Receding meniscus induced docking of yeast Receding meniscus induced - PowerPoint PPT Presentation

The 5 th US-Korea Nano Forum Receding meniscus induced docking of yeast Receding meniscus induced docking of yeast cells for quantitative single- -cell analysis cell analysis cells for quantitative single Min-Cheol Park, 1 Jae-Young Hur, 2 Keon-Woo Kwon, 1 Sang-Hyun Park 2 and Kahp-Yang Suh 1 * 1 School of Mechanical and Aerospace Engineering, Seoul National University, Seoul 151-742, Korea. 2 School of Biological Sciences and Research Center for Functional Cellulomics, Seoul National University, Seoul 151-742, Korea. *e-mail: sky4u@snu.ac.kr http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Single- -cell analysis cell analysis Single Bulk population based analysis misconception from bulk population well plate heterogeneous response population population averaging readout readout Ensemble averaging problem ! http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Why single- -cell analysis? cell analysis? Why single • At single-cell level microwells heterogeneous intensity - Stochastic expression of cell intensity - Observing Single-cell information distance http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Cell chip (single- -cell array) cell array) Cell chip (single Flow Cytometry (FACS) • cannot interrogate individual cells repeatedly • cannot capture cell images with high resolution • cannot observe the spatial localization of fluorescence Automated microscope combined with cell chip • can take hundreds of images of single cells at different time points • can provide quantitative insight into cellular behavior • can be used to determine heterogeneity among a population of single cells High-throughput and high-content single-cell analysis http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Single- -cell docking cell docking Single Bhatia et.al., Nat. Methods (2005) Folch et.al., Lab. Chip (2005) Dielectrophoresis Sedimentation and so on .. Voldman et.al., Anal. Chem. (2002) Optical tweezers Microfluidic patch clamp Physical structure Luke Lee et.al., PNAS (2005) Grier, Nature (review) (2003) Luke Lee et.al., Lab. Chip (2006) http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Our approach • Receding meniscus induced cell docking (Lab Chip, 6, 988, 2006) 30 um wells 10 um wells http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Microw ell Patterning PDMS stamp PDMS pre-polymer + curing agent PU A glass substrate negative silicon master PDMS pouring PDMS stamp PDMS replica silicon master glass substrate PDMS curing at 70 ℃ , for 1h PDMS stamp glass substrate capillary molding for capillary molding K. Y. Suh et al., Adv. Mater. 2001. http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Patterned Microfluidic Channel PDMS microfluidic mold PDMS microfluidic mold Plasma treating glass substrate PDMS substrate PDMS microfluidic mold PDMS microfluidic mold glass substrate PDMS substrate hydrophilic surface Ali K., K. Y. Suh et al., Anal. Chem. 2004. http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Receding m eniscus induced docking • Schematic diagram PDMS microfluidic mould inlet reservoir outlet reservoir microchannel patterned glass substrate Introducing a yeast cell suspension by capillary filling Capturing the cells by lateral capillary force Generating a receding meniscus by evaporation Receding meniscus induced docking http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Receding m eniscus induced docking • Surface tension driven capillary flow inlet reservoir http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Receding m eniscus induced docking • 10 µm width, 1 µm depth microwells http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum High-throughput single-cell analysis 8 µm diameter, 8 µm depth microwells well density: 3,906 wells/mm 2 about 400,000 wells/cm 2 → similar to Affymetrix GeneChip http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum High-throughput single-cell analysis Large area patterning of yeast cells at single-cell resolution Efficiency is more than 90% Diameter : 8 ㎛ , Depth : 8 ㎛ http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Quantitative single- -cell analysis cell analysis Quantitative single Experiment Flow Single-cell docking Automated microscope Image acquisition intensity time Data analysis and modeling Data handling Image analysis http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Quantitative single- -cell analysis cell analysis Quantitative single Yeast strain construction http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Quantitative single- -cell analysis cell analysis Quantitative single Yeast strain construction DNA mRNA signal protein transcription transmission translation SH129 (P Fus1 -EGFP, P Gpd1 -Tdimer2) alpha factor Fus1 promoter activation EGFP mRNA synthesis EGFP signal transmission transcription translation KCl Gpd1 promoter activation Tdimer2 mRNA synthesis Tdimer2 signal transmission transcription translation http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Quantitative single- -cell analysis cell analysis Quantitative single Automated time-lapse fluorescent imaging Live cell imaging EGFP (green) Tdimer2 (red) Merged http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum High-throughput single-cell analysis • Automated microscope Time-lapse image at a point (120 min) http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum High-throughput single-cell analysis Mating signal response (alpha factor) GFP is a reporter of mating signal response (120 min) http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum High-throughput single-cell analysis Salt-stress signal response (KCl) RFP is a reporter of salt-stress signal response (120 min) http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Quantitative single- -cell analysis cell analysis Quantitative single Image analysis – ImagePro TM (Media Cybernetics, Inc.) http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Quantitative single- -cell analysis cell analysis Quantitative single Image analysis (SH129) P Fus1 -EGFP P Fus1 -EGFP P Fus1 -EGFP P Fus1 -EGFP 10 nM α -factor 10 pM α -factor 100 pM α -factor 1 nM α -factor P Fus1 -EGFP P Fus1 -EGFP P Fus1 -EGFP P Fus1 -EGFP 10 uM α -factor 100 uM α -factor 100 nM α -factor 1 uM α -factor http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Stochastic dynamics Stochastic dynamics 25000 cell 1 dynamics 20000 cell 2 dynamics cell 3 dynamics 15000 cell 4 dynamics cell 5 dynamics 10000 ….. 5000 “stochastic dynamics” (pattern analysis) 0 15 30 45 60 75 90 105 120 135 “noise” ≈ Std. dev. / Avg. (point analysis) intensity intensity intensity intensity time time time time http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Stochastic dynamics Stochastic dynamics Single-cell recovery (SH129) weak strong capillary tube http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Stochastic dynamics Stochastic dynamics Single-cell recovery (SH129) weak strong - Same stochastic behavior was observed - Survival strategy of cells: flexible response to sharp changes in environments http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Summar y � A receding meniscus induced cell docking scheme was developed. � Cell response at single cell level was analyzed with statistical significance � Stochastic cell response was observed at different conc. of signaling molecules � Stochastic cell response is intrinsically programmed to survive various changes in cell environments. http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Acknowledgements � Micro Thermal System Research Center of Seoul National University � Minuta Technology � Ministry of Science and Technology through the Bio Tool R&D Project for Cell Research http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.

The 5 th US-Korea Nano Forum Thank you for your attention! Thank you for your attention! http://nftl.snu.ac.kr Cell on a Chip Nano Fusion Technology Lab.