How do you synchronize a microscope with a microfluidic perfusion system?

To create a solution that enables synchronization of a microscope with a microfluidic perfusion system, Elveflow joined forces with the Biozentrum Imaging core facility at the University of Basel and Olympus France.

Introduction | When microscopy meets microfluidics

Collaborating to create adaptable, integrated imaging solutions

To keep pace with the current and future needs of researchers, imaging core facilities worldwide are looking to increase automation and device inter-connectivity – ensuring that investigators can capture an ever-wider range of dynamic cellular processes.

To facilitate the transition to more advanced imaging systems, equipment suppliers are engaging in collaboration – working with customers and other suppliers alike to develop novel solutions and systems that meet the changing needs of researchers.

It is in this spirit of collaboration that Dr. Kai Schleicher, Advanced Microscopy Specialist at the Biozentrum Imaging Core facility at the University of Basel, Dr. Sebastien Peter, Sales Specialist at Olympus, and Elveflow – a microfluidic instruments specialist, came together to create a solution that enables coordination of confocal imaging and microfluidic flow control using cellSens imaging software from Olympus. As well as providing the capability for imaging cells in perfusion conditions, this system supports rapid and precise liquid handling – a crucial benefit for experiments requiring drug treatments or buffer changes.

Presentation

This application note describes how to combine and synchronise liquid perfusion and imaging using an Olympus spinning disc confocal microscope together with an Elveflow pressure-driven flow controlled microfluidic system.

The initial motivation of such a system comes from the need of researchers from various fields of research (Biology and Chemistry) to synchronize liquid handling and imaging during their experiment.

For biologists, this translates into being able to flush through a perfusion chamber containing cells of various media and coordinate the microscopic acquisition with the Olympus microscope.

The development of such microscopic and flow controlled setup will allow researchers to have a full and simultaneous control over their imaging acquisition and their experiment. This platform was designed in an imaging core facility where many users with various backgrounds share instruments to perform their research. Most users using the facility do not have the time to develop such engineered platforms to carry out their experiments. Therefore, the second challenge of this collaboration was to make this dual setup accessible, flexible, reliable and easy to use to ensure highly reproducible and versatile experiments.

Applications

The detailed experimental protocol presented here can be used for  applications such as:

• Dynamic cell culture and perfusion experiment that require a continuous supply of liquid and steady flow rate during perfusion.
• Long term perfusion experiment
• Multiple media injection for dynamic cell culture at various inlets
• Several buffers injected with different pressures to regulate mixing
• Drug screening
• Antibiotic resistance – mother Machine

Setup

Working principle of the dual setup: When microscopy meets microfluidics

Elveflow OB1 pressure-driven flow controller was selected among other flow control systems available on the market because it can be connected via its control box to the RTC (Real Time Controller) Olympus microscope real-time controller via BNC cable (miniature quick connect/disconnect radio frequency connector used for coaxial cable) connexion and TTL (Transistor to Transistor Logic, type of serial interface) ports to send trigger signals.

Figure 3: Picture of the TTL ports from the Elveflow OB1 pressure-driven flow controller control box (right) and the RTC Olympus microscope real-time controller connected via BNC cables (left).

Practically speaking, the OB1 flow controller “sends” or “listens” to the signal emitted or received via the trigger port and acts accordingly based on the trigger sent by the microscope acquisition software.

The set of action triggered by the reception of a signal was defined prealably on the OB1 ESI software with the sequence scheduler. A set of actions also called “sequence” can be easily defined and saved in a template on the ESI software. In this specific note, three templates developed by Dr. Kai Schleicher and Dr. Sebastien Peter, will be introduced.

In a few numbers:

• Triggering works by sending and receiving “High” and “Low” voltages between 0 and 5 V

High and Low states on the Olympus SpinSR (when sending a signal)

• Olympus real time controller: High = 0 V (Default)

• Olympus real time controller: Low = 5 V

High and Low states on the Elveflow controller EXT

• Elveflow controller OB1 EXT TRIG: High = 3.4 V

• Elveflow controller OB1 EXT TRIG: Low = 0 V (Default)

TIP: The Voltage set to define the High and Low signal level is device (microscope) dependent. Thus, it is recommended to check initially with a Voltmeter the the respective “High” and “Low” voltages

When sending a signal, always use a pulse so that the device goes back into its default state, e.g.: from Cellsens to Elveflow INT: In CellSens, use digital port low + wait 100 ms + digital port high. From Elveflow EXT to CellSens: use TRIG: High pulse (100 ms)

Time for setup completion: 0,5 day

Materials

Hardware

1. OB1 flow controller with two channels 0/2000 mbar

2. 1 x Kit starter pack Luer Lock

3. Reservoirs

4. Microfluidic chip

5. The Olympus IXplore SpinSR Spinning Disc Confocal Microscope and Real Time Controller (RTCE) and the Olympus Cellsens software.

6. Fast camera to register (optional)

7. Male-to-Male BNC cables

Note: the Olympus real time controller can send as well as receive a signal on the same port.

Optional: Voltmeter with BNC female adapter (e.g. Digital Multimeter from CEN-TECH)

Figure 4: Picture of the Elveflow OB1 pressure-driven flow controller (Left) and a reservoir connected to the OB1 with a tubing containing the solution to inject through the microfluidic chip.

How to trigger an Elveflow pressure-driven flow controller with the CellSens microscope software?

Quick start guide

Instrument connection

1. Connect your OB1 pressure controller to an external pressure supply using pneumatic tubing, and to a computer using USB cable. For detailed instructions on OB1 pressure controller setup, please read the “OB1 User Guide”. Short end of the black tubing goes into the “pressure inlet” in the OB1 controller, the long end into the “pressure out” of the air-compressor. Plug in Power supplies for the OB1 controller and the air-compressor
2. BNC INT: receives signals connect to an Olympus triggerbox (=RTC) digital I/O port, e.g. 1 (you can choose any of the 4 available ports)
3. BNC EXT: sends signals connect to an Olympus triggerbox digital I/O port, e.g. 2
4. Press Add instrument \ choose OB1 \ set as MK3+, set pressure channels if needed, give a name to the instrument and press OK to save changes. Your OB1 should now be in the list of recognized devices.
5. OB1 calibration is required for the first use. Please refer to the “OB1 User Guide”.
6. Connect USB to the microscope control computer
7. Start Microscope, computer and CellSens
8. Launch the Elveflow software. The Elveflow Smart Interface’s main features and options are covered in the “ESI User Guide”. Please refer to those guides for a detailed description.
9. Turn on the Elveflow OB1 pressure-driven flow controller by pressing the power switch.
10. Open the OB1 Window. In the Elveflow software, load the sequence of your choice (see below).
11. In CellSens, load the corresponding experimental design (see below)
12. Choose a template (Elveflow sequence + Cellsens experiment design)
13. To run an experiment:First, start the Elveflow sequence. Second, start the acquisition in CellSens.

Template definition

First, this section will take you through the steps required to create a template containing a sum of actions forming a sequence that will perform the ESI software automatically.

As an example, if you wish to inject sequentially and automatically three different solutions into a microfluidic chip and you wish to inject the solutions/media/reagents at different pressure or flow rate as described below:

• Channel 1: pressure 40 mbar for 1 hour

• Channel 2: pressure 1000 mbar for 5 minutes (to replace the previous medium as fast as possible)

• Channel 2: pressure 40 mbar for 1 hour

• Channel 3: pressure 1000 mbar for 5 minutes (to replace the previous medium as fast as possible)

• Channel 3: pressure 40 mbar for 1 hour

To translate the following actions and to create and automate a sequence, the following steps are required:

1. Start the ESI Software

2. Open the Sequencer panel

   

    

3.  You can either create a sequence from scratch with the set of tools on the left panel or load a template describing a sequence prealably created.
   

   

    

4. To create a sequence from scratch, You can either define the set of actions you wish to connect in “live” directly via the OB1 main panel.
   

   

    

   1. The first option is to use the left panel to define the configuration directly by clicking on the “OB1” and defining the channel and the desired pressure and save it accordingly as shown in Figure 7.

   2. The second option is create the set of actions “live”: On the main OB1 panel, you can edit the OB1 conditions of pressure for instance (40 mbar steady pressure as shown in Figure 8) and save it by clicking on “Config” on the left top corner of the window and “Save As”. Use a clear name for the file (exp: 40 mbar). Similarly, for the second pressure (1000 mbar) required in the sequence and save it as “1000 mbar”.
      

      

       

   3. To connect the various steps saved prealably, use the left panel as shown in Figure 7 to define the time for each step, the selected instrument in the step, to set the switch between the valves and the related solutions…
      You can keep adding steps according to the strategy you defined for your experiment. In this example, the complete sequence should look like the following Figure 9:

      

       

   4. To end your set of actions, it is recommended to add configuration with the OB1 pressure set at 0 mbar to easily end your experiments in addition to an “END” step as shown in Figure 9.
   5. Once you’re satisfied with the sequence, make sure you saved your sequence properly with the “Save” button. Please note that the sequence you have created is instrument dependent, which means, you cannot reuse or load the same sequence on a different OB1 instrument. You will have to recreate it from scratch on another instrument.
   6. To test the sequence you have created and saved, you can use the button “Play” to check that your sequence is defined correctly.
   7. To run your sequence, load the template previously saved.

Template selection

For this specific collaboration, three templates were defined based on the Elveflow Sequencer and the CellSens experimental design:

Template 1: Cellsens Elveflow (Once, e.g START)

• Olympus real time controller trigger state is High per default

• Elveflow listens indefinitely every 10 ms to its trigger INT as long as its High

• When Cellsens sends a “low” pulse, the Elveflow controller performs the following sequence as described in the following screenshot.

1/ Starts a user set profile immediately (e.g. channel 1, 500 mbar)

2/ Runs the profile for a user set period of time,

3/ Loads a profile which closes all channels on the pump system

4/ Ends

Template 2: CellSens & Elveflow (twice, e.g START/STOP)

• Olympus real time controller trigger state is High per default

• Elveflow listens indefinitely every 10 ms to its trigger INT as long as its High

• When CellSens sends a “low” pulse, the Elveflow controller starts a user set profile immediately (e.g. channel 1, 100 mbar), then listens indefinitely every 10 ms to its trigger INT as long as its High

• When Cellsens sends “low” again, the Elveflow controller loads a profile which closes all channels on the pump system, then ends

Template 3: Elveflow & CellSens (e.g START/FEEDBACK)

• Olympus real time controller trigger state is High per default

• Elveflow listens indefinitely every 10 ms to its trigger INT as long as its High

• When Cellsens sends a “low” pulse, the Elveflow controller starts a user set profile immediately (e.g. channel 1, 500 mbar), then runs the profile for a user set period of time, then loads a profile which closes all channels on the pump system, sends a “High” pulse (100 ms) via its EXT TRIG port to Cellsens, then ends

  

   

Note: Elveflow controller OB1 EXT TRIG is Low (0 V) by default

Acknowledgments

We would like to thank Dr. Kai Schleicher, Advanced Microscopy Specialist at the Biozentrum Imaging Core facility at the University of Basel, Dr. Sebastien Peter, Sales Specialist at Olympus and Simon Van Vliet and Hector Arturo Hernandez Gonzalez from the Jenal lab at the Biozentrum for kindly providing us with materials.

Article also published on olympus-lifescience.com