Localization microscopy and flow control for multiplexing 

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.  

1- 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 their sequential labelling workflow. 

2- 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 run either towards it or towards the waste to flush the tubing between two successive injections. This valve is also useful for automated calibration and priming. 

3- 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. 

4- SDK and software 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, Aria. Aria software gathers all the above functions, and provides an easy-to-use interface, allowing users to add flow management functions to create a complete fluidic protocol, seamlessly.