Automating multimodal microscopy with NanoJ-Fluidics

Abstract

Fluorescence microscopy can reveal all aspects of cellular mechanisms, from molecular details to dynamics, thanks to approaches such as super-resolution and live-cell imaging. Each of its modalities requires specific sample preparation and imaging conditions to obtain high-quality, artefact-free images, ultimately providing complementary information. Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows involving multiple sample preparation steps. We present a robust fluidics approach to automate complex sequences of treatment, labelling and imaging of live and fixed cells. Our open-source NanoJFluidics system is based on low-cost LEGO hardware controlled by ImageJ-based software and can be directly adapted to any microscope, providing easy-to-implement high-content, multimodal imaging with high reproducibility. We demonstrate its capacity to carry out complex sequences of experiments such as super-resolved live-to-fixed imaging to study actin dynamics; highly-multiplexed STORM and DNA-PAINT acquisitions of multiple targets; and event-driven fixation microscopy to study the role of adhesion contacts in mitosis.

NanoJ: a high-performance open-source super-resolution microscopy toolbox

“…NanoJ-Fluidics is a hardware and software framework for precise and accurate automated liquids exchange [ 15 ]. It was developed to enable automation of sample treatment and labelling of live or fixed specimens directly on the microscope stage [ 15 , 43 ].…”

“…NanoJ-Fluidics is a hardware and software framework for precise and accurate automated liquids exchange [ 15 ]. It was developed to enable automation of sample treatment and labelling of live or fixed specimens directly on the microscope stage [ 15 , 43 ]. The NanoJ-Fluidics hardware component is composed of customisable, low-cost and robust LEGO ® syringe pumps and a liquid removal peristaltic pump, all controlled by simple Arduino ® electronics.…”

Super‐beacons: Open‐source probes with spontaneous tuneable blinking compatible with live‐cell super‐resolution microscopy

“…These factors combined open the possibility of designing SBs with bespoke properties for specific applications (eg, imaging probes, as showcased here, or sensors) and/or environments (eg, different salt concentrations or temperatures). Some of these properties such as temperature or chemical environment can be changed dynamically at the microscope to further optimise their photoswitching behaviour while imaging. This flexibility suggests a model‐based design principle could be adopted to reach an optimal structure for a specific application in order to maximally make use of the rapid imaging technologies available.…”

Pinpointing Cell Identity in Time and Space

“…Many of these approaches benefit from specialized LabWare and equipment. There is an increasing ease of access to affordable liquid handling systems, driven by open wet-lab solutions such as OpenLH (Gome et al, 2019) and modular Lego-based and 3D-printed injection pumps (for example Almada et al, 2019). Such systems are essential for high-cycle sequential labeling of tens to hundreds of molecular targets, which can be labeled in a single sample without barcoding, as demonstrated with osmFISH (Codeluppi et al, 2018).…”