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Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope
Published on: April 7, 2014
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Quantitative optical microscopy: measurement of cellular biophysical features with a standard optical microscope
Kevin G Phillips1, Sandra M Baker-Groberg2, Owen J T McCarty3
1Department of Biomedical Engineering, Oregon Health & Science University, School of Medicine; Department of Dermatology, Oregon Health & Science University, School of Medicine; phillkev@ohsu.edu.
Journal of Visualized Experiments : Jove
|April 22, 2014
Summary
Standard optical microscopes can now quantify cellular mass, volume, and density using noninterferometric quantitative phase microscopy (NIQPM) and Hilbert transform differential interference contrast (HTDIC) methods. These accessible techniques are ideal for fixed cellular specimens within specific size and temporal constraints.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Quantitative analysis of cellular properties like mass, volume, and density is crucial for understanding cell biology.
- Traditional methods often require specialized or complex instrumentation.
- There is a need for accessible, quantitative microscopy techniques for cellular analysis.
Purpose of the Study:
- To present two accessible optical microscopy methods for quantitative measurements of cellular mass, volume, and density.
- To detail the principles and applications of noninterferometric quantitative phase microscopy (NIQPM) and Hilbert transform differential interference contrast (HTDIC) microscopy.
- To highlight the advantages and limitations of these techniques for cellular specimen analysis.
Main Methods:
- Utilized a standard optical microscope with bright field and differential interference contrast (DIC) imaging.
- Employed noninterferometric quantitative phase microscopy (NIQPM) based on the paraxial approximation for mass and density measurements.
- Applied Hilbert transform differential interference contrast (HTDIC) microscopy with high numerical aperture (NA) illumination for volumetric measurements.
Main Results:
- NIQPM enables quantitative measurements of total cell mass and subcellular density distribution.
- HTDIC accurately determines cell volume from through-focus DIC image stacks.
- Both methods leverage accessible "off-the-shelf" microscopes, offering technological convenience.
- Limitations include slow z-stack acquisition (1 frame/minute) and size restrictions (0.2-10 μm for NIQPM, 0.2-20 μm for HTDIC).
Conclusions:
- NIQPM and HTDIC provide accessible quantitative biophysical measurements of cellular specimens using standard optical microscopes.
- These methods are well-suited for analyzing fixed cellular specimens that meet specific size and temporal requirements.
- The accessibility and quantitative nature of these techniques advance cellular analysis capabilities.
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