Fig. 1: Design and characterisation of 3D-printed confocal microscope.

a Overview of optical path. The design utilises a single excitation laser, which is focused to a very small (femtolitre) observation volume using a water-immersion objective (Zeiss × 40 or × 63, 1.2 NA). The emitted fluorescence is separated from the excitation fluorescence using a dichroic mirror and further filtered before detection by a single photon avalanche photodiode (SPAD, Micro Photon Devices). b 3D design of the housing for 3D printing; mirrors are pre-aligned within housing and filters, dichroic, and laser line can be easily exchanged. c Photograph of microscope compared with a laptop. d Principle of confocal detection of individual molecules freely floating in solution. e Detection of individual α-syn molecules labelled with Alexa-568 in water (top) compared with water alone (bottom). These measurements were performed on a normal wet bench, in broad daylight. 532 nm excitation, acquired at 10 kHz for 30 s. f Photon counting histograms (450 nm excitation, acquired at 1 kHz for 30 s) of sfGFP (green) and sfGFP-foldon (blue), an sfGFP-tagged trimerisation domain. f Data from main figure fit to Gaussian distribution for N&B analysis, see main text. g Fluorescence correlation spectroscopy (average of 8 × 10 s traces acquired at 1 MHz) of a fluorophore with a known diffusion coefficient (Alexa-488, 10 nm, green) and 10 nm Alexa-488 labelled α-syn in the absence (blue) and presence (red) of 10 mm sodium dodecyl sulphate (SDS) micelles.