Abstract
Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery.
Highlights
Single-molecule measurement techniques have advanced tremendously over the past two decades; such techniques include total internal reflection fluorescence microscopy [1,2], optical trapping forcemetry [3,4], and other techniques simultaneously achieving singlemolecule sensitivity with nanometer and piconewton accuracy [5]
We developed a simple and versatile microscope system for simultaneous observation of multiple fluorescent probes with high spatial precision and high temporal resolution, and we verified the performance of the microscope by tracking multiple myosin molecules
We introduced spectrally resolved tracking microscopy, SRTM, which is a simple and versatile technique for simultaneous tracking of multiple motor proteins even if they are adjacent at the sub-diffraction-limit scale
Summary
Single-molecule measurement techniques have advanced tremendously over the past two decades; such techniques include total internal reflection fluorescence microscopy [1,2], optical trapping forcemetry [3,4], and other techniques simultaneously achieving singlemolecule sensitivity with nanometer and piconewton accuracy [5]. Cutler et al reported slit-scanning confocal fluorescence microscopy with spectroscopic detection of various colors of semiconductor quantum dots (QDs) [20] They realized observation of membrane protein dynamics and colocalization with spatial precision of ~10 nm at the video rate. Since our spectral discrimination method can utilize the time domain for successive record of image frames, it is appropriate for dynamic measurement of multiple mobile molecules such as motor proteins and membrane proteins. This is the major advantage of our method over the temporal discrimination methods [23,24,25,26,27]. We introduce 2DSRTM and how 1D-SRTM can be extended to two-dimensional tracking measurements
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