The eduWOSM

a| The eduWOSM chassis (1) is CNC machined from solid aluminium. A 100x NA 1.3 oil immersion objective (2) screws directly into the block and becomes monolithic with it. The four faces of the block carry identical ports, leading to a common central space beneath the objective. The dichroic block (3) slides into one of the ports and locks in position beneath the objective. The 4-colour LED light engine (4) and the camera (5) fit into 2 other ports. A 3-axis micrometer stage (6) is fixed to the top of the block and carries the sample holder (7). Slides are mounted to the sample holder using a pair of magnets. Z-axis motion of the stage is via a stepper motor (8). b| section through the microscope showing the optical path. Excitation path in magenta, emission path in yellow. c| Dichroic block. d| Keypad e| Light engine f| Microcontroller board.

HeLa cell mitosis

Live imaging of a mitotic HeLa cell with a standard-configuration eduWOSM (100x objective). Representative video frames showing (left to right) mitotic spindle at metaphase, early anaphase, late anaphase and telophase. The top row shows combined tubulin (green) and chromatin (magenta) channels. The rows beneath show the two channels separately at each time point, in reverse contrast for clarity. The full recording consists of 400 frames taken at 4 sec intervals (Supp. Video 1).

Quantifying diffusion in living cells

a| Imaging of a layer of living onion cells using the eduWOSM transmission illumination. Note the large field obtained at the diffraction-limited resolution of the 100x objective. The trajectories of 200 particles, obtained by automated tracking, are marked. Please zoom in for detail. b| Still from example movie (Supp. Video 2). c,d| The automated WOSM tracker macro (Supp. File 1) calculates MSD (mean squared displacement) for all pairs of position-time values along each trajectory and fits expressions for diffusion, subdiffusion and superdiffusion, returning a diffusion constant and an R (goodness-of-fit) value for each. c| example of a superdiffusive trajectory. d| example of a purely diffusive trajectory.

Tracking single fluorophores

Demonstration of single molecule imaging capabilities of the eduWOSM using gliding microtubules, sparsely labelled with HiLyte 647. a| Snapshot of microtubules. Spots corresponds to single HiLyte 647 fluorophores, illuminated by the red LED, exposure time 500 ms. b| Pixel-level view of the region marked. c| Kymograph showing that fluorophores on the marked microtubule glide at a constant velocity of 530 nm/s and that single-step photobleaching occurs (white arrow), indicating that the eduWOSM is successfully tracking single fluorophores. d| Trajectories of all microtubules in the field, constructed by superposing single fluorophore trajectories (Supp. Video 3).