Mar 16, 2026
Nikon Automated Microscopy: High throughput screening of uneven multiwell plates with variable cell densities
- Alex Knowles1,
- Jamie A Adams2
- 1Nikon Healthcare UK;
- 2University of Sheffield
- Nikon Healthcare UK, Branch of Nikon Europe
Protocol Citation: Alex Knowles, Jamie A Adams 2026. Nikon Automated Microscopy: High throughput screening of uneven multiwell plates with variable cell densities. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g79e53vwz/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: May 29, 2025
Last Modified: March 16, 2026
Protocol Integer ID: 219107
Keywords: nikon automated microscopy, variable cell densities an imaging pipeline, screening of uneven multiwell plate, imaging pipeline, uneven multiwell plate, cell culture, autofocus step for each well, variable cell density, well plate, cell, uneven architecture of the plate, tiled image, plate, screening
Abstract
An imaging pipeline to screen cell cultures in a 384 well plates that suffer from uneven architecture of the plate, and variable cell densities, using NIS-Elements software and the Jobs acquisition module. User defined wells are visited, and select channels to image with. Tiled images are acquired of each well, with an autofocus step for each well.
Attachments
Materials
Hardware:
- Nikon Ti2-E motorised inverted microscope
Equipment
pE-800
NAME
Fluorescence LightSource
TYPE
CoolLED
BRAND
https://www.coolled.com/products/pe-800/
SKU
Equipment
Kinetix
NAME
sCMOS Monochrome Camera
TYPE
Photometrics
BRAND
http://www.photomet.com.cn/Kinetix_sCMOS.html
SKU
Software:
- Nikon NIS-Elements 6.10.02
- Jobs smart acquisition module
- GA3 analysis module
Troubleshooting
Set up NIS-Elements Software
Power on the microscope and launch NIS Elements
Import the JOB from the .bin 'Image_Whole_Plate_V1.4' file following the linked protocol
Protocol
CREATED BY
Mark Rigby
Running the JOB
Focus the microscope on the cells in a well using the objective intended for imaging
Note: This routine was optimised with a 10x Plan Apochromat Lambda D objective and the 1.5x tube lens o
In the JOBS Explorer right click on the green play icon next to the JOB protocol to launch the JOB wizards.
In the JOBs GUI firstly choose the channels which you wish to acquire using the capture definition tab.
Note: this requires existing optical configurations or experimental set ups.
Define the plate you are using by clicking the 'Define Plate' Tab. Plates can be selected from a preexisting database or a custom plate can be added.
If the plate has not already been aligned it possible to align the plate from this point using the 'Re-Align Plate Holder' button. Then follow the instructions to manually align the plate.
Note: Once a plate has been aligned for the holder the alignment should hold for multiple plates as long as the holder is not removed
Select the wells which you would like to image by clicking the 'Select Wells' tab and choose the wells by dragging the selection to cover those you wish to image
Define how often the short autofocus will be performed during the tiled image acquisition of the well
plate.
On the bottom right of JOBs GUI click the 'Run' button with green play icon