Apr 01, 2026
Nikon Automated Microcopy: Imaging Regions Across Multiple Coverslips
- Robert Tetley1,
- Alex Knowles1
- 1Nikon Healthcare UK
- Nikon Healthcare UK, Branch of Nikon Europe
Protocol Citation: Robert Tetley, Alex Knowles 2026. Nikon Automated Microcopy: Imaging Regions Across Multiple Coverslips. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4p648lo5/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: March 24, 2026
Last Modified: April 01, 2026
Protocol Integer ID: 313817
Keywords: Automated Microscopy, Nikon, nikon automated microcopy, imaging regions across multiple coverslip, imaging region, low magnification overview of each coverslip, images on region, multiple coverslip, microscope, automatic escaping between each coverslip, imaging protocol, higher magnification rescan, coverslip, low magnification overview, region, slide, image
Abstract
An imaging protocol to perform images on regions over multiple coverslips (slides, or wells), with automatic escaping between each coverslip (slide or well). User defined regions are manually drawn onto a low magnification overview of each coverslip (slide or well), after which I higher magnification rescan is performed over all regions. The microscope will automatically escape and refocus the objective when moving between each coverslip (slide or well).
Attachments
Materials
Equipment
ECLIPSE Ti2 inverted microscope
NAME
Microscope
TYPE
Nikon
BRAND
Ti2
SKU
LINK
Perfect Focus Unit
Imaging light source to match imaging modality (e.g. transmitted light source or LED fluorescence light source).
PC running NIS elements (Version 6 recommended).
NIS elements JOBs module for automated imaging.
Troubleshooting
Set up NIS-Elements Software
Power on the microscope and launch NIS Elements
Import the JOB from the .bin file '3 Slide Overview to High Mag Well Tags_Comments added' following the linked protocol
Protocol
CREATED BY
Mark Rigby
Running the JOB
Focus the microscope on the sample using the objective intended for low magnification scanning
Note: this protocol was optimised for a 4x magnification overview scan
In the JOBS Explorer click on the green play icon next to the JOB protocol to launch the JOB wizards
In the JOBs GUI firstly choose the channels with which you wish to image the low magnification overview
using the capture definition tab.
Next, click the 'Define Plate' Tab. This protocol was set up with a single slide and three coverslips. It is also possible to generate custom well plates to simulate any range of slides, and coverslips. Alternatively, it would be possible to use a standard well plate or dish by selecting this from 'Select from DB'. In all cases align using the 'Re-Align Plate Holder' button if not already aligned.
Using the select wells tab, select the coverslips (or wells if using a plate), that are to be imaged.
Using the select objectives tab, select the objectives for low magnification overview and high magnification rescanning
Define the settings for high magnification rescanning. Please use only the Z and Lambda Tabs.
The 'Z' tab allows a z-stack to be included and the 'Lambda' tab defines which channels will be used. A tick in the box next to the name indicates that these settings will be used.
Once every tab has been visited and checked, click 'Run' at the bottom right of the JOBS GUI to start the acquisition.
Run time control of the JOB
Now follow the instructions on the JOBs window, manually go live to confirm focus and then click 'OK'
Once the first coverslip has been scanned, a new window will appear of the stitched image. Please use draw regions function of the JOBs window to draw regions for high magnification rescanning onto the document.
Note: regions must be drawn onto the newly opened document behind the JOBs window.
Repeat step 12 for each coverslip (or well), following acquisition of the low magnification scans
Once the nosepiece has changed to the high magnification objective, follow the instructions on the JOBs window, manually go live to confirm focus and then click 'OK'.
Repeat step 14 for each coverslip (or well)
Wait for the JOB run to finish