5 th Korea-US NanoForum, Jeju, Korea, April 17-18, 2008 Label-free NanoBio Chemical Imaging of Cells and Tissues for New Bio-medical Applications DaeWon Moon Nano-Bio Fusion Research Center Korea Research Institute of Standards and Science (KRISS) Collaborators: J.Y. Lee, E.S. Lee, T.G. Lee, H.K. Sohn, E.S. Lee, J.E. Gil, W. JeGal, S.H. Kim, (KRISS) J.H. Chung (SNU), J.E. Park (Samsung Medical ), Ann Plant (NIST) Funding: MOST, MOCIE, KRISS, Outline: Our strategy of nano-bio fusion Present status of nanobio imaging methodology at KRISS A case report on Atherosclerosis with cardiovascular lipid, cell adhesion, and collagen ECM imaging Visions in the near future 1/25
How to utilize NT to solve Biomedical Issues through noble methodologies Beyond CMOS Future source source 15 years Non-classical CMOS Gate Gate 1948 drain drain First Transistor Tomorrow Molecular Switches ? Nanowire Transistor ? Today 90 nm Node CMOS pMOS FINFET Nano-Bio Fusion New Materials Strain Enhanced Mobility Solving Bio Issues with NT High throughput STM/AFM, TEM/SEM, XRD, Noble analysis & manipulation PES/AES, SIMS, RBS/MEIS, Raman, ALD, QD, FIB, … .. 2/25
Analysis Demands from Bio- -Medical R&D Medical R&D Analysis Demands from Bio : in in- -vivo/in vivo/in- -vitro, biochemical imaging, dynamics vitro, biochemical imaging, dynamics : sensitivity & selectivity, general methodology sensitivity & selectivity, general methodology Label-free single cells/tissue biochemical imaging for medical & pharmaceutical applications Humanomics Tool Box Systems biology DNA Bioinformatics Phenomics Genomics Physiomics Bottom-Up Top-Down Organ Cell RNA Protein Organomics Cellomics Proteomics nm μm mm m Large Gap between Molecular Biology and Medical Applications 3/25
Label-free Single Cells/Tissue Chemical Imaging R&D at KRISS Polarized Microscopy: Non-linear Optics: SPR imaging CARS microscopy - Cell membrane interface - 3D dynamic biochemical imaging Single Cells Tissue Biochemical Biochemical Imaging Imaging Bio-molecular mass imaging Electrochemical AFM: Scanning ion conductance SIMS/MALDI imaging microscope (SICM) - ex-situ, molecular information - Ion channel monitoring 4/25
CARS (Coherent Anti-Stokes Raman Scattering Energy diagram of Raman Fingerprints of Biological Cells CARS Virtual Levels Virtual Levels ω P ω AS ω L ω S Vibration Label-free biochemical imaging Levels - no biological disturbance Ground Levels high sensitivity (x> 10 4 Raman) high spatial resolution ( 300 nm) 3D dynamic imaging - in-vivo/in-vitro environment 5/25
CARS Microscope at KRISS CARS Excitation Source Stokes Laser 1.5 W @ 1064 nm fixed Pump/Probe Laser 2 W @ 725 – 960 nm Rep. Rate 76 MHz Pulse Width 7 ps Bandwidth 0.38 nm / 6 – 7 cm -1 Raman shift 1500 – 3500 cm -1 coverage ~ 100 mW in total Sample Irradiation Image Acquisition 250 x 250 µ m 2 Imaging Area Pixels 1024 x 1024 1064 nm Modelocked ps laser Frame Rate 10 image/s 750 – 960 nm NIR synchronously pumped ps OPO 500 µ m Z- section Range Laser beam/pulse diagnostics and overlap control 0.1 µ m Z- section Step Dichroic beam coupling and signal decoupling Spatial Resolution Lateral ~ 300 nm Non-descan CARS signal detection Optics Axial ~ 900 nm Relay optics and optimal microscope objective + Multiplex Raman capability : 200 cm -1 ~ 1500 Galvano-mirror laser scan inverted optical microscope 6/25 cm -1
Real Time CARS images of an alive Hela Cell Aliphatic C-H @ ∆ = 2837 cm -1 Dynamic Imaging of Vesicles 7/25
Depth-Resolved Images of an unstained HeLa Cell Bottom Middle Middle Area : 35 x 35 µ m 2 Step : 4 µ m Pixel : 512 x 512 @ 1s 8/25 Top
CARS 바이오 현미경의 현재 @KRISS Single Cells Atherosclerosis Tissues Focal Adhesion Fat Liver Tissue & Migration Skin Stem Cell Stratum Corneum Differentiation µ -CARS Potential Sebaceous Gland HCV-LD Collocalization CARS + AF Hyaloid Vessel Retinal Tissue Live Cell (NIH3T3) 9/25
From Cellular basic studies to Medical interests in Atherosclerosis lipid uptake by imaging plaques and cell-cell, cell-ECM macrophages its stabilization adhesion & migration & its differentiation (CARS & SIMS) (SPR, SIMS, SICM) to foam cells (CARS) US, CT, MRI, PET 10/25
CARS images for lipid vesicle uptake processes in the differentiation of human monocytes (THP-1) to macrophages PMA in 10% serum media duration: 2 hours 11/25
CARS spectra for biochemical characterization of lipids from a mouse atheroma tissue 2wk1_carotid 내 droplet 2wk1_media 1 1 0.8 0.8 CARS intensity CARS intensity 0.6 0.6 0.4 0.4 0.2 0.2 0 0 2600 2700 2800 2900 3000 3100 2600 2700 2800 2900 3000 3100 -1 ) -1 ) Raman Shift(cm Raman Shift(cm 2wk1_lipidrich_deep 2wk_carotid artery 1 1 0.8 0.8 CARS intensity CARS intensity 0.6 0.6 0.4 0.4 0.2 0.2 0 0 2600 2700 2800 2900 3000 3100 2600 2700 2800 2900 3000 3100 -1 ) -1 ) Raman Shift(cm Raman Shift(cm 12/25 - Collaboration with Samsung Medical Center
ex vivo Atherosclerosis Cardiovascular CARS Imaging 3D Reconstruction of 3D Reconstruction of 3D Reconstruction of en face CARS Images en face CARS Images en face CARS Images Cut-Away Side View View Cardiovascular Imaging • in vivo US/SPECT/PET/NIR : - Agents required - Low resolution • ex vivo Biopsy of atheroma tissue : - Cryosection - Foam cell staining with oil red-O dye - Collaboration with Samsung Medical Center Foam cell differentiation/ Atherosclerosis Diagnosis 13/25
Atherosclerosis tissue analysis with multiplex CARS degree of oxidation/saturation of lipids for plaque stabilization analysis ? Multiplex CARS Spectra Necrotic Core Necrotic Core Necrotic Core Fatty Streak Fatty Streak Fatty Streak CARS Image of Atheroma Atheroma Tissue Tissue CARS Image of CARS Image of Atheroma Tissue in the Media Region in the Media Region 5.0 in the Media Region 1 4.5 4.0 Media Media 3.5 CARS Intensity 1 3.0 2 2.5 2 Necrotic Necrotic 2.0 Core Core Large Droplet Large Droplet Pin- Pin -shaped Lump shaped Lump Large Droplet Pin-shaped Lump in the Adventitia in the Adventitia (Unidentified) (Unidentified) 1.5 in the Adventitia (Unidentified) 3 1.0 0.5 4 Lumen Lumen 0.0 3 2700 2800 2900 3000 4 -1 ) Raman shift (cm 14/25
Vision of CARS Laser Microscopy in-vivo Medical and/or Animal model Imaging Endoscopy Biomedicla Imaging & Diagnostics Squeezing CARS Squeezing CARS Microscope Microscope into Optical Fibers into Optical Fibers Animal Model Imaging for Pre-clinicall Screening 15/25
Complementary Use of CARS and SIMS/MALDI imaging Mass Spectrometry (laser/ion beam) CARS : molecular specificity : overview of biochemical imaging : high sensitivity (?) : in-vitro/in-vivo dynamics : high contents biochemical information : poor sensitivity and selectivity : ex-situ, no dynamics Multiplex CARS Lipid structure change C-C skeletal mode @ (~1100 cm -1 ) Mueller et al. JPC B (2002). Chemical Mapping of Tissue Anal. Chem. [Feature Article] (2004) 16/25
Secondary Ion Mass Spectrometry (SIMS) : unique for semiconductor dopant analysis Can SIMS be useful for biochemical imaging of tissues ? Can it beat traditional staining optical microscopy & bio-SEM/TEM ? ION-TOF V at KRISS TOF-SIMS image 17/25
SIMS studies on Photoaging Effects of Skin by UV irradiation + imaging after C 60 ++ cleaning: 25 keV Bi 3 Amino Acid Amino Acid Lipid Lipid (a) (b) C 3 H 9 N C 3 H 9 N C 5 H 15 NO 4 P C 5 H 15 NO 4 P C 4 H 8 NO C 4 H 8 NO Total ion image Total ion image Total ion image Total ion image C 5 H 14 NO C 5 H 14 NO (Trimethyl - (Trimethyl - (Phosphocholine ) (Phosphocholine ) C 3 H 7 43.05 C 3 H 7 43.05 CH 4 N(Gly) 30.03 CH 4 N(Gly) 30.03 C 4 H 6 N(Pro) 68.05 C 4 H 6 N(Pro) 68.05 C 4 H 8 N(Pro) 70.07 C 4 H 8 N(Pro) 70.07 (OH-Pro) (OH-Pro) (Choline ) 104.12 (Choline ) 104.12 ammonium) 60.08 ammonium) 60.08 184.07 184.07 86.06 86.06 Control Control Control Control UV 24h UV 24h UV 24h UV 24h UV 48h UV 48h UV 48h UV 48h UV 72h UV 72h UV 72h UV 72h (collaborations with SNU Medical School, Dermatology, J.H. Chung) Is he happy ? Maybe, No for proteins, Yes for lipids. Good for CV imaging Is he excited ? No. Why ??? >> insufficient molecular ions Complementary use of SIMS & MALDI imaging of tissues with matrix controls 18/25
Surface Plasmon Resonance for cell adhesion & migration imaging SPR applications SPR applications SPR applications quantitative analysis of quantitative analysis of biomolecules biomolecules on surface on surface - biomolecule - biomolecule adsorption dynamics adsorption dynamics - antibody antibody- -antigen, DNA antigen, DNA- -DNA interactions DNA interactions - 50 ㎛ 50 ㎛ A10 SMC on collagen HUVEC on fibronectin 19/25
The Effect of Flow Rate to A10 SMC Adhesion on Collagen 170x 250 μ m 2 flow rate: 1 cm/s flow rate: 27 cm/s flow rate: 1 cm/s 1 hour 5 hours 6 hours 20/25
SPR dynamic imaging of HUVEC adhesion on fibronectin & the Shear Stress Effect no shear stress 0hr 6hr 12hr 18hr 21.5 hr 1.2 Pa shear stress SS on SS 1hr SS 2hr SS 3hr SS 4hr dynamics movies 21/25
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