The apparatus

What it consists of

Illumination

The two stimulus LEDs are switched together via a MOSFET, gated from a Raspberry Pi GPIO pin — the same output the software's LaserController drives in rpi_gpio mode (configurable BCM pin, set in the Setup tab). Measured draw is ~500 mA combined with both LEDs wired in parallel, each individually rated to 700 mA at 2.0–2.4V.

Imaging illumination is handled separately by an IR backlight pad beneath the well plate: an acrylic sheet edge-lit by IR LED strips, diffusing light evenly upward through the plate rather than being switched per-experiment like the stimulus LEDs.

The IR backlight pad, illuminated.

3D-printed parts

Designed in Fusion 360 for the Ender-5 S1 + Player One combination above. STL files for printing are below; Fusion 360 source (.f3d) and step/iges exports are kept in the StentorCam repository's cad/ folder for anyone who needs to modify them.

Base Lens Mount T2-C Adapter Darkfield 24 Darkfield 96

The T2-to-C adapter has tight tolerances — it was designed for and printed on a Bambu X1C at highest layer precision with random seams enabled. Other printers/settings may need adjustment. The lens mount is specific to the Ender-5 S1's extruder mounting arrangement; other printers will need a redesign. The IR backlight pad itself is not a 3D-printed part — it's built from an acrylic sheet and IR LED strips — so there's no STL for it yet; a CAD design for a proper enclosure/mount may be added later.

The darkfield mask

The darkfield mask is the part that makes the imaging itself work, not just the mounting. It implements transmission dark-field imaging for standard multi-well plates using a purely geometric trick — no additional optics required — so that a camera positioned above the plate sees a dark background with only the light scattered by objects in the well (e.g. a swimming Stentor) showing up as bright features. Unscattered illumination is rejected by the mask geometry itself.

An opaque mask beneath the well plate is aligned so each well sits above a corresponding masked region. Illumination from below is blocked by the mask directly under the well and can only emerge from the unmasked area around it, entering the well at oblique angles. Three parameters, tuned jointly, govern whether this works:

• A — mask stop diameter: the opaque region's width beneath each well. Larger A blocks more direct light (better rejection) but throttles throughput; smaller A passes more light but risks direct-illumination leakage into the camera.
• B — well bottom height above the mask: too small and illumination is clipped before reaching the well (weak signal); too large and refraction at the well bottom/liquid meniscus can redirect direct light back into the camera's acceptance cone.
• C — mask thickness: acts as an angular filter — thicker suppresses shallow-angle rays more strongly, but reduces overall throughput and tightens fabrication tolerances.

Because unscattered rays are geometrically excluded from the well walls or the camera's acceptance cone while scattered light is redirected into it, the result is a dark background with only scattering structures visible — without a beam block, condenser, or any additional optical hardware beyond the mask itself. Liquid fill volume matters too: it changes meniscus curvature and optical path length, so consistent well volumes matter for consistent rejection.

From the team's internal writeup, "Dark-Field Imaging Using a Masked Illumination Geometry for Standard Well Plates" (Leonard Chau, PI Dr. Raymond Esquerra, San Francisco State University).

Assembly overview

1. Print the mount, adapter, base, and darkfield mask (see print notes above).
2. Remove the Ender-5 S1's stock extruder/hotend and install the Lens Mount in its place.
3. Attach the T2-to-C adapter to the mount, then attach the C-mount lens.
4. Mount the Player One camera to the lens assembly.
5. Wire the two stimulus LEDs through the MOSFET driver to a Raspberry Pi GPIO pin (set the pin in RoboCam's Setup tab, laser/GPIO mode rpi_gpio).
6. Build the IR backlight pad (acrylic sheet edge-lit with IR LED strips) and clip it to the printer bed using the StentorCam Base's sprung fingers, with the darkfield mask on top.
7. Connect the printer (USB/serial for Marlin) and camera to the machine running RoboCam 3.1, then follow the Docs page to calibrate the four well-plate corners.

Parts cost

Original bill of materials totaled $982 (before tax) with a Raspberry Pi 4 + microSD card ($80 combined) for compute/storage — now upgraded to a Raspberry Pi 5 with an M.2 HAT+ and 512GB NVMe SSD. Total will be updated once the new compute/storage cost is finalized.