Tracking microtubules in live cells with high spatial resolution
Drs. Yicong Wu and Hari Shroff, National Institute of Biomedical Imaging and Bioengineering, and colleagues
Dr. Yicong Wu from the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, lead a team of researchers who developed a microscopy approach that can provide high spatial resolution in all dimensions, high speed imaging and minimal photobleaching and damage. They used this approach to demonstrate a spatial isotropic resolution of 330 nm and also combined the technique with Imaris to track microtubules in live cells.
To achieve high resolution, dual-view plane illumination microscopy switches illumination and detection between two perpendicular objectives in an alternating duty cycle. The resulting volumetric views are then computationally fused. This approach can achieve imaging speed 0.5 s for a 50-plane volume and long-term imaging of up to 14 hours with minimal photobleaching and photodamage.
This dual view inverted selective plane illumination microscopy image shows GFP-EB3–labeled microtubules in a human umbilical vein cell embedded in collagen gel. These example tracks indicate microtubule assembly over a 5-minute volumetric series, and the colors are associated with the speed at which the microtubules were moving. Image courtesy of Dr. Yicong Wu, National Institute of Biomedical Imaging and Bioengineering.
“The imaging ability that comes with dual-view plane illumination microscopy is very desirable for live biological samples that require high resolution, high speed volumetric visualization and/or low phototoxicity, including what we demonstrated - microtubule tracking in live cells, nuclear imaging over 14 hours during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 hours,” Dr. Wu says.
Imaging microtubules in vivo
Imaging in vivo microtubules in three dimensions is challenging because they are only 25 nanometers in diameter and can assemble and disassemble in seconds. Dr. Wu’s research team used dual-view plane illumination microscopy and Imaris to study microtubule assembly dynamics in 3D by acquiring time-lapse images of whole-cell volumes of human umbilical vein endothelial cells.
The cells expressed the fluorescent tip-tracking protein GFP-EB3, which marks the growing ends of microtubules. The researchers deconvolved dual-view plane illumination microscopy volumetric data sets, and then used the ImarisSurpass mode to create a new Spot object for each microtubule track. The manual tracking and auto connect consecutive frames options in ImarisSurpass let them select a Spot and connect the Spot object frame by frame. The researchers selected the microtubule center for each frame from the starting point of the track until the track disappeared or couldn’t be followed. They collected at least 75 tracks for each condition (i.e., thick cell on coverslip, thin cell on coverslip, thick cell in collagen gel) from two or three cells (at least 25 tracks per cell). They also used the ImarisVantage module to calculate the x-y-z track displacements, mean track speeds and track durations, which were then exported to an Excel spreadsheet.
Cell shape influences microtubule assembly
Using this approach, the researchers visualized 3D microtubule assembly dynamics with isotropic resolution and a temporal resolution of 1 volume per second for volumes of approximately 40 X 40 X 20 microns, over extended time periods with little photobleaching. Their results showed that the organization and speed of microtubule assembly depends on the three-dimensional cell shape.
The researchers say that they are not aware of other imaging techniques that enable accurate visualization and quantification of microtubule assembly in 3D. They believe that this new microscopy technique will help reveal mechanisms governing microtubule dynamics in the vascular, nervous, immune and reproductive systems in situ.
The researchers have also used Imaris with inverted selective-plane illumination microscopy for high-speed, 3-D intracellular tracking of influenza viral RNA transport, which was published in a PLoS Pathogens paper.
Additional images were created by the Bitplane team based on the original image data courtesy of Dr Wu. Imaris Surface segmentation and statistical colour coding was used for visualization. Imaris 2D Vantage Plot illustrates the relationship between sphericity and intensity Mean Ch 1
Research paper: Wu Y, Wawrzusin P, Senseney J, Fischer RS, Christensen R, Santella A, York AG, Winter PW, Waterman CM, Bao Z, Colón-Ramos DA, McAuliffe M, Shroff H. 2013. Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat Biotechnol 31(11):1032-1038.