openFrame: A modular, sustainable, open microscopy platform with single‐shot, dual‐axis optical autofocus module providing high precision and long range of operation

Abstract

Abstract“openFrame” is a modular, low‐cost, open‐hardware microscopy platform that can be configured or adapted to most light microscopy techniques and is easily upgradeable or expandable to multiple modalities. The ability to freely mix and interchange both open‐source and proprietary hardware components or software enables low‐cost, yet research‐grade instruments to be assembled and maintained. It also enables rapid prototyping of advanced or novel microscope systems. For long‐term time‐lapse image data acquisition, slide‐scanning or high content analysis, we have developed a novel optical autofocus incorporating orthogonal cylindrical optics to provide robust single‐shot closed‐loop focus lock, which we have demonstrated to accommodate defocus up to ±37 μm with <200 nm accuracy, and a 2‐step autofocus mode which we have shown can operate with defocus up to ±68 μm. We have used this to implement automated single molecule localization microscopy (SMLM) in a relatively low cost openFrame‐based instrument using multimode diode lasers for excitation and cooled CMOS cameras.This article is protected by copyright. All rights reserved

Astigmatism-based focus stabilisation with universal objective lens compatibility, extended operating range and nanometre precision

“…S1, Video S2). 32,43 This laser was delivered through a single-mode fibre to provide a Gaussian beam that could be shaped for axial drift estimation. Some experiments (TIR, 40x and 10x) used a 638 nm multimode laser instead for simpler alignment, but the results were identical (Fig.…”

“…There are similarities between our approach and the recently published openFrame solution. 32 The main difference is that openFrame applies astigmatism near the laser launch position that is very mild, which is suitable for large operating ranges. Ultimately, openFrame achieves a precision of 35 nm and a refresh rate of 4 Hz, whereas our approach achieves a precision of down to 2 nm and a refresh rate of 100 Hz, which is more suitable for high-resolution applications.…”

“…Among beam-based methods, the triangular geometry or total internal reflection (TIR) has been widely adopted, and methods that incorporate open hardware and comprehensive documentation have been developed. 30 Nevertheless, there are limitations of current approaches that can be summarised by: (1) relatively low sampling rates (pgFocus: 30 Hz, 31 openFrame: 4 Hz 32 ); (2): only being compatible with high numerical aperture (NA); (3) electronic components requiring soldering and custom circuit boards and (4): the need to put a focus stabilisation module close to the objective lens as the performance is limited by laser pointing stability. 33 Alternative beam-based methods have recently been introduced, e.g.…”

“…42 However, this issue has recently been resolved in the scattering microscopy field 43,44 and in a recent focus stabilisation method. 32 Here we present PiFocus, a versatile focus stabilisation method implemented on Raspberry Pi, based on monitoring the astigmatic reflection from the coverslip-sample interface. We demonstrate that this method enables <10 nm axial drift control with extended operating range (>10 μm).…”

Innovating in a bioimaging core through instrument development

“…If, however, the system were to be built from scratch with a constrained budget, major efforts would be spent on ensuring the mechanical stability of the detection unit required by SMLM experiments. Modular parts are available to build custom detection units, but OpenFrame 30 solutions can be preferred for modularity, mechanical robustness, and cost-efficiency.…”