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

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.


Introduction
Fluorescence microscopy encompasses a range of biophysical imaging techniques such as epifluorescence, confocal and total internal reflection fluorescence (TIRF) microscopy. Fluorescent samples are excited by a high energy light source at specific wavelength (typically a laser) and dichroic filters are used to separate the excitation light from the fluorescence emission to provide higher contrast than in conventional microscopy. Recently, some labs have provided a gateway into this area for non-specialists by publishing open-source microscopy platforms and software packages. [1] A range of fully open-source microscopy platforms are now currently available including: the miCube, capable of super-resolution microscopy, TIRF and LED brightfield microscopy; [2] the openSPIM, a light-sheet microscope; and the LifeHack microscope, which provides single-molecule localisation microscopy (SMLM) and live cell imaging. [3] Open-source projects also include common microscope components such as microscope fluidic systems, lasers and stages. [4][5][6][7] Open-source software for super-resolution imaging has also been developed. [8][9][10] Previously, we introduced the smfBox, an open source single-molecule FRET microscopy platform to combat the high instrument costs and a lack of open-source hardware that has limited this technique's broad application by non-specialists. [11] In general, the lasers for fluorescence microscopy applications incur a considerable expense. Since multiple fluorophores with different spectra are often used to differentiate biological components, multiple lasers are employed to excite them. However, these laser setups, whether bought commercially or custom-made, often make up a large proportion of the overall cost of modern microscopes. Many fluorescence applications require different excitation specifications (in terms of laser powers and pulsed / alternation time periods) increasing the number of lasers needed. This makes it especially difficult to provide a universal excitation module that is suited to all microscopy applications. Table 1 provides a short list of some of the most common fluorescence applications, and their excitation requirements for normal operation.  Table 1. Common fluorescent applications, their excitation system requirements and primary expense. [12][13][14][15] Over the last decade, a few open source, affordable laser systems have been developed (Table   2). However, these have mainly been targeted towards super-resolution imaging; For example, the low-cost, continuous wave Laser Engine (from Schroder et al.) which provides a homogeneous, scatter-free laser beam through a multi-mode fibre vital for single-molecule localisation microscopy. [5] The NicoLase (from Nico et al.) caters to a broader range of applications with many different lasers in the visible spectrum and provides a full characterisation of the alternation of the lasers required for many single-molecule applications, albeit using higher-end laser diodes which may significantly add to the cost of the setup. [6] Author  Table 2. A list of current open-source laser excitation modules; their properties and their primary application. [5][6] While these open-source excitation modules are available, many researchers have not incorporated them into single-molecule spectroscopy methods as a full characterisation of their properties and suitability for this type of application has been lacking. This is particularly true . [17,18] Here we provide a full excitation module characterisation and explain in depth how to alternate these laser modules to achieve accurate single-molecule FRET (ALEX) and fluorescence correlation spectroscopy (FCS) measurements. The dichroic mirror combined the laser beams before a Fibreport (PAF2A-A15A, Thorlabs) coupled the beam into a single-mode fibre (P3-488PM-FC-10, Thorlabs).

Hardware/ Methods
To control the lasers, a modulation voltage of 5 V was delivered to the lasers at a frequency of 20 KHz. A National Instruments (NI) DAQ board was used to deliver the modulating voltage signal, controlled using the smOTTER software (available on GitHub, https://github.com/craggslab/smfBox). Alternatively, a simple LABVIEW program (supplied in the supplementary material) can be used.

Power and power stability
We conducted a full characterisation of the laser modules to check whether it was able to perform single-molecule experiments. To check whether the module needed excitation filters the wavelength spectrum of both the 520 nm and 638 nm laser modules was recorded ( Figure   2). The wavelength of the green laser diode ranged from 512 nm -526 nm and the spectrum of the red laser diode 630 nm -644 nm ( Figure 2). The resulting spectrum from both lasers was within the range expected and was sufficiently tight for single-molecule experiments without any excitation filters. Since single-molecule measurements are taken on short timescales (ms), inconsistencies in laser power on this timescale can give rise to differences in the amount of fluorescent emission detected. This is particularly important for FCS, where instabilities in laser power can affect the accuracy of results, but also needed for smFRET to obtain good quality data. In the initial design, the laser modules were placed on pedestal posts. A power stability assessment of the red laser module on these posts revealed that the laser power was particularly unstable.
Additionally, the laser module switched off after less than 30 mins, below the acquisition

Alternation rise and fall times for ALEX
To achieve accurate FRET values, alternating the red and green lasers at a frequency of 20 kHz was required. The lasers were alternated by the single-molecule FRET acquisition software (smOTTER). The output of the fibre was coupled to a photodiode to measure the laser power with high (ns) time resolution. Initial results showed that the lasers in the excitation module could indeed be successfully modulated at the required microsecond timescales ( Figure 3A).

smFRET
Since the results of the characterisation of the laser modules were sufficient for single-molecule FRET measurements, the excitation module was used on the smfBox to take smFRET measurements. Three double-stranded DNA samples were used given their well characterised FRET values in the literature. [18] These samples are labelled with ATTO 550 (donor) and ATTO 647N (acceptor) with the donor dye positioned at 23bp, 15bp and 11bp away from the acceptor dye. These three DNA standards have reported FRET Efficiency values of 0.15±0.02, 0.56±0.03 and 0.76±0.015, respectively, as measured in a recent blind multi-lab study. [11,18] The procedure for determining accurate FRET efficiencies for absolute distance To determine these parameters, Jupyter notebooks from the smfBox GitHub along with FRETBursts were used. [19] The correction factors used to find accurate FRET efficiency values were then determined. The first two correction factors α and δ were determined from the donor fluorescence leakage into the acceptor channel and the direct excitation of the acceptor with the donor laser, respectively. Using the average values for both α and δ for all three samples, the correction factors γ and β were determined. The correction factor γ takes into account the different detection efficiencies of the donor and acceptor emission photons, while β describes their different excitation efficiencies by the system (see supplementary information).
To compare the results, Table 3 shows the FRET efficiency values of a benchmark study measuring these duplex DNA strands from 20 independent research groups (Hellenkamp et al., 2018). The table also contains the FRET values of these samples on the smfBox obtained using commercial lasers (Ambrose et al). [11] By implementing the excitation setup on the smfBox FRET efficiency values for the samples 1a, 1b and 1c were 0.198±0.02, 0.599±0.17 and 0.793±0.09, respectively (Figure 4) [18] The samples 1a, 1b and 1c represent low, medium and high FRET samples, respectively. The results from the excitation layer and the FRET histogram of the excitation module are also shown at the top of the figure. These data points have error bars calculated from the standard deviation of the repeats. The 1c sample has error bars that are too small to be seen in this graph. Compared with this is the data from different labs represented by the colours in the legend. The dashed line shows the mean FRET efficiency of the labs in the benchmark study.

FCS
Fluorescence correlation spectroscopy provides insights into the diffusion behaviour and absolute concentration of molecules by recording the fluctuation of fluorescence intensity in the observation volume. We previously measured the diffusion coefficient and molecular brightness for a duplex DNA sample on the smfBox using commercial lasers. [11] To check if the excitation module could reproduce similar results the same DNA duplex sample (1a) was placed on the smfBox. Figure 5 shows the autocorrelation curve of a Rhodamine 6G sample which was used to determine the confocal volume and the diffusion coefficient of the duplex DNA sample. The diffusion coefficient of the duplex DNA was measured as 118 μm 2 /s. This compared well to the previous measurement on the smfBox, using the commercial lasers, of 88 μm 2 /s.

Discussion
Here we present an affordable excitation module (costing > £ 2500) capable of fluorescence applications such single-molecule FRET and fluorescence correlation spectroscopy. This excitation module differs from existing modules, such as Schroder et al. and Nico et al. set out in the introduction, by using low-cost laser diodes inside a setup specifically designed for single-molecule spectroscopy measurements. Our data obtained using this excitation module shows that accurate FRET efficiencies and diffusion coefficients can be measured successfully.
However, unlike the Nicolase and the Laser Engine, which can perform super-resolution imaging techniques, the power output of this module (~20 mW) likely falls short of the more power-hungry super-resolution applications such as STED and single-molecule localization microscopy. Due to the power and alternation requirements of microscopy applications described in the introduction we do anticipate the system will be used for FCCS, light-sheet, TIRF, confocal, SIM and widefield microscopy.
For these lasers to work effectively over the timescales and stability needed for smFRET, a series of experiments stabilising the temperature and allowing faster heat dissipation was key. Here, we were successful by mounting these lasers on heatsinks, one issue we faced was the time the lasers needed to warm-up (~20 minutes), which still arose on the second time they were switched on in close succession. However, characterising these lasers in terms of the rise times, fall times and spectrum demonstrated they could function successfully for single-molecule measurements without further modifications such as excitation filters. A simple improvement could be made by implementing a variable neutral density filter in front of each laser. This will allow the power of the lasers to be controlled.
Since using a single-mode fibre to provide a Gaussian beam is required for confocal single-molecule measurements, a drawback of using this module was the difficulty of aligning both lasers for obtaining high coupling efficiency. Care is also needed to avoid disturbing the alignment due to the sensitivity of the setup. Although, we believe that this could be improved by ensuring the optics are well secured to the breadboard and stored in a solid container. This module also makes use of an expensive NI-DAQ board to drive the microsecond alternation of the lasers. In the future, it would be more economical to implement the software to run from an Arduino or alternative. However, whilst such a solution may provide sufficiently fast electronics for alternating the lasers, the single-photon counting electronics (time-stamping photon arrival times to 10 ns bins) currently remains beyond such devices.
To conclude, this excitation module can be used for accurate single-molecule techniques at a very low-cost. This system performs well for FCS and smFRET experiments providing sufficient power and fast modulation of the lasers. We anticipate this simple to build, cost effective setup will be utilised in future single-molecule FRET and FCS setups, given the significant cost reduction (almost halving the total cost of the smfBox).

Characterisation of the Laser diodes
Characterisation of the wavelength spectrum: The 520 nm and 638 nm lasers

Accurate smFRET Measurements
Three duplex DNA standards were labelled with ATTO 550 23bp, 15bp and 11bp away from were plotted and fitted to obtain the last correction factors γ and β. The corrected FRET efficiencies were then determined using these four correction factors.

FCS measurements
The green laser was turned on and the Rhodamine 6G solution was diluted with water until the smfBox measured counts of ~25 kHz. A 2 minute acquisition was recorded. A known diffusion coefficient of 414 μm 2 /s for Rhodamine 6G was then used as a standard to determine the confocal volume. A dilute (~10 nM) doubly labelled DNA standard as previously described was placed on the smfBox using the same method and was recorded.
The diffusion coefficient and molecular brightness were then calculated in the MATLAB software package PAM. [20] First, the signal from the observation volume was recorded over time and then correlated to provide an autocorrelation curve ( Figure 6) using Equation 3.
The decay of the autocorrelation curve depends on the rate of diffusion of the sample. A 3D diffusion model shown in Equation 4 was then used to fit this curve.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author. Ashley Cadby is a Professor of Biophysics in the Imagine Life consortium at the University of Sheffield. His research interests include Microscopy and Single molecule biophysics. The Cadby Lab sits at the core of the Bio Imaging centre, hosted within the Biophysics Research Group at the University of Sheffield, UK. The group is known for the development of optical systems for biological imaging focusing on their application to real world problems. This may be repurposing existing technology to build a better performing optical system, or the development of novel imaging to solve a specific problem.