 장치를 설계했다. 조명 동적 응답은 며칠에 걸쳐 측정 될 수 있도록 배양 샘플링, 촬상 한 화상 분석이 완전 자동화된다.

미생물 Optogenetic 애플리케이션을위한 설계 및 자동화 일루미, 배양 및 샘플링 시스템의 구현

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

design-implementation-an-automated-illuminating-culturing-sampling

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.6K 조회수.  University of Wisconsin-Madison.  우리는 거꾸로 현미경으로 유출 물에 미생물의 문화와 정기적으로 이미지 세포를 조명하기 optogenetic 시스템과 함께 사용 연속 배양 장치를 설계했다. 조명 동적 응답은 며칠에 걸쳐 측정 될 수 있도록 배양 샘플링, 촬상 한 화상 분석이 완전 자동화된다. 

비디오: Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT : 바이오 매스의 효소 발견과 칵테일 최적화를위한 자동 플랫폼

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

연구

생체공학

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

위한 실크 영화 문화 시스템 체외에서 분석 및 Biomaterial 디자인

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

디자인과 소프트 모듈 형 로봇의 탄성 장치의 제작 최소 침습 수술

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

모듈형 DNA 장치 조립 로봇 액체 처리 자동화

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

높은 처리량의 사용 모델 IgG1 조 세포에서의 생산을 위한 Microbioreactor 시스템 자동화

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

라이브 셀 분석의 전단 응력에 녹 농 균 등 처리량 미세 자동 시스템을 사용 하 여

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

자동화된 미생물체 시스템을 사용하여 중국 햄스터 난소 세포의 단일 클론 항체의 정화 및 분석

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Design and Analysis for Fall Detection System Simplification

This paper presents a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition system that can be easily implemented and adopted. The methodology is based on the configuration of specific types of sensors, machine-learning methods and procedures. The protocol is divided into four phases: (1) database creation (2) data analysis (3) system simplification and (4) evaluation. Using this methodology, we created a multimodal database for fall detection and human activity recognition, namely UP-Fall Detection. It comprises data samples from 17 subjects that perform 5 types of falls and 6 different simple activities, during 3 trials. All information was gathered using 5 wearable sensors (tri-axis accelerometer, gyroscope and light intensity), 1 electroencephalograph helmet, 6 infrared sensors as ambient sensors, and 2 cameras in lateral and front viewpoints. The proposed novel methodology adds some important stages to perform a deep analysis of the following design issues in order to simplify a fall detection system: a) select which sensors or combination of sensors are to be used in a simple fall detection system, b) determine the best placement of the sources of information, and c) select the most suitable machine learning classification method for fall and human activity detection and recognition. Even though some multimodal approaches reported in literature only focus on one or two of the above-mentioned issues, our methodology allows simultaneously solving these three design problems related to a human fall and activity detection and recognition system.

We present a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition system. The goal is to build a system for accurate fall detection that can be easily implemented and adopted.

낙하 감지 시스템 단순화를 위한 설계 및 해석

This paper presents a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

소규모 셀 처리를 위한 자동 카운터플로우 원심 시스템

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...