Fluidics widget user interface

Fluidics Control, Manual Control and Status panels

Startup:

1. Make sure fluidics system hardware is connected and powered on. Click “Initialize” to initialize hardware.

2. Click “Load Sequences” to load the csv file you need to run the experiment. For imaging we can just include the sequences for one round.

   An example:

   sequence_name	fluidic_port	flow_rate	volume	incubation_time	repeat	fill_tubing_with	include
   Flow SSC Buffer	25	500	2000	0	1	0	1
   Flow Probe	1	500	1500	15	1	26	1
   Flow Readout Buffer	26	500	2000	0	1	0	1
   Flow Imaging Buffer	27	500	2000	3	1	0	1
   Imaging
   Flow Stripping Buffer	28	500	2000	2	1	0	1
   Flow SSC Buffer	25	500	2000	0	1	0	1

   merfish-imaging-example.csv

   • We can do priming and cleaning up in the Fluidics Control panel, so we don’t need to include priming and cleaning up sequences in the CSV file.
   • The fluidic_port value in “Flow Probe” sequence can be any integer from 1 to 24. This value will be replaced with actual port value specified in “Fluidic Rounds” during imaging.
   • Add a line labeled "Imaging" in your CSV file to indicate where imaging should occur.

3. Introduction for control features

   • Priming and cleaning can be done in the widget
     • Ports: Specify which ports you want to prime or clean using this format: e.g., 1,2-10,12-14 If left blank, all available ports defined in config.json file will be used.
     • Fill tubing with: The last port of reagent that you want to use for filling the tubing to the flow cell. For example you may use wash buffer here when doing priming.
     • Volume: The volume for drawing liquid from “fill tubing with” port.
     • Repeat: Specifies how many times to run the wash cycle.
   • Manual control: flow liquid from a specified port with specified flow rate and volume

Priming:

1. Fill source containers with reagents needed
2. Remove the fitting from the syringe pump's extract port (port 2) and connect the inlet side of the flow cell connector to it
3. In “Prime Ports”, fill in ports needed to prime, and the last port and volume you want to use for filling the system. Click “Start” and wait for it to finish.

Load sample:

1. Mount the sample in flow cell as instructed by the flow cell manufacturer
2. Reattach the original fitting to the syringe pump extract port and securely connect the flow cell connector
3. Using “Manual Control”, manually flow the common buffer you used to prime the system through the flow cell with specified flow rate and volume. Fill the flow cell with this buffer, verify the connection and eliminate any bubbles.
4. Mount the flow cell onto the stage adapter and place it on the microscope.
5. Place the lid to block light, ensuring the tubings pass through the openings on its sides.

Imaging:

1. Use hardware trigger and “glass slide” sample format. Find interested area in 405 nm channel with lower magnification objective. Adjust the shape/size of the area to be imaged.

   

2. Switch to higher magnification (60x) in software and save coordinates in “Wellplate Multipoint” tab

   

3. Switch to higher magnification objective (e.g., 60x silicone) on the scope and apply silicone oil