BBSRC–STFC FAF · APP88756

BBSRC · In-chamber fluorescence for Crossbeam + Katana

In-chamber fluorescence for the Crossbeam and Katana volume-EM stack. The selected optical baseline is now NA 0.55 with Firefli Red. The active 3D model and Blender sensor results now use the NA 0.55 candidate. Live Onshape remains at Rev G.

Updated 18 September 2026 · NA 0.55 model and sensor simulations regenerated · Original Rev G retained as history

Purpose

The instrument needs a small fluorescence channel that fits the chamber envelope, uses a long-working-distance objective, and does not collide with the microtome, detectors or stage travel. The design feeds a horizontal camera arm through a dichroic cube and a 45° prism above the objective, with reciprocal LED excitation on the same shared bore.

The active simulation uses the selected NA 0.55 objective with the existing downstream aperture geometry. The updated model is an optical simulation candidate; real optics and whole-assembly chamber clearance still require validation.

Latest baseline — NA 0.55 & Firefli Red

Decision dated 10 September 2026 · Applied to local 3D and Blender simulations 18 September · Live Onshape unchanged

The starting objective is now NA 0.55. The working candidate is the Mitutoyo M Plan Apo 50× with 13 mm working distance and a 4 mm focal length. Its identity and physical envelope must be confirmed before mechanical changes. With the existing ideal 100 mm tube lens, the nominal system magnification is 25×.

Previous model versus updated simulation
Parameter	Previous Rev G	Updated simulation
Objective catalogue magnification	20×	50× candidate
Numerical aperture	0.42	0.55
Working distance	20 mm	13 mm
System magnification with ideal f100 tube	10×	25×
Object pixel size	0.345 µm	0.138 µm
Object field	662.4 × 414 µm	264.96 × 165.60 µm
Objective-front datum	Z73.58 mm	Z66.58 mm

The initial sample is Firefli Red-labelled polystyrene, with reported excitation/emission peaks at 542/612 nm. It supersedes the provisional Nile Red channel for initial testing. Fluorescence has been reported after preparation, with high background; quantitative performance is still unverified.

Provisional spectral targets are a green LED around 530–545 nm, excitation around 540/20 nm, a 565–570 nm dichroic transition, and emission around 610/40 nm. These are targets for evaluation, not a purchased or validated filter set. The former 505 nm long-pass dichroic is unsuitable for routing green excitation in this configuration.

The model now includes the 7 mm shift toward the sample, an 82 mm-long conservative objective envelope and the 4.4 mm exit pupil. The recomputed aperture audit passes all 21,654 sampled rays. This does not validate the actual lens, lid or knife clearance, spectral performance or a print release.

Objective and channel decision

New sample-image review

The supplied EVOS image combines red-pseudocoloured RFP fluorescence and transmitted light, acquired with a 20× / NA 0.45 objective. Visible red signal supports testing the selected NA 0.55 objective, but the merged 8-bit RGB image cannot establish fluorescence background, photon SNR or detection limits.

Next measurements need separate original fluorescence and transmitted-light frames, an identically prepared blank, the installed filter-cube identity and particle size. Compare contrast above measured background before choosing the final spectral components.

Sample review and acquisition limitations

Historical Rev G geometry and aperture corrections

Earlier layout

SEM fluorescence layout schematic — Earlier concept schematic — objective, prism, dichroic, camera arm and illumination arm relative to the microtome lid.

Historical mechanicals — Rev G

Rev F vignetted off-axis image and illumination bundles at the shared/image bore, illumination approach and dichroic clear aperture. Rev G enlarges those openings and shifts the tube-lens / camera and LED seats so the sampled pupil rays clear. The 6 September audit records no solid–solid interferences among the 34 assembled parts after the correction; the nine-part print layout regenerated cleanly. These are recorded checks, not a fresh validation of the live CAD.

Rev G mounting datums (mm) — NA 0.42 configuration

Z0 (mount)

0.00

Sample

53.58

Obj. front

73.58

Optical axis

188.58

Item	Rev F	Rev G
Shared / image bore	Ø23 mm	Ø40 mm
Illumination approach bore	Ø24 mm	Ø40 mm
Dichroic clear rectangle	32 × 22 mm	46 × 32 mm
Dichroic glass envelope	35.6 × 25.2 × 1.1	50.8 × 35.6 × 1.1
Tube-lens / filter envelopes	Ø25–30 mm	Provisional Ø50.8 mm
Field stop	Ø1.8 mm	Ø3.6 mm @ f40 conjugate

Rev G Blender 3D overview — Rev G — Blender overview of housing envelopes and optical axis.

Rev G ray bundles — Rev G — image (orange) and reciprocal excitation (blue) clear the enlarged bores.

Rev F ray bundles showing vignette — Rev F (historical) — same sample shows corner vignette; red curves mark blocked diagnostic paths.

Updated 3D model — NA 0.55 / WD 13 mm

The active model now uses the selected Mitutoyo 50× candidate: NA 0.55, 13 mm working distance and a 4.4 mm exit pupil. The objective is represented by an 82 × Ø34.5 mm conservative envelope, with its front at Z66.58 mm and the shoulder retained at Z148.58 mm. It is not a detailed supplier barrel model or a live Onshape revision.

Updated NA 0.55 objective envelope and recomputed folded optical paths — New objective envelope and recomputed ray paths. Full housing covers and knife geometry are not included.

NA 0.55 ray audit with horizontal camera and 13 millimetre working distance — 21,654 sampled rays pass the downstream apertures; 594 Blender mesh tests found no aperture collisions.

Orange identifies the image branch and blue the reciprocal excitation branch. Colours do not predict fluorescence colour or intensity. Pupil positions and ideal lens transfer remain assumptions.

Image Excitation Failed Drag to orbit · scroll to zoom · Blender Z-up → Three.js (x, z, −y)

Model from assets/na055/na055_optical.glb; optional rays in mm. Geometric clearance only.

Blender simulations — updated NA 0.55 sensor view

The regenerated Blender scene uses NA 0.55 / f4 / WD13, a 4.4 mm pupil and nominal 25× transfer to the designed sensor. It includes focus, +10 µm defocus, LED-off and uniform-layer controls. The synthetic target was rearranged for the smaller field; this is not a fixed-specimen brightness comparison against Rev G.

Nominal magnification

25×

Object field

264.96 × 165.60 µm

Object sampling

0.138 µm/pixel

Sensor image

1920 × 1200 px

Blender simulation comparison of an in-focus fluorescent target, a target raised by 10 micrometres, an LED-off control and a uniform fluorescent layer — Same display scale: focus, +10 µm sample displacement, LED off and uniform-layer illumination. The specimen is synthetic; these are simulated images, not measurements of microplastics.

Focus: a 20 µm reference dot measures 145 pixels (20.01 µm), checking the implemented image scale.
Defocus: raising the sample 10 µm spreads the signal and reduces the smallest test-dot peak from about 0.6365 to 0.00931 in relative linear units.
LED off: all pixels are zero in the model’s dark control. Real detector noise is not included.
Illumination: the assumed LED and diffuser produce a calculated relative irradiance range of 0.913–1.027 across the sample grid, normalised to the centre.

The simulation uses ideal optical transfer and assumed LED/diffuser properties. It does not predict absolute brightness or detection limits, and does not include diffraction, real-lens aberrations, spectral response or camera noise. The new aperture-clearance audit is a separate check; none of these results is a real-lens or whole-assembly validation.

In-focus image +10 µm defocus LED-off control Uniform layer

Updated model & simulation downloads

Updated Blender optical model Updated Blender sensor scene Updated 3D model (GLB) Model scope and results Numerical ray audit Sensor checks

Open the updated sensor scene in Blender 4.5 or later and press F12. LED Power 0 gives the dark control; SAMPLE Location Z +0.010 mm gives +10 µm defocus. Restore Power to 1 and Location Z to 0 for the reference image.

Original CAD and historical files

The links below still refer to Rev G (NA 0.42) and require project access. The live Onshape assembly and print layout have not been changed by this simulation update.

Assembled Onshape model Nine-part print layout Rev G audit report Sensor study & controls Historical Rev G sensor scene Historical Rev G optical audit scene

Caveats

This is a geometric clearance result, not a validation of image quality, LED radiance, field uniformity, photon budget, spectral filters, ghosts, MTF, print tolerance, thermal drift or vacuum compatibility. Ø50.8 lens and filter solids are provisional envelopes — not purchased parts. Pupil planes used in the sensitivity sweep are assumptions; Mitutoyo’s proprietary prescription is unavailable. Do not treat ideal thin-lens focus as evidence of real-lens performance. Real tube-lens BFD, relay prescription and SEM/vacuum fit remain open before final print release.