Abstract
A new single-aperture 3D particle-localization and tracking technique is presented that demonstrates an increase in depth range by more than an order of magnitude without compromising optical resolution and throughput. We exploit the extended depth range and depth-dependent translation of an Airy-beam PSF for 3D localization over an extended volume in a single snapshot. The technique is applicable to all bright-field and fluorescence modalities for particle localization and tracking, ranging from super-resolution microscopy through to the tracking of fluorescent beads and endogenous particles within cells. We demonstrate and validate its application to real-time 3D velocity imaging of fluid flow in capillaries using fluorescent tracer beads. An axial localization precision of 50 nm was obtained over a depth range of 120μm using a 0.4NA, 20× microscope objective. We believe this to be the highest ratio of axial range-to-precision reported to date.
Highlights
Diffraction is perhaps the major factor limiting high-resolution microscopy of biological samples
Point sources can be localized with a precision that can be much better than the diffraction limit and this is the basis of recent rapid progress in localization microscopy techniques such as stochastic optical reconstruction microscopy (STORM) [1] and photo-activated localization microscopy (PALM) [2]
Results and discussions experimental results are presented, including examples of translations of the Airy-beam point-spread function (PSF) with defocus, characterization of PSFs over an extended depth range, examples of 3D particle localization and particle-tracking velocimetry of fluid flow seeded with fluorescent beads within fluorinated ethylene propylene (FEP) capillaries
Summary
Diffraction is perhaps the major factor limiting high-resolution microscopy of biological samples. Precise localization using conventional microscopy is limited by diffraction to thin planes of about a micron thick, which prevents localization of points in three dimensions over extended depth ranges. Reported techniques suffer from a fundamental limit of microscopy: high transverse resolution requires a high NA for the objective, which necessarily results in a small depth-of-field (DOF), and a small depth range over which adequately precise localization is possible. For a typical system employing a 20× objective lens to yield localization precision of better than 100 nm, the axial range is limited to less than 10 μm [15], which is comparable to cell dimensions and is smaller than many biological structures of interest
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