PT  - JOURNAL ARTICLE
AU  - Dylan R. George
AU  - Benjamin Ambrose
AU  - Ashley J. Cadby
AU  - Timothy D. Craggs
TI  - An Open-source, Cost-Efficient Excitation Module for Single-molecule Microscopy
AID  - 10.1101/2022.02.28.482236
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.02.28.482236
4099  - http://biorxiv.org/content/early/2022/03/01/2022.02.28.482236.short
4100  - http://biorxiv.org/content/early/2022/03/01/2022.02.28.482236.full
AB  - In fluorescence microscopy, scientific-grade laser diodes are key components and contribute a considerable expense to the total cost of the microscope setup. Existing open-source laser excitation modules are mainly designed for super-resolution imaging and have not been fully characterised to meet the requirements of single-molecule spectroscopy measurements. In this paper we introduce an open-source, cost-efficient excitation module that can be used for confocal, accurate single-molecule FRET, fluorescence correlation spectroscopy (FCS) and low-power super-resolution techniques. The module delivers two diode lasers (520 nm and 638 nm) via a single-mode fibre, with stable power output (<1% deviation), which can be modulated on the microsecond timescale. Here, we benchmark these lasers against smFRET standards, and recover consistent diffusion coefficients from FCS measurements, thereby demonstrating their suitability for a range of single-molecule spectroscopic experiments, whilst reducing the overall costs of our open-source smFRET instrument (the smfBox) by ~50%, making these types of experiments even more accessible to the widest possible userbase.Competing Interest StatementThe authors have declared no competing interest.