Imaging Eukaryotic Cells with the Double Helix Point Spread Function

October 7th, 2019 by DHO Team

In this blog posting, we would like to highlight research from the Lee Lab[1] at University of Cambridge using the Double Helix point spread function in the study of T-cells for whole-cell visualization, reconstruction, and 3D Tracking. In the article, titled Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function,” researchers from the Lee Lab used the double-helix point spread function (DH-PSF) phase masks offered by Double Helix Optics to capture images of eukaryotic cells, specifically whole-cell reconstruction of a T cell, visualization of large-scale reorganization of the outer membrane of Jurkat T cells, and 3D single-particle tracking (SPT) of proteins on the surface and within the cytoplasm of T cells as well as proteins within the nucleus of ES cells.

Using the DH-PSF in conjunction with single-molecule imaging, the researchers were able to gain unprecedented detail of the eukaryotic cell and to capture more depth per axial plane.  Capturing detailed whole-cell data for eukaryotic cells has proven challenging because of the depth range required.  These cells do not fit within the limited depth of field capabilities of sub-diffraction-limit localization methods. Adding the DH-PSF, the standard PSF generated is reshaped to allow for high localization precision with extended depth of field.

This unique ability of the DH-PSF to maintain precision with extended depth makes this technology well-suited for imaging and tracking of complex biological structures for two reasons. 

  • First, the extended depth of field means that imaging a large cell can be accomplished faster and using fewer layer images.
  • Second, the speed of data acquisition, the extended depth of field, and the volumetric position information enable the tracking of high-speed molecules in three dimensions.

The paper presents several remarkable results from using the DH-PSF to explore the features of eukaryotic cells.

Super-resolved whole-cell reconstruction of a T cell using the DHPSF.
Super-resolved whole-cell reconstruction of a T cell using the DHPSF.

Reconstruction of the three-dimensional composition of a set of several T cells was accomplished using only three to five layers. The images confirmed that fixed Jurkat T cells appeared predominantly spherical without significant differences among all the cells tested.

Visualization of large-scale reorganization of the outer membrane of Jurkat T cells on activating coated surfaces by mesh fitting to 3D localization data
Visualization of large-scale reorganization of the outer membrane of Jurkat T cells on activating coated surfaces by mesh fitting to 3D localization data

Observation of a T cell over a ten-minute span showed a substantial difference in morphology between resting cells, which are largely spherical, and those exposed to an activating antibody, which flatten and extend against a surface.

Many additional observations are described in the paper. Together, these results demonstrate how the DH-PSF extends the ability to study biophysical phenomenon down to the nanoscale organization of proteins. Specifically, the DH-PSF:

•  reduces 3D imaging experimental complexity
•  enables simultaneous imaging of multiple particles in 3D
•  extends the detectable range of single-particle trajectories in 3D
•  increases the ability to track fast-moving particles in 3D

The Double Helix SPINDLE® integrates seamlessly with existing microscope and camera systems, adding PSF engineering capabilities, including DH-PSF, that enable 3D imaging and SPT with a depth and precision unmatched in optics today. Modular phase masks give even more choices of PSFs, such as single-helix, tetrapod, and multicolor, for a variety of applications.

Interested in learning how Double Helix Optics can enhance your research? Contact one of our engineers and discover how you can “explore what no one has seen before.”

[1]Alexander R.Carr, Aleks Ponjavic, Srinjan Basu, James McColl, Ana Mafalda Santos, Simon Davis, Ernest D. Laue, David Klenerman, Steven F. Lee