Biology Reference
In-Depth Information
CHAPTER
6
Determining the Structure-
Mechanics Relationships
of Dense Microtubule
Networks with Confocal
Microscopy and Magnetic
Tweezers-Based
Microrheology
Yali Yang * and Megan T. Valentine * ,{
* Department of Mechanical Engineering, University of California, Santa Barbara, California, USA
{ The Neuroscience Research Institute, University of California, Santa Barbara, California, USA
CHAPTER OUTLINE
Introduction .............................................................................................................. 76
6.1 Protocols........................................................................................................... 78
6.1.1 Preparation of Samples ..................................................................... 78
6.1.1.1 Preparation of Tubulin Proteins ...................................................78
6.1.1.2 Preparation of MT Networks Embedded with Magnetic Beads......79
6.1.2 Structure Determination.................................................................... 80
6.1.2.1 Steady-state Network Morphology Determined by Confocal
Microscopy .............................................................................................80
6.1.2.2 Dynamic Network Formation Determined by Confocal
microscopy .............................................................................................81
6.1.2.3 Analysis of Network Mesh Size....................................................81
6.1.2.4 Analysis of Average Filament Length ...........................................82
6.1.3 Magnetic Tweezers Devices for Microscale Manipulation ...................... 83
6.1.3.1 Principles of Magnetic Tweezers Technologies ............................83
6.1.3.2 Calibrations ................................................................................85
6.1.3.3 Particle Tracking.........................................................................86
6.1.3.4 Fast Force Switching...................................................................87
6.1.3.5 High-force Implementation..........................................................88
6.1.3.6 Ring-shaped Magnets Enable Oscillatory Measurements..............89
6.1.3.7 Portable Magnetic Tweezers Enables Simultaneous Imaging
and Manipulation ....................................................................................90
 
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