AFM time-lapse imaging Yuri Lyubchenko University of Nebraska Medical Center

Time-lapse imaging of molecular dynamics Lyubchenko and Shlyakhtenko (1997) PNAS

Scanning speed 1000 times faster than regular AFM - video rate has been achieved The instrument operates in liquid Gentle touch of the sample - oscillation amplitude ~ 1nm High speed AFM design of Toshio Ando, Kanazawa University, Japan High speed AFM design of Toshio Ando, Kanazawa University, Japan Schematic drawing of the HS-AFM head integrated with an inverted type of optical microscope. Z-piezo scanner (1), sample stage (2), cantilever (3), counter weight (4), x- piezo scanner (5), illuminator (6), stepper motor (7), piezo for exciting cantilever (8), objective lens (9), dichroic mirror (10), quarterwave plate (11), polarization beam splitter (12), laser diode (13), focusing lens (14), split photodiode (15), and CCD camera (16).

Dynamics of SSB-DNA complexes: video - one frame/720 ms. Dissociation- association events of SSB are observed with the use of APS-mica. Single stranded DNA binding proteins Shlyakhtenko et al (2012a) Biochemistry

The SSB hops between the targets. High speed AFM of SSB-DNA complexes Frame 115Frame 120Frame 135 Frame 146 Frame 160 Frame 164Frame 165 Frame 113 Shlyakhtenko et al (2012a) Biochemistry The SSB dissociation.

(A) Individual frames are shown every 2.5 s. (B) the change in DNA length over a time period of 20s measured in 0.5s intervals. The translocation of 300 bp DNA segment occurs within 15 s. (A) Individual frames are shown every 2.5 s. (B) the change in DNA length over a time period of 20s measured in 0.5s intervals. The translocation of 300 bp DNA segment occurs within 15 s. EcoRII translocation: video - one frame/500 ms. Visualization of protein mediated DNA translocation Novel finding: DNA translocates without rotation Gilmore et al (2009) Biochemistry

Direct visualization of the DNA cleavage The enzyme cleaves DNA concertedly at the APS-mica surface/liquid interface. DNA cleavage with SfiI enzyme: video - one frame/500 ms. SfiI binding The complex was preassembled in the presence of Ca cations and they were replaced with Mg cations to start the cleavage Suzuki et al (2011) Biophysical J.

Dynamics of Mononucleosomes Lyubchenko & Shlyakhtenko (2009) Methods Shlyakhtenko et al (2009)

Dynamics of nucleosomes: High speed AFM Miyagi et al (2011) Biochemistry

Reversible sliding of nucleosome. Scan rate 1 fps Miyagi et al (2011) Biochemistry