計算機控制的自動微吸管系統

計算機控制的自動微吸管系統

該系統是一種安裝在正常倒置顯微鏡上的計算機控制的微量吸管系統，量化細胞形變、細胞間粘附作用和細胞與基質之間的粘附作用，可同步實現熒光觀測、單細胞捕獲、分選和分離和圖像視頻處理

一個可在已有顯微鏡上搭建的，可同步實驗吸吮加載、圖像視頻處理與熒光觀測的細胞力學裝置。可建立微管吸吮力學加載-熒光觀測耦合的分子-細胞動力學實時原位觀測系統。
單個細胞粘附力測定模式圖：
細胞與細胞之間粘附力測定模式圖：
通過施加負壓將細胞的一部分（或整體）吸入微管中,測量一定負壓吸吮作用下細胞的變形及其時間歷程或細胞粘附分離的臨界負壓來評價細胞的變形特性，同時記錄細胞的吸入量。
圖像處理技術和力學模型，量化測量細胞形變、細胞對之間的相互作用以及黏附特性、基質附著細胞測定細胞硬度等力學特性。
壓電微管吸吮模塊：對樣品池內細胞的捕獲、分選、吸吮和微操控；
集成倒置熒光相差顯微鏡：用于對所述細胞進行熒光激發；
信號采集模塊：用于對微弱熒光信號的采集；
控制模塊：用于對微管吸吮和熒光采集同步觸發，并進行數據分析處理

特點：

• 1)High throughput single cell sorting directly from the Petri dish

• One single cell arrives to each PCR tube

• 10 PCR strips containing 80 tubes can be filled in a cycle

• Glass cover slip for testing single cell deposition in situ

• Drop volume less than 1 ul for adherent cells

• Pick up volume of ~1 nl for suspended cells

• 15-20 seconds per cell. When collecting multiple cells, sorting speed is 1 cell/second.

• Number of cells picked up in a single run: 1-1000.

• Isolates a subpopulation of live adherent cells expressing fluorescent or luminescent markers

• Both unlabeled and fluorescentcells are recognized by computer vision

• Viable cells after sorting

• Any adherent and non-adherent cell type can be sorted

• Cell culture needs minimal preparation before sorting

• Average sorting process takes only a few minutes

• Multichannel detection using the fluorescent filter setup of the microscope

應用范圍：

1）計算機控制的自動化微吸管系統測定單細胞黏附力（Single Cell Adhesion Assay Using Computer Controlled Micropipette）

2）計算機控制的自動化微吸管系統測定細胞與細胞之間的黏附力（Cell-cell adhesion force assay Using Computer Controlled Micropipette）

1. R. Salánki et al. : Single cell adhesion assay using computer controlled micropipette, PLoS ONE 9(10): e111450 (2014) Open access paper.

2. P. K. Jani et al.: Complement MASP-1 enhances adhesion between endothelial cells and neutrophils by up-regulating E-selectin expression, Molecular Immunology 75, 38–47 (2016)

3. N. Sándor et al.: CD11c/CD18 Dominates Adhesion of Human Monocytes, Macrophages and Dendritic Cells over CD11b/CD18, PLoS ONE 11(9), e0163120 (2016) Open access paper.

其他應用：

3）單細胞分離與抓取分選系統 （Single Cell Isolation and Sorting）

1. Z. K?rnyei et al.: Cell sorting in a Petri dish controlled by computer vision Nature Scientific Reports 3, Article number: 1088 (2013) Open access paper.

2. R. Salánki et al.: Automated single cell sorting and deposition in submicroliter drops, Appl. Phys. Lett. 105, 083703 (2014)

3. R. Salánki et al.: High-throughput image based single cell isolation, Microscopy and Analysis, January issue, S10-13 (2015) Open access paper.

4. R. Ungai-Salánki et al.: Automated single cell isolation from suspension with computer vision, Scientific Reports 6, Article number: 20375 (2016) Open access paper.

4）循環腫瘤細胞（CTC）分離 （Circulating tumor cell (CTC) isolation）

Marnie Winter et al.: Isolation of Circulating Fetal Trophoblasts Using Inertial Microfluidics for Noninvasive Prenatal Testing, Advanced Materials Technologies 1800066 (2018)

5）單細胞RNA測序 （Single Cell RNA Sequencing）應用文獻

1. Mia Palmkvist: Malaria and polypeptides of plasmodium falciparum at the infected erythrocyte surface, PhD Thesis, Karolinska Institutet, Stockholm (2016)

2. A. Kozlov et al.: A screening of UNF targets identifies Rnb, a novel regulator of Drosophila circadian rhythms, The Journal of Neuroscience  7, 3286-16 (2017)

3. M. Ngara et al.: Exploring parasite heterogeneity using single-cell RNA-seq reveals a gene signature among sexual stage Plasmodium falciparum parasites, Experimental Cell Research (2018)

6）蛋白工程（Potein Engineering)

1. K. Piatkevich et al. : A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters, Nature Chem Biol, doi:10.1038/s41589-018-0004-9 (2018)