Advancements in S PM instrumentation for bio-applications S ang-il Park
• Non Contact AFM Outline • Conclusions • Introduction • SICM
cantilever PSPD -x AFM Laser y Z x mirror sample
Advantages of AFM • High Resolution: ~nm lateral, <nm vertical • Quantitative 3-D information • Operates in air, liquid, and vacuum – Ability to study in physiological buffer • Does not depend on electrical conductivity – No requirement for Au/Pd or C- sputter coating • Can measure mechanical, electrical, optical, and other physical properties • Manipulation of specimen in nanometer scale
Microscopy in Biology -1 -10 Scale Small (10m) Large (10m) non-invasive atom molecule structure cell tissue organ visual, x-ray, ultrasound in vivo ... optical microscopy Imaging in vitro live Atomic Force Microscopy SPM in vitro dead Scanning EM destructive Transmission EM isolated structures x-ray chrystalography, cryoelectron tomography purified molecules
Common Problems in Conventional AFM � Piezo tube is not an orthogonal 3-D actuator � Non-Contact Mode not possible due to Slow z-servo response Even after software flattening, flat surface does not “look” flat.
AFM Technology Innovation stacked piezo � Independent z scanner from x-y scanner z-scanner � Precision Nanometrology � True Non-Contact AFM cantilever sample → x-y flexure scanner Single module parallel- kinematics x-y scanner
Tapping vs. True Non-Contact Mode Tapping Mode True Non-Contact Mode Constant Tip-sample Distance Destructive Contact by non-contact between tip and sample surface → Ultimate Resolution of AFM! → Tip Wear and Sample Damage!
Microscopy in Biology -1 -10 Scale Small (10m) Large (10m) non-invasive atom molecule structure cell tissue organ visual, x-ray, ultrasound in vivo ... optical microscopy Imaging Non-Contact mode AFM in vitro live SPM in vitro dead Tapping mode AFM Scanning EM destructive Transmission EM isolated structures x-ray chrystalography, cryoelectron tomography purified molecules
1 × 1µm Dried, NC-AFM DNA 3 × 3µm
1 × 1µm Plant Virus Dried, NC-AFM 1 × 1µm
S pontaneous assembly of Viruses on multilayered polymer surfaces Topography Phase image 2 × 2µm 2 × 2µm Dried, NC-AFM
Bacteria as Chemical Factories - vitamins - therapeutic agents - pigments - amino acids - viscosifiers - industrial enzymes - PHAs (biodegradable plastics)
PHAs (Polyhydroxyalkanoates) In-Liquid, NC-AFM Sample provided by Kumar Sudesh
tructure of Cell Membrane Complex S
Inside the cell membrane Imaging the Inside of Cell Membrane ultra-sonication
TEM image of Hela Cell Inside (8 μ m)
AFM image of Hela Cell Inside In-Liquid, NC-AFM Sample provided by Jiro Usukura
AFM image of Hela Cell Inside Clathrin Coated Vesicle Microtubule Actin In-Liquid, NC-AFM Sample provided by Jiro Usukura
Clathrin Coated Vesicle Model In-Liquid, NC-AFM Sample provided by Jiro Usukura
Imaging the Muscle Fibers AFM image Confocal microscopy (Bar: 50 μ m)
Imaging the Muscle Fibers TEM AFM image In-Liquid, NC-AFM (5 × 5µm) AFM By Noemi Rozlosnik
F-d curves on Muscle Fibers By Noemi Rozlosnik
Collagen Fibers from the Connective Tissue In-Liquid, NC-AFM Fat cells in the background By Noemi Rozlosnik
ICM OM/ S NS
PM Heads for Bio Imaging SICM HEAD for NSOM & Raman Optical HEAD Exchangeable S 25µm AFM HEAD
NS OM: Kidney Cell (293 T) AFM Topography 50 X 50um 100 X 100um NSOM image
canning Ion Conductance Microscopy S
S canning Ion Conductance Microscopy Ag/Agcl electrode Current Amp. Nano Pipet Z Control Scanner System Live Cells X-Y Scanner
S canning Ion Conductance Microscopy DC Control Distance-modulated Control Shao, Korchev Hansma (2001) (1989)
ICM of Live Cell: C2C12(mouse muscle) Current Topography S
Mechanical S timulation with a Nanopipet Negative Positive Pressure Pressure No Pressure Feedback On Feedback On Feedback On Feedback Off d=0
“ S mart ” Patch-clamp
Multi-Component Graded Deposition of Biomolecules with a Multi-Barreled Nanopipet
Microscopy in Biology -1 -10 Scale Small (10m) Large (10m) non-invasive atom molecule structure cell tissue organ visual, x-ray, ultrasound in vivo SICM ... optical microscopy Imaging Non-Contact mode AFM in vitro live SPM in vitro dead Tapping mode AFM Scanning EM destructive Transmission EM isolated structures x-ray chrystalography, cryoelectron tomography purified molecules
Biological Applications of S PM • Biological Sample Imaging – Cell – Membrane & Membrane Protein – DNA • Molecular Interaction – Protein-protein interaction – DNA-protein interaction – Cell to cell interaction – Single-molecule force spectroscopy • Biological system dynamics – Cell dynamics – Vesicle dynamics – Phase transition of phospholipid membrane • Manipulation – Biomolecular nanolithography (protein, nucleotide) – Bio-Manipulator
Conclusions • SPM is a very powerful tool for nano-bio science and technology. • The new generation AFM with true Non-Contact mode was developed. • SICM is becoming the new driving force in the field of nano-bio science.
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