Automated Sequential Injection System

To save time and minimize reagents consumption, Aria loads its internal and external tubing with the successive solutions to inject in the chip.

Working principle:

If the protocol commands to inject 40 µL of solution A, incubate for 1 hour, inject 50 µL of solution B, incubate for 1 hour then wash with buffer for 5 minutes and the total volume of tubing ( internal + external) is of 300 µL Aria will prefill tubing as follows:

As a consequence, calibration of the tubing length and related volume is necessary.

Here is a description of all the functions available to optimize protocol writing in Aria

Calibration

Calibration is automated and the software assists the user to determine the total volume of his step up (internal volume + L1+L2). Calibration values are recorded for future experiments.

Prefill

Initially all tubing inside Aria are dry. The “Prefill” function is recommended as a first step to load all solutions inside Aria without injecting air in the chip or chamber. This function can be deactivated if the user performed the loading manually. Fluigent strongly recommends using this function.

Perfusion/injection

Perfusion can be set in terms of volume (ex: step 1) or duration (ex: step 2) of injection.

The user selects the reservoir of the solution to inject (ex: reservoir 1), sets the flow rate (ex: 100 µL/min) at which the solution will enter the flow cell and the volume (ex: 100 µL) or duration (ex: 1min) of injection. The flow rate  range is from 40 µL/min to 1 mL/min. The software automatically calculates the time at which the solution should enter the flow cell.

Incubation/wait

Incubation time can be easily set by entering the duration of the incubation (ex: 1h30) in the ‘wait’ function.

The ‘wait for user’ function is a variation of the ‘wait’ function. It is particularly useful if the user has to perform a manual operation like preparing cells before injection. The system waits until the user notifies it can proceed to the next step. In absence of notification by the user, the system proceeds to the next steps after 12 h.

Clear tubing

To prevent contamination between successive injections, the tubing can be cleared with buffer using ‘Flush tubing’ function. The user selects the reservoir containing the wash solution (ex: reservoir 10) and the flow rate at which the tubing is flushed (ex: 100 µL/min).

Aria pushes all the residual fluids contained in the L1 tubing to waste, and fills the L1 with wash solution. Buffer contained in L1 will also be directed to the waste. This operation does not involve flow to the  chip as neither residual fluids nor buffer enter the chip. However, the residual fluid contained in L2 will not be cleared. For this reason, Fluigent  recommend the user to keep L2 as short as possible.

External synchronization

Aria is equipped with TTL signalling and can send and receive external TTL signals.

• Sending TTL

Each step is flanked by two bells () : at the front and back of the settings (see below for ‘volume injection step).

By clicking on each of both of the bells they get activated (). Aria will then send a ttl signal either when the step starts (example below) or ends or both.

Fluigent recommends using this function to synchronize perfusion and imaging.

• Receiving TTL

The function ‘wait for TTL’ puts Aria on hold until it gets an external TTL signalling to proceed with the next steps of the protocol. If the Aria does not get any signalling it automatically proceeds with the next steps after 12h.

Minimum volume reservoir

Aria is designed to minimize reagent consumption. A small additional volume of liquid (36µL) is necessary to preload the system and to ensure that some residual liquid is still present in reservoir after injection to prevent delivery of air to the system.

As the user writes his protocol, the software calculates the minimum volume that should be placed in each reservoir to successfully run the protocol.

Write, load, save sequence

To design a new sequence, click on .

Before running a sequence, the software will automatically ask the user to save it. All saved sequences are accessible and can be loaded by clicking on .

Sequences can be saved at any time by the user by clicking on the saved data .

Recorded data

For each experiment, Aria automatically records and saves the flow rate, pressure, the reservoir from which the solution is withdrawn if solution is delivered to the chip or waste. All data is accessible by clicking on .