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bioRxiv · 10.1101/2022.02.28.482236

An Open-source, Cost-efficient Excitation Module for Single-molecule Microscopy

George, D. R.·Ambrose, B.·Cadby, A. J.·Craggs, T. D.
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Abstract

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.

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George, D. R., Ambrose, B., Cadby, A. J., Craggs, T. D.. 2022-03-01. An Open-source, Cost-efficient Excitation Module for Single-molecule Microscopy. https://doi.org/10.1101/2022.02.28.482236

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