研究目的
To monitor the dynamic reorganization of the collagen network during mechanical stretching in biological tissues, specifically in ex vivo murine skin dermis, using a fast polarization-resolved SHG microscope.
研究成果
The fast P-SHG microscope enables accurate monitoring and quantification of collagen reorganization in dynamic samples, showing a linear decrease in entropy with increasing stretch ratio. This provides a robust method for studying biomechanical properties at the microscopic scale, with potential applications in various tissues and processes.
研究不足
The acquisition time is relatively long (150 s for full images), which may not capture very fast tissue deformations. The method relies on sufficient SHG signal strength, which can be low in some tissues or depths due to scattering. Calibration is necessary due to polarization distortions from microscope components. Accuracy is affected by residual ellipticity and sample heterogeneities.
1：Experimental Design and Method Selection:
A custom-built upright laser scanning microscope is used for multiphoton imaging. Fast polarization-resolved SHG (P-SHG) is implemented with an electro-optical modulator (EOM) for line-to-line polarization switching synchronized with scanning. Calibration is performed to ensure accurate polarization states.
2：Sample Selection and Data Sources:
Ex vivo murine skin dermis from eight wild-type mice (129sv) is used. Samples are prepared by depilation, epidermis removal, and cutting into a dog-bone shape for uniaxial tensile loading.
3：List of Experimental Equipment and Materials:
Femtosecond laser (Mai-Tai, Spectra-Physics), water-immersion objective lens (20×,
4：95 NA, Olympus), photon-counting photomultiplier tubes (P25PC, Electron Tubes), EOM (M350-210-02, Conoptics), half-waveplate (ACWP-700-1000-06-2, CVI), quarter-waveplate (ACWP-700-1000-06-4, CVI), fast power supply (302A, Conoptics), custom-built uniaxial traction device, immersion gel (Lacrygel, Europhta). Experimental Procedures and Operational Workflow:
Skin samples are stretched at a slow strain rate (2 μm/s) while force is measured. P-SHG imaging is performed with 18 incident polarizations (0-170° every 10°) at each stretch ratio step (
5：05 increments). Image stacks (375 × 375 × 50 μm3) are acquired at 100 kHz pixel rate. Data Analysis Methods:
Collagen orientation is determined pixel-wise using FFT algorithms on P-SHG data. Entropy of orientation distribution is calculated and plotted against stretch ratio. Linear fitting is used to quantify reorganization. Morphological filtering is also applied for comparison.
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femtosecond laser
Mai-Tai
Spectra-Physics
Provides excitation for multiphoton imaging at 860 nm wavelength.
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objective lens
20×, 0.95 NA
Olympus
Focuses the laser beam onto the sample for imaging.
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photon-counting photomultiplier tube
P25PC
Electron Tubes
Detects multiphoton signals in epi-detection geometry.
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electro-optical modulator
M350-210-02
Conoptics
Achieves fast rotation of incident linear polarization for P-SHG imaging.
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half-waveplate
ACWP-700-1000-06-2
CVI
Sets incident polarization at 45° from EOM axis.
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quarter-waveplate
ACWP-700-1000-06-4
CVI
Compensates for ellipticity introduced by microscope components.
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fast power supply
302A
Conoptics
Powers the EOM for fast polarization switching.
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immersion gel
Lacrygel
Europhta
Ensures optical contact with the objective lens and prevents skin dehydration.
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