✓Set up and prepare 3D hydrogel microenvironments for live-cell imaging
✓Perform FRET imaging on cells within hydrogels using epifluorescence microscopy
✓Calculate and analyze ratiometric FRET data with linear calibration
Protocol
Förster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.
Difficulty
advanced
Total time
~4–6 hours (hydrogel preparation ~1–2 hrs; imaging and analysis ~2–4 hrs per sample)
Model organism
Cell culture (mammalian cells; forskolin-treated cells shown in results)
Biosafety
BSL-1
Steps
1
Prepare three-dimensional hydrogel substrate
Synthesize and crosslink hydrogel material according to protocol specifications. This creates the physiologic 3D microenvironment for subsequent cell embedding.
▶ 00:47
2
Acquire FRET images in three-dimensional hydrogels
Perform epifluorescence microscopy on cells embedded in hydrogels using donor and acceptor fluorophore channels to capture real-time FRET signals.
▶ 02:30
3
Calculate FRET ratio with linear calibration
Process acquired images to compute ratiometric FRET values from donor/acceptor intensity pairs, applying analysis to ensure linear response across the activation range.
▶ 04:35
4
Validate FRET imaging with pharmacological treatment
Apply forskolin or comparable stimulus to cells and confirm FRET signal changes, demonstrating functional readout of cellular activation in 3D hydrogels.
▶ 07:11
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