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

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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

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SLIDE 1

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Receding meniscus induced docking of yeast Receding meniscus induced docking of yeast cells for quantitative single cells for quantitative single-

  • cell analysis

cell analysis

Min-Cheol Park,1 Jae-Young Hur,2 Keon-Woo Kwon,1 Sang-Hyun Park2 and Kahp-Yang Suh1*

1School of Mechanical and Aerospace Engineering,

Seoul National University, Seoul 151-742, Korea.

2School of Biological Sciences and Research Center for

Functional Cellulomics, Seoul National University, Seoul 151-742, Korea. *e-mail: sky4u@snu.ac.kr

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SLIDE 2

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Single Single-

  • cell analysis

cell analysis

Bulk population based analysis

population readout well plate heterogeneous response population readout

Ensemble averaging problem !

averaging

misconception from bulk population

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SLIDE 3

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

intensity distance heterogeneous intensity microwells

  • At single-cell level
  • Stochastic expression of cell
  • Observing Single-cell information

Why single Why single-

  • cell analysis?

cell analysis?

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SLIDE 4

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Cell chip (single Cell chip (single-

  • cell array)

cell array)

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
  • f single cells

High-throughput and high-content single-cell analysis

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SLIDE 5

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Single Single-

  • cell docking

cell docking

Dielectrophoresis

Voldman et.al., Anal. Chem. (2002) Bhatia et.al., Nat. Methods (2005)

Optical tweezers

Grier, Nature (review) (2003)

Microfluidic patch clamp

Luke Lee et.al., PNAS (2005)

Sedimentation

Folch et.al., Lab. Chip (2005)

Physical structure

Luke Lee et.al., Lab. Chip (2006)

and so on ..

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SLIDE 6

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Our approach

  • Receding meniscus induced cell docking (Lab Chip, 6, 988, 2006)

30 um wells 10 um wells

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SLIDE 7

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Microw ell Patterning

for capillary molding PDMS pouring PDMS replica PDMS pre-polymer + curing agent PDMS curing at 70℃, for 1h negative silicon master silicon master PDMS stamp PU A glass substrate PDMS stamp PDMS stamp glass substrate glass substrate

capillary molding

  • K. Y. Suh et al., Adv. Mater. 2001.
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SLIDE 8

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Patterned Microfluidic Channel

glass substrate PDMS substrate PDMS microfluidic mold PDMS microfluidic mold

Plasma treating

glass substrate PDMS substrate PDMS microfluidic mold PDMS microfluidic mold

hydrophilic surface

Ali K., K. Y. Suh et al., Anal. Chem. 2004.

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SLIDE 9

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Receding m eniscus induced docking

PDMS microfluidic mould patterned glass substrate inlet reservoir

  • utlet reservoir

microchannel

Introducing a yeast cell suspension by capillary filling Generating a receding meniscus by evaporation Capturing the cells by lateral capillary force Receding meniscus induced docking

  • Schematic diagram
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SLIDE 10

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Receding m eniscus induced docking

inlet reservoir

  • Surface tension driven capillary flow
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SLIDE 11

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Receding m eniscus induced docking

  • 10 µm width, 1 µm depth microwells
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SLIDE 12

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

High-throughput single-cell analysis

8 µm diameter, 8 µm depth microwells well density: 3,906 wells/mm2 about 400,000 wells/cm2 → similar to Affymetrix GeneChip

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SLIDE 13

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

High-throughput single-cell analysis

Large area patterning of yeast cells at single-cell resolution Diameter : 8 ㎛ , Depth : 8 ㎛ Efficiency is more than 90%

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SLIDE 14

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

time intensity

Single-cell docking Automated microscope Image acquisition Image analysis Data handling Data analysis and modeling

Quantitative single Quantitative single-

  • cell analysis

cell analysis

Experiment Flow

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SLIDE 15

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Quantitative single Quantitative single-

  • cell analysis

cell analysis

Yeast strain construction

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SLIDE 16

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Quantitative single Quantitative single-

  • cell analysis

cell analysis

Yeast strain construction

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

SH129 (PFus1-EGFP, PGpd1-Tdimer2)

DNA mRNA protein

transcription translation

signal

transmission

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SLIDE 17

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Quantitative single Quantitative single-

  • cell analysis

cell analysis

Automated time-lapse fluorescent imaging

EGFP (green) Tdimer2 (red) Merged Live cell imaging

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SLIDE 18

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Time-lapse image at a point (120 min)

High-throughput single-cell analysis

  • Automated microscope
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SLIDE 19

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

High-throughput single-cell analysis

Mating signal response (alpha factor)

GFP is a reporter of mating signal response (120 min)

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SLIDE 20

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

High-throughput single-cell analysis

Salt-stress signal response (KCl)

RFP is a reporter of salt-stress signal response (120 min)

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SLIDE 21

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Quantitative single Quantitative single-

  • cell analysis

cell analysis

Image analysis – ImageProTM (Media Cybernetics, Inc.)

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SLIDE 22

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Quantitative single Quantitative single-

  • cell analysis

cell analysis

Image analysis (SH129)

PFus1-EGFP 10 pM α-factor PFus1-EGFP 100 pM α-factor PFus1-EGFP 1 nM α-factor PFus1-EGFP 10 nM α-factor PFus1-EGFP 100 nM α-factor PFus1-EGFP 1 uM α-factor PFus1-EGFP 10 uM α-factor PFus1-EGFP 100 uM α-factor

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SLIDE 23

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Stochastic dynamics Stochastic dynamics

5000 10000 15000 20000 25000 15 30 45 60 75 90 105 120 135

“noise” ≈ Std. dev. / Avg. (point analysis)

cell 1 dynamics cell 2 dynamics cell 3 dynamics cell 4 dynamics cell 5 dynamics …..

“stochastic dynamics” (pattern analysis)

intensity time intensity time intensity time intensity time

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SLIDE 24

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Stochastic dynamics Stochastic dynamics

Single-cell recovery (SH129)

weak strong capillary tube

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SLIDE 25

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

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
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SLIDE 26

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

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.

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SLIDE 27

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

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

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SLIDE 28

The 5 th US-Korea Nano Forum

Cell on a Chip

http://nftl.snu.ac.kr Nano Fusion Technology Lab.

Thank you for your attention! Thank you for your attention!