Abstract
We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.
Highlights
We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation
The first challenge for force spectroscopy studies is the low throughput of most single-molecule approaches, which is just starting to be addressed with recently developed multiplexed methods[6,7,8,9,10,11,12,13]
We have met all of these challenges with spatiotemporally multiplexed force spectroscopy, a combination of massively parallel spatial multiplexing with repeated interrogation enabled by self-assembled nanoscale devices
Summary
We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. The ability to mechanically manipulate single molecules is leading to insights throughout biomedical research, from the action of molecular motors in replication and transcription to the role of mechanical forces in development[1,2,3,4,5] While in principle these approaches enable the full characterization of individual molecular complexes and the study of population heterogeneity at the single-molecule level, in practice key challenges exist. Similar to our first prototype device[18], an entire microscope imaging system is rotated to observe microscopic objects subjected to uniform centrifugal force (unlike earlier ‘spinning disk’ centrifuge microscopes[19,20]) This inexpensive design brings new features including temperature control and high-resolution particle tracking (B2 nm). The rich and relatively large data sets provided by our technique could complement other analysis techniques for statistical deconvolution[23,24]
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