We are pleased to highlight the work of Stijn van Veen, Emiel W. A. Visser, and Lorenzo Albertazzi, who recently shared an innovative study on an automated DNA-PAINT workflow for nanoparticle characterization. Their publication presents a modern approach that makes super resolution imaging more accessible, more reproducible, and much easier to run at scale. It shows how researchers can move from slow, manual steps to a streamlined automated system where a pump, a fluidic setup, and smart software coordinate the full process.
The study explores what automated DNA-PAINT means in practice. DNA-PAINT is a fluorescence imaging technique that reaches very high localization precision by using transient binding between complementary DNA strands. These short binding events generate single molecule signals that can be accumulated over time, creating images with nanoscale resolution. The challenge is that this method often requires many imaging cycles, probe exchanges, and careful control of imager concentration. Automation solves most of these issues, enabling stable conditions, lower background noise, and a consistent workflow for everyone, including non expert users.
The authors demonstrate how simulations, fluidic control, and imaging analysis can be combined into a full multiplexed DNA-PAINT workflow. The result is an integrated tool that improves performance and reduces the manual burden usually associated with single molecule localization microscopy. This publication is an excellent example of how microfluidic control and precise liquid handling can support high resolution imaging in biology, nanoscience, and materials research.
Overview of the automated DNA-PAINT workflow. From van Veen et al, DOI: 10.1101/2025.08.05.668442.
The publication presents a complete and automated DNA-PAINT workflow designed to increase throughput and simplify super resolution imaging for nanoparticles. The authors start from a simple question: how can researchers obtain high quality single molecule localization microscopy data without spending hours on manual probe exchanges, buffer changes, and parameter tuning. Their answer is a combination of fluidic automation, simulation guided optimization, and an analysis pipeline that works as a single, connected system.
To understand the value of this work, it is useful to recall how DNA-PAINT functions. In this technique, very short transient binding events between complementary DNA strands create single fluorescent bursts. Each burst corresponds to a binding event between a mobile imager strand and a fixed docking strand. When these events are collected over time, the microscope reconstructs the precise positions of molecules with nanoscale localization precision. This is why DNA-PAINT is known as a super resolution imaging technique, because it relies on the accumulation of individual photon events rather than classical diffraction limited detection.
Principle of DNA-PAINT showing transient binding between imager and docking strands for super resolution imaging. From Yuanyuan Liu et al, In Situ Super Resolution Imaging of Telomeres with DNA-PAINT, DOI: 10.1021/acsomega.2c05752.
However, this impressive resolution comes with challenges. The density of these binding events depends strongly on the imager concentration, buffer conditions, and the number of targets on a particle. If the concentration is too high, events overlap and the image becomes difficult to interpret. If it is too low, the signal is weak and the analysis loses accuracy. In manual workflows, researchers test different concentrations one by one, which consumes time and reagents. The authors solve this with a simulation approach, generating synthetic time traces to predict the best conditions before starting the experiment. This step removes guesswork and ensures that the imaging runs under optimized parameters.
Once the imaging conditions are defined, the study introduces a fully automated flow system that controls washes, probe exchanges, and imaging cycles. The slide is connected to a multi inlet valve and a syringe pump, allowing different imager strands, buffers, and washing solutions to be introduced in a controlled sequence. Because the microscope and the pump communicate through software, the entire sequence runs without manual intervention. This automation also reduces errors linked to timing, flow speed, or incomplete washing, giving more consistent results across samples, users, and laboratories.
Example of multiplexed DNA-PAINT imaging with probe exchange. From van Veen et al, DOI: 10.1101/2025.08.05.668442.
The authors then extend the workflow to multiplexed DNA-PAINT imaging, where several targets are detected one after another using different DNA sequences. With automation, the system can exchange each imager strand, capture the required data, and return to the same field of view with high reproducibility. This capability is essential for applications that require simultaneous detection of multiple targets, such as molecular mapping, nanoparticle functionalization, or advanced biomedical research. It is the same principle used in many spatial biology techniques, where multiple markers are visualized within a single sample to understand complex biological structures.
After acquisition, the publication describes an automated analysis pipeline. It aligns the different color channels, performs drift correction, clusters the localizations, and extracts quantitative information such as the number of binding sites per particle. This step is crucial because DNA-PAINT does not only provide images, it offers molecular counting capabilities. This means that the workflow can estimate how many DNA strands are attached to each nanoparticle, revealing heterogeneity that cannot be detected with classical ensemble methods.
The publication concludes by showing the flexibility of this system, first with polystyrene nanoparticles and then with liposomes. While liposomes are more difficult to functionalize, the study highlights how DNA-PAINT can reveal differences in labeling efficiency, offering important insights for drug delivery and nanomedicine research. The authors also show that the method can easily be adapted to other structures, such as antibodies, chromatin features, or DNA origami, making the approach suitable for many nanoscale applications.
Overall, this work demonstrates how an automated DNA-PAINT technique can drastically improve efficiency, reduce manual workload, and increase reproducibility. It transforms a complex super resolution workflow into a streamlined, accessible tool for researchers who want precise, stable, and scalable single molecule imaging. This publication positions automation as a key factor for the future of high content imaging, especially as laboratories move toward integrated and self driven systems.
The publication brings together three complementary methods to create a full automated DNA-PAINT workflow. Each method solves a specific challenge linked to high resolution single molecule imaging, and together they form an efficient system that improves accuracy, stability, and reproducibility across the entire experiment. The authors show how simulation tools, automated liquid handling, and structured data processing can work as one integrated approach.
The first method is the use of simulation guided optimization to prepare the experiment. Because DNA-PAINT relies on transient binding between complementary DNA strands, the speed and density of these binding events strongly influence the final image. These events depend on the imager concentration, the number of docking sites, and the buffer conditions. Instead of testing each condition manually, the authors generate thousands of synthetic time traces that mimic the real fluorescence signals. These simulations predict the expected event density and the resulting localization precision, helping researchers select the right measurement time and concentration before they begin. This predictive step removes trial and error and ensures the experiment starts under optimized conditions.
Simulation guided optimization of DNA-PAINT parameters. From van Veen et al, DOI: 10.1101/2025.08.05.668442.
The second method is the automated fluidic setup, which is essential for performing long and multiplexed DNA-PAINT experiments. In the study, the imaging slide is connected to a multi inlet valve and a high precision syringe pump. They use AMF’s LSPone for this purpose, a compact and programmable microfluidic pump designed for precise low volume flow. The LSPone enables accurate control of flow speed, stable liquid displacement, and reliable switching between different solutions. These capabilities are crucial for DNA-PAINT, where each imager strand must be introduced and removed cleanly to avoid cross contamination between the different color channels. The LSPone drives the automated sequence of buffer washes, probe exchanges, and imaging cycles, allowing the workflow to run for hours without human intervention. By controlling the liquid handling with high precision, the pump ensures stable signal quality, minimal background, and consistent experimental conditions, which are key for super resolution imaging.
Automated fluid exchange enabling consistent multiplexed imaging. From van Veen et al, DOI: 10.1101/2025.08.05.668442.
This automated configuration also supports multiplexing, because the pump can move from one imager solution to another with exact timing. When combined with the microscope’s automated position control, the system successfully returns to the same field of view after each exchange. This precise coordination between fluidics and imaging is one of the strongest points of the method, because it ensures that all targets are recorded on the same particles across several imaging channels.
The third method is the automated analysis pipeline, which processes the large amount of data produced by the imaging steps. The authors combine drift correction, density filtering, and clustering algorithms to clean and organize the localization dataset. Fiducial markers are used for alignment, ensuring that the fluorescent signals remain accurately registered across the different multiplexed rounds. The pipeline then applies qPAINT to calculate the number of docking strands per particle, revealing how many targets are present and how they are distributed. This form of molecular counting is one of the main advantages of DNA-PAINT, because it provides information unavailable through classical bulk analysis. The analysis also extracts structural features such as particle size, shape, and heterogeneity, giving deeper insights into nanoparticle preparation and functionalization. Together, these three methods create a powerful automated DNA-PAINT technique. The combination of predictive simulations, precise liquid handling with the LSPone, and a structured data analysis workflow transforms DNA-PAINT into a stable, high performance imaging tool. The study shows that this approach can be applied to polystyrene nanoparticles, liposomes, and many other biological or synthetic systems that require accurate nanoscale characterization. It demonstrates how modern automation can enhance efficiency, improve reproducibility, and support advanced biomedical research.
A central part of this publication is the transition from a manual DNA-PAINT process to a fully automated workflow, and this automation relies directly on the fluidic hardware used in the setup. The authors integrate AMF’s LSPone, a high precision microfluidic syringe pump, as the core liquid handling component that manages buffer exchanges, imager strand introduction, and washing steps. Its accuracy, stability, and low internal volume make it an ideal match for DNA-PAINT, where every change in concentration or flow rate affects the final localization precision.
The LSPone is used to drive the entire fluidic sequence required for multiplexed DNA-PAINT. During imaging, the system must remove one imager solution, wash the sample, and introduce the next imager strand without disturbing the structure or the fluorescent signals. With manual handling, this process can easily introduce variability, leftover imager molecules, or mechanical shifts in the sample, which degrade resolution. The automated control provided by the pump solves this problem by delivering consistent flow speeds, precise volumes, and reliable timing. As a result, the particle remains perfectly positioned while the sample environment changes around it, preserving the stability needed for single molecule localization microscopy.
Another important contribution is the ability of the LSPone to manage several channels through a connected microfluidic valve.
This multi input setup allows researchers to store different imager solutions, PBS washes, or other buffers, then switch between them through software commands. Because the pump supports programmable actions, the full sequence runs seamlessly as a coordinated protocol: flow imager, acquire images, wash, switch channel, and repeat. For multiplexed DNA-PAINT, this automated switching is essential, since each target is identified by a specific DNA sequence, and the imaging cycles must be reproducible across all rounds.
The pump’s precision also reduces background noise by ensuring that imager strands are fully removed before the next cycle begins. This improves image quality, enhances molecular counting accuracy, and prevents fluorescent carryover between targets. In addition, its controlled low pressure operation helps preserve delicate biological samples such as liposomes, ensuring that the imaging setup can be used not only for synthetic nanoparticles but also for biological structures that are more sensitive to flow.
In this study, the integration of the LSPone highlights the role of advanced microfluidics in enabling high performance super resolution imaging. By automating sample handling, the pump supports long acquisition times, large datasets, and repeated imaging cycles without manual intervention, effectively transforming DNA-PAINT into a more scalable and accessible imaging technique. The authors show that this level of automation is a key requirement for future high content imaging systems, especially as laboratories move toward integrated, self driven, and machine guided workflows.
The LSPone is AMF’s compact and programmable microfluidic syringe pump designed for precise, stable, and repeatable liquid handling at the microliter scale. It is built for applications that require tight control over flow speed, smooth delivery, and minimal internal volume, making it an ideal fit for single molecule imaging, nanofluidic experiments, and long automated workflows such as DNA-PAINT. Its mechanical design reduces dead volume and ensures accurate fluid displacement, which is essential when working with fluorescent probes and sensitive DNA based imaging techniques. The pump delivers precise control over liquid movement, enabling stable sample conditions, optimized imager exchange, and consistent buffer handling, all of which directly improve image quality and reproducibility.
In addition to its fluidic performance, the LSPone features a simple and robust communication interface that makes automation easy to implement. The device can be controlled through standard ASCII based serial commands, among other available communication protocols, making it easy to connect with custom scripts, third party software, or automated microscopy platforms. In the publication, the authors used a MATLAB based interface to send commands to the pump and coordinate its actions with the microscope. This setup allowed the imaging protocol to run smoothly: the pump received instructions to switch channels, withdraw or dispense solutions, and follow precise timing steps, while the microscope captured data at the correct moments. This form of software integration is one of the key advantages of the LSPone, because it allows laboratories to combine fluidics, imaging, and analysis into a single automated system without requiring complex coding or proprietary environments.
By combining reliable mechanics with a flexible control interface, the LSPone provides a strong foundation for advanced imaging setups. It supports demanding applications where precise flow, minimal noise, and repeatable conditions are required, such as multiplexed DNA-PAINT, qPAINT analysis, or nanoscale mapping of biological structures. This balance of performance and simplicity is what makes the LSPone a powerful tool for researchers working in imaging, diagnostics, nanotechnology, or any field where high resolution and controlled liquid handling are essential.
Alongside the LSPone, AMF has recently introduced the LSPone HD, our new high definition version designed for even greater precision and smoother control of low volume flows. The HD model brings improved mechanics, higher resolution positioning of the plunger, and enhanced stability for demanding single molecule and super resolution imaging workflows. It is built for laboratories that need tighter control, better repeatability, and a more refined fluid handling experience in automated setups such as multiplexed DNA-PAINT. The LSPone HD keeps the same compact footprint and easy communication interface while delivering a new level of performance for advanced microfluidic applications.
This work offers a detailed view of how simulation, automation, and advanced analysis can transform DNA-PAINT into a practical and scalable imaging approach. If you would like to explore the full methodology, the experimental details, and the results presented by the authors, you can consult the complete publication. It includes all data, figures, code references, and extended explanations on the workflow, the imaging setup, and the analysis pipeline.
If you have questions about how AMF’s microfluidic components, including the LSPone and the new LSPone HD, can support DNA-PAINT, SMLM, or other precision imaging workflows, our team is available to help.
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