Apr 03, 2025
Multiple Colour Combinations Well-plate Imaging
- Joe Airey1
- 1Nikon healthcare UK
- Nikon Healthcare UK, Branch of Nikon Europe
Protocol Citation: Joe Airey 2025. Multiple Colour Combinations Well-plate Imaging . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz684xgx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 03, 2025
Last Modified: April 03, 2025
Protocol Integer ID: 126102
Keywords: imaging different color combination, different color combinations in different well, multiple colour combinations well, multiple imaging channel, plate imaging, nikon ti2, imaging, focus, further color, well plate, different well
Abstract
The following JOB will guide the user through how to align a well-plate on a Nikon Ti2, find the focus and then setup up multiple imaging channels for imaging different color combinations in different wells on a well-plate.
The JOB can be easily expanded to add further colors and is annotated within to describe how this can be done.
Materials
Diff_Colour_Rows.bin25KB The file above is the .bin file containing the JOB.
Nikon Ti2-E microscope
Imaging well-plate (e.g. 96 well-plate)
Fluorescent sample
Imaging light source with multiple colours (e.g. laser bed or LED light source).
PC running NIS elements (NIS5 or 6 recommended).
NIS elements JOBs module for automated imaging.
Troubleshooting
Start-up
Power the microscope and open NIS elements software
Place the well-plate onto the stage holder, select an objective and focus on the first well to be image with the required magnification (e.g. 4x objective).
Note: for scanning large numbers of wells an air objective or water objective with water immersion dispenser is advised.
Running the JOB
open 'JOBS' explorer, click 'new' and then 'import'. Import the JOB (in material section of this protocol) from wherever you have saved the .bin file.
Run the JOB by clicking the 'play' icon next to the JOB in the JOB explorer window. This will open the JOB GUI and guide you through setting up the colors and rows to be imaged.
Select the color combinations required for each row of the well-plate by selecting combinations of optical configurations. Note that the first capture definition (contain multiple or single OCs) will be used on the first row and so on.
Define the well-plate being used by selecting from the database in NIS and align the well-plate by following the guidance in the alignment GUI.
Select the rows or columns of wells to be imaged with each successive capture definition.
Click 'run'
Note by clicking 'edit' on the JOB prior to running, annotations on what each step contains and how these can be duplicated to add >3 color combination and >3 rows / columns can be found: