Long-axial-range 3D imaging with Double Helix ePSFs
Using DHO phase masks, the Gustavsson Lab at Rice University and colleagues have demonstrated the superior performance of Double Helix ePSFs at short- and long-depth-capture ranges.
Why this is important
Single-molecule localization microscopy (SMLM) enables the visualization of biological structures beyond the diffraction limit of light. Typically, extending SMLM to 3D imaging involves capturing and computationally stacking multiple axial slices, especially when imaging thick samples like entire mammalian cells. This multi-slice procedure is slow and increases phototoxicity, making it unsuitable for live-cell imaging or particle-tracking applications.
In this study, Nakatani et al. benchmark the localization precision of DHO’s Double Helix engineered point spread functions (DH-PSFs) under various imaging conditions. They demonstrate that DH-PSFs surpass conventional 2D clear-aperture imaging and limited-range astigmatic PSFs by providing isotropic, consistently high localization precision across an extended axial range, up to 12x the standard depth of field of traditional systems.
The science
Conventional clear aperture and astigmatic PSF (cylindrical lens) SMLM imaging techniques have restricted axial sensitivity (~1 µm for astigmatic PSFs) and exhibit significantly declining localization precision – and, consequently, reduced resolution – beyond this limited range. Conversely, DH-PSFs encode axial position information directly within their distinct shape, simplifying analysis and maintaining robust performance over extended depths. Historically, however, DH-PSFs have been restricted by axial ranges inadequate for capturing thick biological samples without scanning.
Nakatani et al. systematically evaluated DH-PSFs with axial ranges up to 12 µm, quantifying localization precision using fluorescent beads. They confirmed that while clear aperture and astigmatic PSFs experienced sharp declines in localization precision outside ±0.5 µm, DH-PSFs provided stable, superior precision throughout their entire extended range. Further validating these results, the authors employed DNA-PAINT super-resolution imaging of lamin B1 – a nuclear envelope protein – in U-2 OS cells. Their findings show that the use of DH-PSFs significantly simplifies imaging workflows, allowing efficient, stitching-free 3D imaging of entire mammalian cells with enhanced precision and minimized computational complexity.
How Double Helix Optics made it possible
The extended-depth, 3D imaging capabilities demonstrated by this study were enabled by DHO’s patented Double Helix ePSF phase masks, which were integrated into a high-NA fluorescence microscope. DHO's 3DTRAX software efficiently facilitated localization analysis, achieving high-precision 3D localizations across significantly extended axial ranges (up to 12 µm).