Mar 30, 2026
Nikon Automated Microscopy: Label-free timelapse imaging in well plates with Volume Contrast
- Akihiro Suto1
- 1Nikon Healthcare UK
- Nikon Healthcare UK, Branch of Nikon Europe
Protocol Citation: Akihiro Suto 2026. Nikon Automated Microscopy: Label-free timelapse imaging in well plates with Volume Contrast . protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9r28qv3e/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: December 10, 2024
Last Modified: March 30, 2026
Protocol Integer ID: 114670
Keywords: Live imaging, Wellplate imaging, Volume Contrast, Brightfield imaging, Light Microscopy, fluorescence microscopy techniques such as confocal imaging, nikon automated microscopy, visibility of cell boundary, brightfield imaging, fluorescence imaging, brightfield contrast technique, robust segmentation of cell boundary, nikon volume contrast, addition of any brightfield contrast technique, cell boundary, confocal imaging, fluorescence image quality, microscopy technique, brightfield image, segmentation of the cytoplasmic compartment, live imaging within monolayer, fluorescence, cell cytoplasm, cultured cell, extra fluorescence channel, cytoplasmic compartment, phase contrast, free timelapse imaging in well plate, contrast mechanism, combining brightfield, volume contrast, cell, wellplates with volume contrast, live imaging, observing cellular growth, brightfield, methods like differential interference contrast, differential interference contrast, timelapse imaging, membrane dye, workflow for timelapse imaging,
Disclaimer
Caution is advised when using the JOB, as stage movement is unsupervised. Setting z-drive limits is strongly recommended.
Abstract
Brightfield imaging is a label‑free and gentle method for observing cellular growth. However, robust segmentation of cell boundaries in brightfield images remains challenging, particularly when combining brightfield with high‑resolution fluorescence microscopy techniques such as confocal imaging. While methods like differential interference contrast (DIC) and phase contrast can improve visibility of cell boundaries, they introduce optical components and contrast mechanisms that can degrade fluorescence image quality, making them less suitable when high‑quality fluorescence imaging is required.
Nikon Volume Contrast allows the conversion of a small z-stack in brightfield into a fluorescence-like image which highlights the cell cytoplasm, without the addition of any brightfield contrast techniques. This allows for segmentation of the cytoplasmic compartment of a cell, and may be used to replace cytoplasmic or membrane dyes, thus freeing up an extra fluorescence channel.
This protocol incorporates a workflow for timelapse imaging in wellplates with volume contrast for live imaging within monolayer cultured cells.
Materials
Timelapse with Volume Contrast v3.bin7KB Above is the JOB file to be imported into NIS elements to run this protocol.
This protocol was developed on the following Nikon Microscope:
Nikon Eclipse Ji
Other materials necessary to conduct this protocol:
- Imaging grade well-plate (e.g. 96 well-plate)
Standard tissue culture wellplates will require long working distance objectives with working distance >2mm, typically 4x and 10x objectives
- PC running NIS elements (Version 5 or higher recommended).
- NIS elements JOBs module for automated imaging.
Troubleshooting
Position wellplate onto microscope stage
Import .bin file into the JOBs explorer (only first time)
Open JOBs Explorer, from View Menu (top of screen) > Analysis Controls > JOBs Explorer
Import .Bin file from this protocol - found in materials or below:
Timelapse with Volume Contrast v3.bin7KB For detailed instructions on how to import JOBs .bin files, refer to this protocol:
Protocol
CREATED BY
Mark Rigby
Detect plate in Sample Navigation
The sample navigation window is available on the left-hand side of the Compact layout
In the advanced layout, it can be found under:
View (top menu) > Acquisition controls > Sample Navigation in other layouts.
Different layouts are shown on the top left of the NIS-Elements window
Open up sample navigation
Sample Navigation Pane
Select Well plate holder, and the well plate checkbox.
Sample navigation plate selection
Select "Detect" in Sample Navigation Well Plate Tab
Label and select wells to image
Choose whether to label the wells of your wellplate
Wellplate labelling interface
Select the wells to image in the Sample Navigation Wellplate tab
In the sample navigation window, press scan.
Then, select Custom Job, and choose the previously imported JOB, and scan.
Check scanning settings
Select the correct Optical configuration for Brightfield
Check scanning settings
Select number of time points and intervals you wish to capture
Select the number of images per well you wish to capture.
Note that brightfield illumination can be variable towards the edges of wells, due to a meniscus of water distorting the brightfield light path. Due to this, it is recommended to limit the working area to the centre of the wells.
Run the JOB
Example Results
Expected result
Monolayer cells at 20x in brightfield
Volume contrast at 20x