advancements in spm instrumentation for bio-applications · pdf filespm microscopy in biology...
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Advancements in SPM instrumentation for bio-applications
Sang-il Park
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Outline
• Introduction• Non Contact AFM• SICM• Conclusions
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AFM
cantilever
Lase
r
PSPD
mirror
x y -x
Z
sample
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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
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SPM
Microscopy in Biology
ScaleSmall (10m)-10
opticalmicroscopy
Large (10m)-1
in vivo
isolatedstructures
purifiedmolecules
in vitro livein vitro dead
atom molecule structure cell tissue organ
Imag
ing
dest
ruct
i ve
non-
inva
s ive
Transmission EMScanning EM
visual,x-ray,
ultrasound...
x-raychrystalography,
cryoelectrontomography
Atomic Force Microscopy
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Common Problems in Conventional AFM
Piezo tube is not an orthogonal 3-D actuatorNon-Contact Mode not possible due to Slow z-servo response
Even after software flattening, flat surface does not “look” flat.
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AFM Technology Innovation
Single module parallel-kinematics x-y scanner
stacked piezoz-
scan
ner
x-y flexure scanner
sample
cantilever
Independent z scanner from x-y scannerPrecision NanometrologyTrue Non-Contact AFM
→
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Tapping vs. True Non-Contact Mode
Destructive Contact
between tip and sample surface
→ Tip Wear and Sample Damage!
Constant Tip-sample Distance
by non-contact
→ Ultimate Resolution of AFM!
Tapping Mode True Non-Contact Mode
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SPM
Microscopy in Biology
ScaleSmall (10m)-10
opticalmicroscopy
Large (10m)-1
in vivo
isolatedstructures
purifiedmolecules
in vitro livein vitro dead
atom molecule structure cell tissue organ
Imag
ing
dest
ruct
i ve
non-
inva
s ive
Transmission EMScanning EM
visual,x-ray,
ultrasound...
x-raychrystalography,
cryoelectrontomography
Tapping mode AFM
Non-Contact mode AFM
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DNA
3 × 3µm 1 × 1µmDried, NC-AFM
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Plant Virus
1 × 1µm 1 × 1µmDried, NC-AFM
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Topography Phase image
Spontaneous assembly of Viruses on multilayered polymer surfaces
2 × 2µm 2 × 2µmDried, NC-AFM
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Bacteria as Chemical Factories
- vitamins- therapeutic agents- pigments- amino acids- viscosifiers- industrial enzymes- PHAs (biodegradable plastics)
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PHAs (Polyhydroxyalkanoates)
In-Liquid, NC-AFMSample provided by Kumar Sudesh
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Complex Structure of Cell Membrane
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Imaging the Inside of Cell Membrane
ultra-sonication Inside the cell membrane
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TEM image of Hela Cell Inside (8μm)
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AFM image of Hela Cell Inside
In-Liquid, NC-AFM
Sample provided by Jiro Usukura
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Microtubule Actin Clathrin Coated Vesicle
In-Liquid, NC-AFM
AFM image of Hela Cell Inside
Sample provided by Jiro Usukura
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In-Liquid, NC-AFM
Clathrin Coated Vesicle
Sample provided by Jiro Usukura
Model
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Confocal microscopy(Bar: 50 μm)
AFM image
Imaging the Muscle Fibers
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AFM imageTEM AFM
In-Liquid, NC-AFM
Imaging the Muscle Fibers
By Noemi Rozlosnik
(5 × 5µm)
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F-d curves on Muscle Fibers
By Noemi Rozlosnik
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Fat cells in the background
Collagen Fibers from the Connective Tissue
By Noemi Rozlosnik
In-Liquid, NC-AFM
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NSOM/SICM
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Exchangeable SPM Heads for Bio Imaging
25µm AFM HEAD Optical HEAD for NSOM & Raman
SICM HEAD
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100 X 100um 50 X 50um
NSOM: Kidney Cell (293 T)
AFM Topography
NSOM image
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Scanning Ion Conductance Microscopy
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Control System
Current Amp.
ZScanner
Nano Pipet
Ag/Agclelectrode
Live Cells
Scanning Ion Conductance Microscopy
X-Y Scanner
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DC Control
Scanning Ion Conductance Microscopy
Hansma(1989)
Distance-modulated Control
Shao, Korchev(2001)
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SICM of Live Cell: C2C12(mouse muscle)
Topography Current
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Feedback OnFeedback On
Feedback On
Feedback Off
d=0
PositivePressure
NegativePressure
NoPressure
Mechanical Stimulation with a Nanopipet
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“Smart” Patch-clamp
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Multi-Component Graded Deposition of Biomolecules with a Multi-Barreled Nanopipet
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SPM
Microscopy in Biology
ScaleSmall (10m)-10
opticalmicroscopy
Large (10m)-1
in vivo
isolatedstructures
purifiedmolecules
in vitro livein vitro dead
atom molecule structure cell tissue organ
Imag
ing
dest
ruct
i ve
non-
inva
s ive
Transmission EMScanning EM
visual,x-ray,
ultrasound...
x-raychrystalography,
cryoelectrontomography
Tapping mode AFM
Non-Contact mode AFM
SICM
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Biological Applications of SPM• 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
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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.