Apr 28, 2026
Calcium Imaging of iNeurons and iDA Neurons Using Spinning Disk Confocal Microscopy
- Felix Kraus,
- Harper JW1
- 1harvard university
Protocol Citation: Felix Kraus, Harper JW 2026. Calcium Imaging of iNeurons and iDA Neurons Using Spinning Disk Confocal Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkzrzwl5r/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 15, 2026
Last Modified: April 28, 2026
Protocol Integer ID: 313301
Keywords: ASAPCRN, cell calcium imaging in induced neuron, calcium imaging of ineuron, cell calcium imaging, calcium imaging, using spinning disk confocal microscopy, disk confocal microscopy, spinning disk confocal microscopy this protocol, confocal microscopy, induced calcium dynamic, calcium dynamic, dopaminergic neuron, calcium indicator, neuron, fluorescence, ida neuron, induced neuron, ineuron, lapse imaging, cellposesam
Funders Acknowledgements:
Aligning science across Parkinson's
Grant ID: 024268, 025160
Abstract
This protocol describes live-cell calcium imaging in induced neurons (iNeurons) and induced dopaminergic neurons (iDA neurons) using the calcium indicator Fluo-4 and spinning disk confocal microscopy. Neurons differentiated in glass-bottom multiwell plates are loaded with Fluo-4 and imaged at physiological temperature and CO₂ conditions. Time-lapse imaging captures spontaneous and stimulus-induced calcium dynamics. Image sequences are subsequently processed using custom machine learning segmentation models implemented in CellposeSAM, followed by fluorescence trace extraction and network activity analysis in R.
Guidelines
- Maintain stable environmental conditions (37 °C, 5% CO₂) throughout imaging.
- Avoid photobleaching by minimizing laser exposure prior to recording.
- Perform all fluorescence handling under low-light conditions**.
- Maintain consistent imaging parameters across samples for quantitative comparison.
- Ensure medium changes are performed gently to avoid detaching neurons.
Materials
**Reagents**
- Fluo-4 calcium indicator dye
- PBS (pre-warmed)
- ND2 neuronal culture medium
- Potassium chloride (KCl)
- CNQX
- D-AP5
**Consumables**
- μ-Slide 8-well glass-bottom plates (ibidi, #80807)
**Equipment**
- Nikon Eclipse Ti2-E motorized microscope
- Yokogawa CSU-W1 spinning disk confocal system
- Nikon Plan Lambda 20× / 0.75 NA air objective
- Environmental chamber for 37 °C and 5% CO₂
- Hamamatsu ORCA-Fusion BT CMOS camera
- NIS-Elements acquisition software
- Image analysis workstation with Python and R installed
Safety warnings
⚠️ Fluo-4 dye may be harmful if inhaled or ingested. Handle with appropriate PPE.
⚠️ CNQX and D-AP5 are neuroactive compounds; avoid skin contact and inhalation.
⚠️ Dispose of chemical and biological waste according to institutional biosafety guidelines.
Before start
1. Differentiate iNeurons or iDA neurons to the desired time point in μ-Slide 8-well glass-bottom plates (ibidi, #80807).
2. Pre-warm:
- ND2 neuronal culture medium
- PBS
3. Prepare Fluo-4 dye solution according to manufacturer recommendations.
4. Prepare stimulation and inhibition solutions:
- 200 mM KCl
- 50 μM CNQX
- 20 μM D-AP5
5. Pre-equilibrate the microscope environmental chamber to 37 °C and 5% CO₂.
Before Start
Differentiate iNeurons or iDA neurons to the desired time point in μ-Slide 8-well glass-bottom plates (ibidi, #80807).
Pre-warm:
ND2 neuronal culture medium
PBS
Prepare Fluo-4 dye solution according to manufacturer recommendations.
Prepare stimulation and inhibition solutions:
200 mM KCl
50 μM CNQX
20 μM D-AP5
Pre-equilibrate the microscope environmental chamber to 37 °C and 5% CO₂.
Procedure
Differentiate iNeurons or iDA neurons in μ-Slide 8-well glass-bottom plates until the desired experimental time point.
Remove culture medium from cells.
Add Fluo-4 dye solution to each well.
Incubate cells for 30 minutes at 37 °C.
Remove the dye solution.
Wash cells gently with pre-warmed PBS_.
Add fresh ND2 medium to each well.
Place the imaging plate in the microscope environmental chamber.
Allow cells to equilibrate to 37 °C and 5% CO₂.
Select the 20× / 0.75 NA air objective.
Acquire time-lapse images using 2×2 pixel binning mode.
Imaging parameters:
Objective: 20× / 0.75 NA air
Pixel binning: 2×2
Image size: 785 × 570 pixels
Laser: 488 nm
Laser power: 5%
Exposure: 100 ms
Frame rate: 10 frames/s
Frames per recording: 1000
Record calcium dynamics under one of the following conditions:
Basal activity
Depolarization stimulus: 200 mM KCl
Synaptic inhibition: 50 μM CNQX + 20 μM D-AP5
Save image sequences in ND2 format for downstream analysis.
Data Analysis
Time-lapse images are analyzed using CellposeSAM with custom machine learning models.
Resources:
https://github.com/sauerkrausi/neuroLSD
https://doi.org/10.5281/zenodo.16733440
ND2 files are imported into Python.
Time-lapse stacks are segmented using custom-trained CellposeSAM models.
Segmentation masks identify regions of interest (ROIs) corresponding to individual neurons.
Fluorescence intensity is extracted for each ROI across all time points.
Signal Processing
Fluorescence traces are baseline corrected to generate ΔF/F signals.
Signals are binarized to detect calcium events.
Event detection enables quantification of:
Calcium spike frequency
Event amplitude
Event duration
Network Analysis
Batch processing enables extraction of per-cell calcium traces.
Pairwise correlations between neuronal activity traces are calculated.
Network-level properties such as synchrony and coordinated firing are quantified.
Troubleshooting
Weak fluorescence signal
Possible Cause: Insufficient dye loading
Solution: Increase Fluo-4 incubation time
High background fluorescence
Possible Cause: Residual dye
Solution: Perform additional PBS washes
Photobleaching
Possible Cause: Excessive laser power
Solution: Reduce laser intensity or exposure
Poor segmentation
Possible Cause: Low contrast or dense cultures
Solution: Retrain Cellpose model or adjust preprocessing
Acknowledgements
Data Analysis section references:
- https://github.com/sauerkrausi/neuroLSD
- https://doi.org/10.5281/zenodo.16733440
Analysis and visualization are performed using R.
Expected Results:
- Individual neurons should exhibit detectable Fluo-4 fluorescence fluctuations corresponding to intracellular calcium dynamics.
- KCl stimulation should induce strong synchronous calcium responses across neurons.
- CNQX + D-AP5 treatment should suppress excitatory network activity.
Typical outputs include:
- Per-cell calcium traces
- Calcium event detection metrics
- Network synchrony measurements
- Correlation matrices.
**Troubleshooting**
| Problem | Possible Cause | Solution |
|----------------------------|---------------------------------|-------------------------------------------|
| Weak fluorescence signal | Insufficient dye loading | Increase Fluo-4 incubation time |
| High background fluorescence | Residual dye | Perform additional PBS washes |
| Photobleaching | Excessive laser power | Reduce laser intensity or exposure |
| Poor segmentation | Low contrast or dense cultures | Retrain Cellpose model or adjust preprocessing |