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Detection of volume changes in calcein-stained cells using confocal microscopy
Allyson Fry Davidson1, Adam Z Higgins
1School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR 97331-2702, USA.
Journal of Fluorescence
|March 8, 2013
Summary
Calcein fluorescence response to cell volume changes depends on confocal microscopy settings. Adjusting the pinhole diameter alters optical section thickness, reversing fluorescence trends during cell shrinkage.
Area of Science:
- Cell biology
- Microscopy techniques
- Fluorescence imaging
Background:
- Calcein is a widely used intracellular fluorescent probe for monitoring cell volume.
- Previous studies reported conflicting fluorescence responses of calcein to cell shrinkage depending on microscopy setup.
- Wide-field microscopy typically shows decreased fluorescence, while confocal microscopy shows increased fluorescence upon shrinkage.
Purpose of the Study:
- To investigate the impact of optical setup on calcein fluorescence response to cell volume changes.
- To elucidate the role of optical section thickness in calcein-based cell volume measurements.
- To determine the specific confocal microscopy parameters influencing calcein fluorescence.
Main Methods:
- Utilized confocal microscopy to image calcein-stained endothelial cells.
- Systematically varied the confocal pinhole diameter to alter optical section thickness.
- Induced cell shrinkage and monitored changes in calcein fluorescence intensity.
- Correlated fluorescence response with specific pinhole diameters and corresponding optical section thicknesses.
Main Results:
- Confocal microscopy with large pinhole diameters (thicker optical sections) showed decreased calcein fluorescence upon cell shrinkage.
- Confocal microscopy with small pinhole diameters (thinner optical sections) demonstrated increased calcein fluorescence with cell shrinkage.
- A transition in fluorescence response was observed at a pinhole diameter of 285 μm, corresponding to an optical section thickness near the cell height.
Conclusions:
- The optical section thickness, controlled by the confocal pinhole size, critically influences calcein fluorescence response to cell volume changes.
- Experimental design and interpretation of calcein-based cell volume studies must account for confocal microscopy settings.
- Precise control of optical parameters is essential for accurate cell volume measurements using calcein.
