Jones Lab:Protocols:Kup

Photek Luciferase imaging

To capture data

1) Focus camera in brightfield mode
I do this in the dark, with the door open. Remember to set gains high!

2) Click 'Run TSI' from Circadian toolbar

3) Change settings as appropriate- time is in seconds
Remember to save the TRI file, set ND filter to 100% (ie. no filter)

4) Once data collection is complete load TRI from menu
Click first file in sequence

5) Check overview of data using CRT
TRI -> Show Count Rate Trend

To create a sequence file

1) Click on 'Load frames' on Circadian toolbar

2) Choose first image in folder

3) Open into a buffer

4) Doubleclick to open buffer

To annotate image with OAPs

Right click to cancel at any time

1) Click 'Circle OAP'

2) Tap on image to add OAP

3) Resize to suit

4) Click to confirm size

5) Click to confirm position

6) Label

7) Copy using copy command

To extract data

1) Click 'Extract OAP' command on circadian toolbar

Make sure selection = all frames (eg. selection 1-48)

2) Export to Excel

Andor Luciferase imaging

To capture data

1) Load Micromanager
Select Camera + LEDs as configuration option

2) Set lights as required using pull down menu and dimmer switches

3) Turn off LEDs and room light
I do this in the dark, with the door open.

4) Click 'Live' on Micromanager window, and focus.

5) Stop live imaging

6) Open Script panel
Tools...>Script Panel

7) Select program you want to run
Red, Blue, Red+Blue or Dark

8) Hit 'Run'

9) Come back to save your file after data collection

Analysing Andor data

Use Maloof lab method [1]