Advancements in S PM instrumentation for bio-applications S - - PowerPoint PPT Presentation

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Advancements in S PM instrumentation for bio-applications S - - PowerPoint PPT Presentation

Sep 09, 2022 206 likes •594 views

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

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SLIDE 1

Advancements in S PM instrumentation for bio-applications

S ang-il Park

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SLIDE 2

Outline

  • Introduction
  • Non Contact AFM
  • SICM
  • Conclusions
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SLIDE 3

AFM

cantilever Laser PSPD mirror x y

  • x

Z

sample

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SLIDE 4

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
  • ther physical properties
  • Manipulation of specimen in nanometer scale
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SLIDE 5

SPM

Microscopy in Biology

Scale

Small (10m)

  • 10
  • ptical

microscopy Large (10m)

  • 1

in vivo

isolated structures

purified molecules

in vitro live in vitro dead atom molecule structure cell tissue organ

Imaging

destructive

non-invasive Transmission EM Scanning EM

visual, x-ray, ultrasound ...

x-ray chrystalography, cryoelectron tomography

Atomic Force Microscopy

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SLIDE 6

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.

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SLIDE 7

AFM Technology Innovation

Single module parallel- kinematics x-y scanner stacked piezo

z-scanner

x-y flexure scanner

sample cantilever

Independent z scanner from

x-y scanner

Precision Nanometrology True Non-Contact AFM

→

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SLIDE 8

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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SLIDE 9

SPM

Microscopy in Biology

Scale

Small (10m)

  • 10
  • ptical

microscopy Large (10m)

  • 1

in vivo

isolated structures

purified molecules

in vitro live in vitro dead atom molecule structure cell tissue organ

Imaging

destructive

non-invasive Transmission EM Scanning EM

visual, x-ray, ultrasound ...

x-ray chrystalography, cryoelectron tomography

Tapping mode AFM Non-Contact mode AFM

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SLIDE 10

DNA

3 × 3µm 1 × 1µm Dried, NC-AFM

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SLIDE 11

Plant Virus

1 × 1µm 1 × 1µm Dried, NC-AFM

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SLIDE 12

Topography Phase image

S pontaneous assembly of Viruses on multilayered polymer surfaces

2 × 2µm 2 × 2µm Dried, NC-AFM

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SLIDE 13

Bacteria as Chemical Factories

  • vitamins
  • therapeutic agents
  • pigments
  • amino acids
  • viscosifiers
  • industrial enzymes
  • PHAs (biodegradable plastics)
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SLIDE 14

PHAs (Polyhydroxyalkanoates)

In-Liquid, NC-AFM Sample provided by Kumar Sudesh

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SLIDE 15

Complex S tructure of Cell Membrane

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SLIDE 16

Imaging the Inside of Cell Membrane

ultra-sonication Inside the cell membrane

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SLIDE 17

TEM image of Hela Cell Inside (8μm)

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SLIDE 18

AFM image of Hela Cell Inside

In-Liquid, NC-AFM Sample provided by Jiro Usukura

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SLIDE 19

Microtubule Actin Clathrin Coated Vesicle In-Liquid, NC-AFM

AFM image of Hela Cell Inside

Sample provided by Jiro Usukura

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SLIDE 20

In-Liquid, NC-AFM

Clathrin Coated Vesicle

Sample provided by Jiro Usukura Model

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SLIDE 21

Confocal microscopy

(Bar: 50 μm)

AFM image

Imaging the Muscle Fibers

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SLIDE 22

AFM image

TEM AFM

In-Liquid, NC-AFM

Imaging the Muscle Fibers

By Noemi Rozlosnik (5 × 5µm)

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SLIDE 23

F-d curves on Muscle Fibers

By Noemi Rozlosnik

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SLIDE 24

Fat cells in the background

Collagen Fibers from the Connective Tissue

By Noemi Rozlosnik In-Liquid, NC-AFM

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SLIDE 25

NS OM/ S ICM

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SLIDE 26

Exchangeable S PM Heads for Bio Imaging

25µm AFM HEAD Optical HEAD for NSOM & Raman SICM HEAD

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SLIDE 27

100 X 100um 50 X 50um

NS OM: Kidney Cell (293 T)

AFM Topography NSOM image

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SLIDE 28

S canning Ion Conductance Microscopy

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SLIDE 29

Control System Current Amp. Z Scanner Nano Pipet Ag/Agcl electrode Live Cells

S canning Ion Conductance Microscopy

X-Y Scanner

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SLIDE 30

DC Control

S canning Ion Conductance Microscopy

Hansma (1989)

Distance-modulated Control

Shao, Korchev (2001)

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SLIDE 31

S ICM of Live Cell: C2C12(mouse muscle)

Topography Current

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SLIDE 32

Feedback On Feedback On Feedback On Feedback Off

d=0

Positive Pressure Negative Pressure No Pressure

Mechanical S timulation with a Nanopipet

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SLIDE 33

“S

mart” Patch-clamp

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SLIDE 34

Multi-Component Graded Deposition of Biomolecules with a Multi-Barreled Nanopipet

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SLIDE 35

SPM

Microscopy in Biology

Scale

Small (10m)

  • 10
  • ptical

microscopy Large (10m)

  • 1

in vivo

isolated structures

purified molecules

in vitro live in vitro dead atom molecule structure cell tissue organ

Imaging

destructive

non-invasive Transmission EM Scanning EM

visual, x-ray, ultrasound ...

x-ray chrystalography, cryoelectron tomography

Tapping mode AFM Non-Contact mode AFM

SICM

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SLIDE 36

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

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SLIDE 37

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.