PUMA microscope by Dr Paul J. Tadrous · brightfield · 20x/0.40 · eyepiece 10x/20

Looking through the PUMA at a tardigrade

Every image on this page is computed, none is painted. Light from a white LED travels as a wave through a real, nanoCT-scanned tardigrade. It then passes lenses whose data come from open patents: the objective, the eyepiece and a model eye. The colour fringes, blur and diffraction you see all come out of that calculation.

Simulated view through the eyepiece
20 µm

Drag inside the ring: roll the animal · on the ring: turn the slide · wheel, pinch or ↑ ↓: focus · ← →: turn · double-click: back to the start view

The instrument

Drag to rotate, scroll or pinch to zoom. Built from the original PUMA CAD parts (FreeCAD → Blender → glTF), assembled by hand from the PUMA build guide, so part positions are approximate.

The animal walking

Simulated view of the tardigrade walking, stopping to probe and turning, seen through the microscope
work in progress 10.5 s of walking, a 2 s stop to probe and a head-led turn, focal plane 36 µm below the dorsal surface, through the ray-traced PUMA optics (5 wavelengths, 37 condenser points), 8 frames per second. An articulated rig deforms the 3D refractive-index volume: the trunk bends along a midline that follows the head's path (follow-the-leader), each lobopod leg bends and telescopes from base to tip, and every claw stays fixed on the slide while it is in stance (inverse kinematics). Gait from Nirody et al. 2021 and Anderson et al. 2024 (back-to-front stepping wave, duty factor ~0.7, legs IV mostly holding on). The specks are detritus lying on the slide. The motion is still being refined against measurements from real footage.

The optical path

  1. White LED, 6500 KPUMA docsBlue InGaN peak at 452 nm plus phosphor. 9 wavelengths from 420 to 680 nm.
  2. Illumination: mirror or Köhler condenserselectableThe PUMA Foundation scope lights the slide with a plane mirror (modelled as NA ≈ 0.05). From 20x up the PUMA docs call for a condenser (measured up to NA 0.70). The Köhler aperture stop can be set to NA 0.15, 0.30 or 0.40.
  3. Tardigrade Hypsibius exemplarisnanoCT, CC BY152 µm long, 270 nm voxels, 11 segmented organs. Refractive index per tissue is a literature-based assumption.
  4. Cover glass 0.17 mm, waterSchott D 263Focusing deeper into the water adds spherical aberration. The calculation includes it.
  5. Plan achromat 20x/0.40Olympus patentUS 4,212,515, 7 elements, glasses S-LAH66, S-FPL51, S-LAL12 and others. A generic objective rather than a specific retail part, as the PUMA intends.
  6. Tube 160 mm, field stop 20 mmPUMA docsFinite DIN system without a tube lens.
  7. Eyepiece 10xAO patentUS 3,888,567, f = 24.9 mm. The PUMA only specifies "WF 10x/20".
  8. EyeNavarro 1985Aspheric model eye with dispersion of the ocular media. The exit pupil is 1.05 mm.

What is computed

  • Wave propagation through the 3D animal in 134 slices (multi-slice), with diffraction, refraction and multiple scattering.
  • Partially coherent illumination as an incoherent sum over condenser source points (7 to 91, depending on the aperture) × 9 wavelengths.
  • Longitudinal colour as a wavelength-dependent sharp plane, found by ray tracing from the specimen to the retina. At 450 nm it lies 11 µm deeper than for green.
  • Residual aberrations (spherical aberration, spherochromatism) as a pupil phase from the traced wavefront.
  • Colour through the CIE 1931 colour-matching functions, white-balanced like an eye adapted to the lamp.

What is assumed or simplified

  • Tissue refractive indices: cytoplasm ≈ 1.355, storage cells ≈ 1.39, cuticle ≈ 1.46. Algae in the gut absorb with a chlorophyll-like spectrum.
  • Only the central 207 µm are simulated. The rest of the 1 mm field of view is empty slide in the simulated background colour.
  • Aberrations are those on axis. Field curvature towards the edge of the field is not included.
  • Scalar wave optics, no polarisation. The detail view is a digital zoom on the same data.
  • The scanned animal was critical-point dried and is therefore smaller than a living one (living adults are 250–500 µm).
Optical layout, longitudinal colour and point spread functions
The ray-traced optical path. Left: the secondary spectrum, i.e. the sharp plane in the specimen per wavelength. Right: point spread functions at green focus.
Refractive-index volume of the tardigrade
The tardigrade as a 3D refractive-index volume, sections at 589 nm.

Sources and licences. Tardigrade scan: Gross, V. et al. (2019) "X-ray imaging of a water bear offers a new look at tardigrade internal anatomy", Zoological Letters 5:14, CC BY 4.0. The PUMA microscope was invented and designed by Dr Paul J. Tadrous (@TadPath): github.com/TadPath/PUMA (CAD GPL-3.0, docs GFDL); paper: J. Microsc. 283:259–280 (2021), doi:10.1111/jmi.13043. This page is an independent project built on PUMA and is not endorsed by its author. Objective: US 4,212,515 (Itaya, Olympus 1980). Eyepiece: US 3,888,567 (Shoemaker, American Optical 1975). Eye: Navarro, Santamaría & Bescós, JOSA A 2 (1985). Ray tracing: Optiland (MIT). Glass data: Ohara and Schott catalogues. 3D viewer: model-viewer (Apache-2.0).