The increased use of open science, 3D printing and single-board technologies like Raspberry Pi and Arduino have democratized technology by making it easier and cheaper for researchers to create prototypes and make automated lab tools. It also facilitates knowledge sharing, allowing other researchers to improve designs and applications. Numerous open-sourced designs for low-cost 3D-printed optical microscopes have been developed. Among them, OpenFlexure laboratory-graded automated microscope designed using Raspberry Pi is considered superior owing to its high position precision. By allowing anyone to contribute to the OpenFlexure project, the open-source philosophy contributes to the scientific advancement of the project, thus benefiting the community.
In some fields like life science, specimens are often thicker than the focus depth of the objective lens of the microscope, such that both the in-focus and blurred out-of-focus planes contribute to image formation. In such cases, obtaining higher resolution and contrast requires developing techniques that can accurately obtain sectioned images only from the in-focus planes. Structured illumination microscope (SIM) is an example of such technique. It uses a predefined spatially varied illumination, made by adding space patterns and a grid of line at the conjugate plane, to modulate the wide-field illumination. The advantages of SIM include its wide-field technique that does not require scanning focused spot of light and the use of a simple mathematical process to computationally obtain optically sectioned images. However, despite its practical implications, there are limited studies on the fabrication of high-resolution imaging systems with printed parts.
Inspired by the open-source philosophy, Professor Tatsunosuke Matsui and Mr. Daigo Fujiwara from Mie University reported a new low-cost fabrication of SIM with optical sectioning capabilities as one way of contributing to the community-driven development of low-cost and highly effective microscopes. The design of the proposed SIM system was based on the concept of OpenFlexure Microscope families. Particularly, the SIM system was upgraded by adding several additional 3D-printed parts. Their work is currently published in the journal, Optics Express.
The authors obtained optically sectioned images by projecting single-spatial-frequency grid patterns onto the specimen and using the grid patterns to record three images at various spatial phases, followed by post-processing using simple mathematics expressions. The main body of the microscope was based on the OpenFlexure Delta Stage because it allows independent motorization of the sample stage. Raspberry Pi camera and off-the-shelf LED were used as image sensors and illumination sources, respectively. Similarly, instead of expensive piezo stages, low-cost stepper motors and flexural mechanisms provided by the printed plastics were utilized for actuation and sample stage positioning. Moreover, the modification of the OpenFlexure Block Stage allowed precise actuation of the grid.
The proposed SIM system achieved optical sectioning capability strength of a few microns depth resolution, comparable to that of confocal microscopes. The flexible plastics and flexural mechanism ensured a highly precise positioning control of up to tens of nanometers. The main advantage of SIM system was the low-cost fabrication costs. This was attributed to the utilization of freely accessible STL and OpenSCAD files and the open-source nature of the OpenFlexure Microscope projects. Furthermore, the Raspberry Pi not only allowed automation of the system operation but also allowed remote operation from PC via a local area network.
In summary, Professor Tatsunosuke Matsui and Mr. Daigo Fujiwara demonstrated the fabrication of a cost-effective 3D-printed robotic SIM with optical sectioning capability. The results illustrated the underlying capability of modifying the original OFM families to realize multiple imaging modes. For example, the basic concept of fabricating SIM presented here is versatile and can also be adapted for 3D fluorescence imaging, and the applications of the SIM are not restricted to optical sectioning but also other functionalities like surface profiling. In a statement to Advances in Engineering, Professor Tatsunosuke Matsui explained that the open-source philosophy will continue democratization technology and accelerating the advancement of microscopy technologies.
Acknowledgment: The authors acknowledge using open-source 3D-printable designs. They have made STL files freely available in their GitHub repository.
Matsui, T., & Fujiwara, D. (2022). Optical sectioning robotic microscopy for everyone: The structured illumination microscope with the OpenFlexure stages. Optics Express, 30(13), 23208-23216.