UCsim2: 2D Structured Illumination Microscopy using UC2

Abstract

State-of-the-art microscopy techniques enable the imaging of sub-diffraction barrier biological structures at the price of high-costs or lacking transparency. We try to reduce some of these barriers by presenting a super-resolution upgrade to our recently presented open-source optical toolbox UC2. Our new injection moulded parts allow larger builds with higher precision. The 4× lower manufacturing tolerance compared to 3D printing makes assemblies more reproducible. By adding consumer-grade available open-source hardware such as digital mirror devices (DMD) and laser projectors we demonstrate a compact 3D multimodal setup that combines image scanning microscopy (ISM) and structured illumination microscopy (SIM). We demonstrate a gain in resolution and optical sectioning using the two different modes compared to the widefield limit by imaging Alexa Fluor 647- and SiR-stained HeLa cells. We compare different objective lenses and by sharing the designs and manuals of our setup, we make super-resolution imaging available to everyone.

Super-resolution structured illumination microscopy: past, present and future

“…However, wider dissemination of SIM has been curtailed by the complexity of instruments and its proneness to reconstruction artefacts when sample properties, system calibration and parameter settings are not carefully matched [49]. Thus, to lower the activation energy for research labs to venture into super-resolution SIM, many recent developments aim at further performance/resolution enhancement and 'democratizing' SIM by -using nonlinear SIM approaches [50][51][52][53] -implementing the technique into smaller and more cost-efficient set-ups [64][65][66], -making the method more robust against artefacts using alternative illumination schemes [67][68][69] and/or intelligent data processing [70,71], -increasing its application range, e.g. by the implementation of adaptive optics [72][73][74], and cryo-imaging [47,75].…”

“…In parallel, the rapidly evolving field of computational microscopy is paving the way to circumvent the resolution limit without the necessity of specialized hardware reducing the complexity of instrumentation. For example, machine learning (ML) assisted denoising has been demonstrated to improve SIM reconstructions by reducing artifacts and boosting the effective resolution for 2D-SIM [65,66,70,71,[81][82][83][84]. Thus, by combining physical data acquisition with smart image processing algorithms, previously inaccessible information can be recovered.…”

Answers to fundamental questions in superresolution microscopy

“…Inexpensive/simple SIM as well as inexpensive ISM have recently been demonstrated in our laboratory [34]. Consumer-devices such as DMDs are a big help to this aim.…”

An Open-Source Modular Framework for Automated Pipetting and Imaging Applications

“…Material S1) offers a static sample well, moves the camera in XYZ to generate large-scale microscope images and uses a white-light LED to realize transmission brightfield imaging at cellular resolution. A more generic solution, where customized imaging schemes such as fluorescence or multiple objective lenses for different magnification and optical resolutions are desired, is provided by the UC2-based 20,32 system "Hi2" (Figure 1d). We extend the functionality of our recently introduced open-source modular optical toolbox UC2 with the capability of large-scale fluorescence microscopy with the development of a laser engraver-based XY stage (Figure 2a) and a novel Z-focusing unit (Figure 2b).…”