Live projection imaging of sub-cellular dynamics at high speed via shear warp angled projection microscopy

Session

Stream 6 (Frontiers): Quantifying Dynamic Movement in Living Cells

Authors

James Manton (2), Bo-Jui Chang (3), Etai Sapoznik (3), Theresa Pohlkamp (3), Tamara Terrones (3), Erik Welf (3), Vasanth Murali (3), Philippe Roudot (3), Kayley Hake (1), Lachlan Whitehead (4), Andrew York (1), Kevin Dean (3), Reto Fiolka (3)

Affiliations

1. Calico Life Sciences
2. MRC Laboratory of Molecular Biology
3. UT Southwestern
4. Walter and Eliza Hall Institute of Medical Research

Keywords

projection, light sheet, spinning disk, fast, volumetric, 3D, sub-cellular, dynamics

Abstract text

Conventional approaches to recording and presenting data from 3D samples require the acquisition of a stack of images, which are then rendered in a 2D display via projection. We introduce a simple, cost-effective and easily implementable scan unit that converts any camera-based microscope into a 'live projection' imaging system. In combination with a microscope having optical sectioning, this enables the direct recording of high-contrast and blur-free projection images with uncompromised lateral resolution.

Live projection provides intuitive renderings of entire samples in real-time, improving microscope usability and reducing data overhead by factors in excess of 100-fold. Furthermore, by imaging the sample from multiple perspectives, our approach permits real-time stereoscopic visualisation as well as 3D particle localisation throughout a cellular volume, from just two images.

Developed in a trans-Atlantic collaboration, we present the theoretical and mathematical underpinnings of the 'shear warp angled projection' approach underlying the live projection system, along with the practical implementation and experimental data from both light sheet and spinning disk microscopes. Experimental demonstrations include imaging lysosomal and mitochondrial dynamics in HeLa cells, calcium signalling in cultured neurons at 119 Hz, three-dimensional localisation of genetically encoded nanoparticles, and simultaneous orthogonal views of an embryonic zebrafish heart.