Lattice light-sheet microscopy extends the capabilities of motor-PAINT to map the organization and orientation of microtubules in three-dimensional samples.

Microtubules are one of the main components of the cytoskeleton and are directly involved in many critical biological processes, including transport of intracellular cargo such as membrane vesicles and organelles. This cargo is moved by motor proteins using microtubules similarly to train tracks. The direction that the motors move depends on the orientation of the microtubules.

Numerous methods have been developed to study the orientation of microtubules and intracellular traffic flow, particularly in neurons. Motor-PAINT is one new superresolution microscopy-based method that enables the observation of microtubule polarity. While this method has advantages over previous electron microscopy-based methods, it relies on TIRF microscopy, thus limiting sample types to thin cells with observations of microtubules occurring within ~100 nm of the coverslip.

A group led by Dr. Lukas Kapitein at Utrecht University, the Netherlands, has adapted the motor-PAINT technique to use lattice light-sheet microscopy with ZEISS Lattice Lightsheet 7. Their protocol is published in Single Molecule Analysis. Methods in Molecular Biology, vol. 2694 (2024) and uses the entire volume of T-cells to demonstrate how lattice light-sheet motor-PAINT can be used to map complex three-dimensional microtubule arrays across large volumes.

The biggest advantage of lattice light-sheet motor-PAINT is the ability to perform single molecule imaging in the whole cell volume. It is not limited by the distance from the coverslip and can be performed with a user-friendly commercial microscope in an automated manner.

A raw recording of one trajectory of a single kinesin molecule walking on a microtubule is shown (left). By inducing astigmatism (optical transformation), the research team was able to encode the z-coordinate of the molecule into its vertical or horizontal shape stretch. On the right, the molecule's XY coordinates (center of the spot) together with Z (determined from the shape) are shown.

The movement of kinesin molecules on microtubules can be tracked over time. These observations are collected to build a time-accumulated reconstruction. Shown here is one plane of kinesin molecule movement within an epithelial cell. The data are color coded for depth (Z position).

The time accumulated movements of kinesin molecules along individual planes are then used to reconstruct the microtubule cytoskeleton in a whole cell. This creates a 3D single molecule, superresolution image of the microtubule cytoskeleton.

The lattice light-sheet motor-PAINT method enables superresolution imaging of microtubules along with their orientation throughout the entire cell volume, as shown here using a T cell (Jurkat cell).

In addition to superresolving the microtubule structure itself, lattice light-sheet motor-PAINT gives the microtubule cytoskeleton orientation, which dictates the direction of intracellular cargo transport. This is highly relevant in studies of cell organization.

With lattice light-sheet microscopy, the research team under Dr. Lukas Kapitein was able to expand the capabilities of motor-PAINT to achieve single molecule, 3D superresolution imaging of the microtubule cytoskeleton in entire cell volumes. This method maps both microtubule organization and orientation.

The authors believe this method could benefit the cell biology community. Microtubules are present in almost every cell type and change their network organization depending on cell function. Moreover, many differentiated cells are polar and have unique cytoskeleton architecture. The authors feel this technique will allow users to map the transport network of recently developed 3D models such as epithelial sheets and spheroids, organoids, brain tissue slices, pancreatic islets, and more. This will provide a new angle on the study of transport disorders associated with cancer, as well as cardiovascular, intestinal, diabetic, and neurodegenerative diseases.

Read their article to learn the necessary steps to purify, label, use, and image kinesin motors for motor-PAINT and review the analysis pipeline used to visualize the resulting data.