Atomic Force Microscope

Gerrit L. Heuvelman

Kirchhoff-Institut fr Physik Molekulare Biophysik (F15)

Scanning Probe Microscopy

STM: scanning tunneling microscope

tunneling of electrons between probe and surface y x

AFM: atomic force microscope

measuring of the force on the probe

MFM: magnetic force microscope

AFM with magnetical probe

SNOM: scanning near-field optical probe is a fiber; microscope tunneling of fotonen

STM: scanning tunneling microscope

piezoelement

tunneling of electrons through air between probe and surface

ee eeeeee-

nA
probe

e-

< 1nm

x-y stage

only conducting material

STM: scanning tunneling microscope

Itip

piezo-element (changes length at different voltages)

nA
R

Icontrol

I I -> V transfer

AFM: atomic force microscope SFM: scanning force microscope

photodiode laser

AFM probe scans over the surface (in contact)

probe

QuickTime and a GIF decompressor are need ed to see this picture.

piezo-element

e.g. living cells, chromatin fibers

MFM: magnetic force microscope

laser

photodiode

AFM with magnetic probe

magnetic tip

piezo-element

e.g. hard disc, tape

SNOM: scanning near-field optical microscope

metal-coated fiber tip

fiber

tunneling of photons between probe and surface

sample

lens
filter detector

shows the amount of light that is absorbed/transmitted for different colors

e.g. fluorescent molecules

Atomic Force Microscope

photodiode laser

cantilever piezo x z y cantilever tip

Atomic Force Microscope

mirror laser beam photodiode SPM tip fluid in tipholder sample piezo translator motor control O-ring fluid out

fluid cell

x,y,z piezo translator

sample

in air and in buffer solutions

movement of the AFM tip along the sample

AFM tip

superhelical DNA plasmid path of AFM tip DNA double helix

Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+

negatively charged mica surface

AFM image of a 6.8 kb superhelical plasmid

AFM tip

AFM image of a nucleosome on a 614 base pair DNA

2 m x 2 m overview scan

zoom of mononucleosome complexes

Kepert, F., Fejes Tth, K., Caudron, M., Mcke, N., Langowski, J. & Rippe, K., manuscript in preparation

AFM image of a nucleosome on a 614 base pair DNA

2 m x 2 m overview scan

zoom of mononucleosome complexes

Kepert, F., Fejes Tth, K., Caudron, M., Mcke, N., Langowski, J. & Rippe, K., manuscript in preparation

DNA entry-exit angle of nucleosomes

60 160 140 120 100 80 60 40 20 0 60 60 50 50 40 30 20 10 0 40 30 20 10 0 10 0 70 30 20 50 40

DNA entry-exit angle

DNA entry-exit angle

movement of a 600 bp DNA fragment by AFM

different levels of chromatin packing

A B

22 nm nm

D E

300 nm 300 nm

11 nm 11 nm

700 nm

700 nm

30 nm 30 nm

F 1400nm 1400 nm

chromatin structure
400 nm x 400 nm

chromatin structure
400 nm x 400 nm

chromatin structure

spectrin
molecule that contributes to the mechanical properties, especially the elasticity of the cells

C-terminal

Helix C Helix A Helix B

measurement of its mechanical stability provides information about the physiological function

N-terminal

stretching spectrin with an AFM

4 repeated spectrin domain cantilever tip surface

1. adhesion force between cantilever tip and surface

2. dissociation from the folded state to the intermediate unfolded state 3. dissociation from the intermediate to the total unfolding state

force

2 3

distance

stretching spectrin with an AFM

100 80 60 40 20 0 -20 0 20 40 60 80

force (pN)

0.20 0.15

probability

distance (nm)

0.10 0.05 0.00

10

20

30

40

50

unfolding force (pN)