Localization microscopy and flow control for multiplexing 

The microscopy and imaging industry is continually evolving, and the improvement and development of techniques brings a whole scope of new applications in cell biology. For some of those applications, stable and reproducible flow control is required, and microfluidics is often the technology used as it allows for high-performance multiplexing. Learn about localization microscopy and how Fluigent’s microfluidic technology is used in the field, below.

What is single-molecule localization microscopy? 

Single-molecule localization microscopy (often called SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard microscopy techniques and can image biological structures at the molecular scale​1​.  

In single-molecule localization microscopy, individual fluorescent molecules are computationally localized, and the localizations are used to generate a super-resolution image. Sub-applications include oligo and refresh STORM, DNA PAINT, multiplexed antibody labeling, cell biology, neuroscience research, and others.  

Why is microfluidics used for SMLM? 

A typical single-molecule localization microscopy workflow includes fluorescent labeling, sample preparation, and image acquisition in fixed and live cells.  

Increase labeling targets 

Applications are developing in localization microscopy where the number of targets for imaging far outstrips the number of probes that can be chromatically separated. Sequential labeling with microfluidics allows for a much larger number of targets to be addressed.  

Reduce protocol failure and secure reproducibility 

In addition to being extremely time-consuming, manual injection or standard pipetting can harm biological samples for such applications: 

  • Disparate injections increase variability (5.7% intra-individual imprecision and 8.1% inter-individual imprecision for pipetting 10 µL) 
  • Turbulent flow can damage the sample 
  • Touching container sides can cause contamination 
  • Pipetting with an angle, failure to pre-wet tip, and tip wiping can increase chances of under-delivery and loss of sample. 

Microfluidics allows users to overcome the above limitations as it permits the delivery of highly controlled microliter flow rates (down to a few nL/min, with an accuracy < 5% m.v) in a fully automated manner.  

How Fluigent empowers localization microscopy 

Building a microfluidic multiplexing system for sequential labeling requires expertise in microfluidic fluid management, electronics, and software, which demand dedicated engineering resources, time, and money. For instance, for sequential injection, after evaluating the physics of flow control, users must gather fluid delivery components, microfluidic valves,electronics, and other equipment. In addition, software is required to automate the flow control for microscopy.  

Fluigent has developed custom, on-demand multiplexing systems and OEM Automation Workflows dedicated to localization microscopy and live cell imaging.  

The different functionalities developed that are related to our development are described below. They can be reused as bricks in a custom project and/or combined with our other modules. See why customers chose Fluigent for automating flow control for multiplexing  

Improved flow performance via Fluigent’s patented pressure-driven flow management 

Strong stability to ensure sample viability 

Our devices include Fluigent’s pressure-based flow control technology. Using pressure-driven flow control, the sample is smoothly injected into a microfluidic system. As there are no mechanical parts in contact with the fluids, pressure controllers can establish pulseless flows that cannot be obtained with peristaltic pumps, or even the most accurate syringe pump. Using Fluigent controllers, pressure stability with < 0.1% CV is obtained. This allows a new level of stability that is required for emerging applications.  The graphs below show perfusion comparisons between Fluigent controllers and peristaltic and syringe pumps. 

The user can choose the flow rate, the volume, and the time of delivery of each solution independently. Then, the system autonomously performs the protocol and can stay stable for long-term processes.  

Flow rate features dedicated to microscopy applications  

The addition of an on-off valve in the device guarantees that the flow is stopped when the user orders it to perform imaging. As illustrated by the adjacent graph, when using cut flow technology, the flow rate immediately stops when the desired volume is injected, preventing backflows.  

Preventing backflow during incubation steps enables the microfluidic sequential injection system to switch quickly between injection and incubation steps, which is advantageous for microscopy studies. Another valve at the output just before the chip or the chamber allows the flow to lead either towards the valve or towards the waste to flush the tubing between two successive injections. This valve is also useful for automated calibration and priming. 

High-multiplexing capabilities through in-house microfluidic valve integration & automation 

To offer multiplexing capabilities to our microscopy users, we developed automated sequential injection systems based on the integration and automation of several microfluidic valves (such as 11-port/10-way rotary valve, 3/2-way valves). They are accompanied by tailor-made electronics that ease connectivity and maintenance and mechanical integration that minimizes risks such as bubble formation and improves ergonomics. They are all controlled by our algorithm to work together with our flow control components in the most efficient and performant way and are integrated into a setup to obtain a fully automated system. 

With such systems, several buffer reservoirs can be swapped out during an experimental workflow, enabling the multiplexing of many labels in one experiment. This makes it optimal for localization microscopy applications. 

valves for lolcalization microscopy technology
integration of a distribution valve in a microfluidic sequential injection system
Figure 1: Example of integration of a distribution valve in a microfluidic sequential injection system. 

We have developed custom products that can manage up to 20 solutions but also developed systems that can be interfaced with 96 well-plates for even further multiplexing capabilities.  

Custom software and SDK for saving time, ease of use, and seamless OEM integration 

Fluigent has developed software functions dedicated to multiplexing applications that allow users to fully automate liquid handling sequences for microscopy use. The perfusion device can automatically perform any perfusion protocol, offering the following benefits: 

  • Ability to fit any experimental design for accurate and reproducible preparation of samples for fluorescence microscopy 
  • Intuitive, time-saving, and easy to use: iIndividual methods can be created, saved, and recalled quickly for a run 
  • Enables long-term perfusion studies such as multicolor PAINT imaging 

The features include: 

  • Flow management functions: volume injection, timed injection, reservoir selection, liquid flushing, etc. 
  • Sequencing functions: wait, wait for user, wait for TTL, Group, Loop 
  • Protocol loading & saving functions 
  • Data recording function 
  • TTL and TCP/IP functions 
  • Communication with external systems and wait for their signals as part of the protocols to perform other imaging processes while the perfusion protocol is paused 
  • Error functions (feedback on errors encountered during the protocol) 

The standard software version is included in our end-user automated sequential injection system, the Aria.  

In addition, an SDK library gathering all the above functions is available for users that wish to integrate our liquid handling functions into their own software for OEM automation workflow, allowing them to have a single interface to actuate all components that are part of their fluorescence microscopy system (fluid management unit, imaging unit, heating devices, incubator, etc.).  

Other custom functionalities can be added according to your needs and the modules included (temperature control, liquid mixing, flow sensors and more).

Other available features:  

  • Local control thanks to a control panel  
  • Night mode to allow its use in a dark room for fluorescent microscopy 
Custom software and SDK


​​Lelek, M. et al. Single-molecule localization microscopy. Nature Reviews Methods Primers vol. 1 Preprint at https://doi.org/10.1038/s43586-021-00038-x (2021).​ 

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