Automated Isolation of Single Cells Using High-Resolution and Subcellular Imaging

使用高分辨率和亚细胞成像自动分离单细胞

• 批准号: 9251383
• 负责人: Steven Charles Gebhart
• 金额: $ 81.85万
• 依托单位: CELL MICROSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9251383
• 关键词: Achievement; Agreement; Algorithmic Analysis; Algorithms; Automation; Behavior; Cell Culture Techniques; Cell Separation; Cells; Characteristics; Computer software; Coupled; Coupling; Detection; Doctor of Philosophy; Elastomers; Engineering; Exhibits; Fluorescence; Genomics; Glass; Goals; Human Resources; Image; Image Analysis; Image Enhancement; Incubators; Individual; Interview; Laboratories; Letters; Link; Magnetism; Molecular Analysis; Needles; Neurons; Optics; Organelles; Performance; Phase; Phenotype; Physiology; Plant Resins; Plasticizers; Polymers; Polystyrenes; Production; Property; Proteins; Refractive Indices; Reporter; Research; Research Personnel; Resolution; Retrieval; Running; Signal Transduction; Sorting - Cell Movement; Stem cells; System; Technology; Testing; Time; Universities; Update; Validation; Virginia; Work; base; cellular imaging; commercialization; cost effective; design; elastomeric; high resolution imaging; high throughput screening; imaging capabilities; improved; innovation; instrument; interest; manufacturing scale-up; microsystems; nanoparticle; neoplastic cell; performance tests; phenotypic data; programs; prototype; screening; seal; single cell analysis; stem cell differentiation; transgene expression; user-friendly

PROJECT SUMMARY  Pairing  detailed  phenotypic  characterization  of  cells  with  downstream  molecular  analysis  represents  a  significant challenge, especially at the level of single cells. Cell Microsystems has developed and commercialized  the CellRaft™ System, which is currently the only commercially available means of imaging cells using brightfield  and/or multi-­‐‑channel fluorescence prior to single cell isolation and downstream molecular analysis. Investigators  interested in pairing phenotypic data with genomic analysis at the single cell level have employed the CellRaft  System for a range of innovative studies including stem cell differentiation, tumor cell physiology and screening  of neurons. However, the materials used in the currently commercially available CellRaft Arrays are not optimized  for imaging quality and were selected for other properties such as the re-­‐‑sealing behavior of the elastomer and cell  culture compatibility of the plastic. During the Phase I program, alternative materials for the CellRaft Array were  explored for improved optical properties such as refractive index agreement and reduced autofluorescence. One  particular  alternative  materials  combination  exhibited  favorable  performance  in  both  criteria  leading  to  dramatically  improved  cell  imaging  performance.  Here,  we  refer  to  this  version  of  the  product  as  the  High  Resolution,  or  HR-­‐‑CellRaft.  The  focus  of  our  Phase  II  proposal  is  to  commercialize  the  HR-­‐‑CellRaft  for  users  interested  in  pairing  detailed  phenotypic  imaging  not  possible  on  the  current  CellRaft  Array  (e.g.  subcellular  protein  localization,  organelle  physiology,  and  semi-­‐‑quantitative  fluorescent  signals),  as  well  as  automate  the  CellRaft  System  workflow.  In  a  parallel  program,  we  are  developing  a  highly  automated  instrument,  the  Automated  Isolation  and  Retrieval  (AIR™)  System  for  single  cell  imaging  and  isolation  employing  the  current  CellRaft Array. During Phase II we will implement several design revisions to the optical capabilities of the AIR  System  as  well  as  its  software-­‐‑based  image  analysis  algorithms  to  better  leverage  the  HR-­‐‑CellRaft’s  improved  optical  properties.  The  new  proposed  instrument  is  referred  to  here  as  the  CLEAR  AIR™  System.  Additional  fluorescence detection channels (six instead of three in the AIR™ System), higher magnification objectives (10X,  20X and 40X as opposed to the single objective -­‐‑ 4X or 10X -­‐‑ found on the AIR™ System) and a higher resolution  camera  will  enhance  the  imaging  capabilities  of  the  system.  For  external  validation  of  HR-­‐‑CellRaft  Array  and  CLEAR  AIR  System  performance,  we  have  identified  two  investigators  from  our  Early  Adopter  Program  with  research needs uniquely satisfied by the HR-­‐‑CellRaft and CLEAR AIR System. One will isolate single cells based  on subcellular localization of a reporter protein and the second will isolate cells with varying levels of transgene  expression,  with  both  groups  coupling  these  phenotypic  observations  to  downstream  single  cell  molecular  analysis.  The  HR-­‐‑CellRaft  and  the  improved  optical  subsystem  in  the  CLEAR  AIR  System  meets  an  urgently  unmet need for pairing high-­‐‑resolution phenotypic imaging with downstream molecular analysis of single cells.

项目总结