Optical design and development of a cost-efficient, compact, super-sensitive scanning confocal fluorescence microscope

Current commercial confocal fluorescence detection instruments are typically sophisticated, bulky and expensive, not suitable for rapid clinic routine detection targeting early fatal disease diagnosis, e.g. Alzheimer’s Disease. The detection of those severe and disabling diseases at an early stage is quite difficult due to very low abundant biomarkers to be determined. When miniaturizing and optimizing optical systems containing mass-produced optical components for more versatile and robust point-of-care usages, the optical characteristics should be carefully examined to suit the core requirements of high diagnostic accuracy and sensitivity.
We designed a scanning confocal fluorescence optical system in the framework of a EU-funded FET project called ‘SensApp’ to reach a cutting-edge limit of detection (LOD) below 1pg/ml. By carefully quantifying the spectra and scattering features of the optical components in the detection system, including the laser diode, filter set and sample slide, a comprehensive optical modelling approach was established to evaluate the influence of different components and to determine best optical configurations. The approach enables fast decision on the most performant configuration which greatly reduces the development time and prototyping cost. Two system configurations via tilt illumination and perpendicular illumination are compared to find an optimal design over excitation efficiency, signal-noise-ratio as well as tolerance requirements. Tolerances from typical perturbations, such as element decenters and tilts, are also
analyzed to give a practical consideration of the mechanical holder design and system assembly. Furthermore, the illumination uniformity (e.g. Gaussian beam and top-hat beam) which is often overlooked has been thoroughly considered from Beer’s Law to determine the maximum peak intensity and to avoid potential serious photobleaching at low abundant detection.
As we know signal-noise-ratio (SNR) is the most important criterion for super-sensitive detection, the corresponding noise sources are identified (including autofluorescence and scattered light sources), and various strategies to reduce noise are proposed to improve the SNR. In the end, we have built the confocal fluorescence detection system given all the above-mentioned aspects. To determine the system performance, the samples were made by diluting a series of solutions using dye Alexa Fluor 647 and PBS buffer. Experimental results have demonstrated that the as-built system can reach a limit of detection at a very low abundant concentration such as sub pM/ml.