May 19, 2026
Nikon Automated Microscopy: Calcium Imaging of cultured cells with a single wavelength calcium indicator (e.g. Fluo-4)
- 1Nikon
Protocol Citation: Akihiro Suto 2026. Nikon Automated Microscopy: Calcium Imaging of cultured cells with a single wavelength calcium indicator (e.g. Fluo-4). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxqd7ov8j/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: In development
We are still testing and developing this protocol.
Created: May 29, 2025
Last Modified: May 19, 2026
Protocol Integer ID: 219108
Keywords: Nikon, Ti2, Widefield, Calcium Imaging, Chamber Slide, calcium imaging of cultured cell, calcium imaging, field microscopy, calcium indicator dye, single wavelength calcium indicator, cultured cell, cardiomyocyte
Abstract
This JOB was created in NIS-Elements version 6.10.01, and is designed to image live monolayer-cultured cells (e.g. Cardiomyocytes) stained with a calcium indicator dye such as Fluo-4 using a wide-field microscopy.
The JOB will take multiple timelapse recordings at high framerate from different positions within the well.
Materials
This protocol requires a camera capable of high framerate acquisition, and a frame out timing signal (e.g. Hamamatsu Flash 4.0 camera)
This protocol requires a TTL IO output from the camera to a NIDAQ breakout box, connected to a PCIE card.
These are necessary to allow the precise frame timings from the camera to be saved.
Troubleshooting
Preparation of Microscope
Import JOB definition - as below
Multipoint Calcium Imaging.bin11.7KB Protocol
CREATED BY
Mark Rigby
Preheat the stage/cage incubator to 37°C 1 - 2 hours ahead of imaging
Flush the incubation chamber with CO2 10-20 minutes ahead of imaging.
Ensure the correct holder is used for the incubation chamber, and that there are no large openings for CO2 to escape from the chamber
Imaging: Setting appropriate acquisition parameters
Place chamber slide on microscope stage, select an objective for imaging at the required magnification.
Typically a 20x Air objective will give a sweet spot for magnification and brightness. Immersion media objectives are desirable for greater than 20x magnification, however an objective heater may be necessary to maintain temperature of cells.
Navigate to the centre of your well and focus on your sample
Optional: Search for the perfect focus system (PFS) offset and activate PFS
This will stabilise the microscope against thermal fluctuations, and maintain focus across the sample.
Set acquisition parameters for excitation light intensity
Recommended at least 100FPS for Calcium imaging in cardiomyocytes, an exposure of 10ms or less is required.
Crop the camera sensor to a smaller region of interest (ROI) to achieve higher framerates.
Cropping height with increase framerate, cropping in width will not increase framerate.
The smaller the ROI, the faster the framerate (FPS seen during the live-view in the bottom left corner)
Acquisition menu
Automated acquisition in multiple points
Run JOB File
Set which channel you want to image in
E.g. For Fluo-4, you would choose the Green channel on your Nikon microscope
Set the number of timelapse recordings to capture according to experimental requirements / size of well
Set the duration of each individual recording
Press Run
Ensure the sample is still in focus, then press done to start acquisition.