Label-Free Multiphoton Microscopy: Much More Than Fancy Images
Abstract
:1. Introduction
2. Multiphoton Technique
3. Label-Free Multiphoton Microscopy in Biomedical Research
3.1. Autofluorescence
3.2. Second Harmonic Generation
3.3. Third Harmonic Generation
3.4. Research and Clinical Applications
4. Sample Preparation
4.1. Fixation
4.2. Sectioning
4.3. Mounting
5. Quantitative SHG Image Methods
5.1. Amount and Texture Description
5.1.1. Intensity-Based Analysis
- →
- First-Order Statistics (FOS)
- →
- Second-Order Statistics (Gray Level Co-Occurrence Matrix, GLCM)
5.1.2. Transform-Based Methods
- →
- 2D Fast Fourier Transformation (2D-FFT)
- →
- Wavelet Transformation
5.2. Fibers Orientation
- →
- Forward–Backward SHG-Signal (F-SHG/B-SHG)
- →
- Polarization
- →
- Coherency (C)
5.3. Fibers Waviness
5.4. Fiber Thickness and Distance
6. Limitations and New Perspectives
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
STED | Stimulated Emission Depletion |
STORM | Stochastic Optical Reconstruction Microscopy |
PALM | Photo Activated Localization Microscopy |
NADH | 1,4-DiHydroNicotinamide Adenine Dinucleotide |
SHG | Second Harmonic Generation |
THG | Third Harmonic Generation |
CARS | Coherent Anti-Stokes Raman Spectroscopy |
SRS | Stimulated Raman Spectroscopy |
MPM | Multiphoton Microscopy |
TPE | Two-Photon Excitation |
OPO | Optical Parametric Oscillator |
MPF | Multiphoton Fluorescence |
LFM | Label-Free Multiphoton |
YFP | Yellow Fluorescent Protein |
FAD | Flavin Adenine Dinucleotide |
FA | Formaldehyde |
PFA | Paraformaldehyde |
FOS | First-Order Statistics |
GLCM | Gray Level Co-Occurrence Matrix |
FFT | Fast Fourier Transformation |
IDM | Inverse Difference Moment |
iSCAT | Interferometric Scattering |
PSHG | Polarization-Sensitive Second Harmonic Generation |
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Biomedical Field | Representative Results |
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Regenerative medicine and tissue engineering |
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Cancer |
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Cardiovascular |
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Neuroscience |
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In vitro 3D models |
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Development and embryogenesis |
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Immunology |
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Ophthalmology |
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Respiratory disease |
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Muscle physiology and pathology |
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Kidney, Colon, and Liver |
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Features | Methods | Set-Up Sophistication | Analysis of Complexity |
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Amount and texture description | First-order statistics (FOS) | + | + |
Second-order statistics (gray level co-occurrence matrix, GLCM) | + | ++ | |
2D-Fast Fourier Transformation (2D-FFT) | + | ++ | |
Wavelet transformation | + | +++ | |
Fibers orientation | Forward–backward SHG signal (F-SHG/B-SHG) | ++ | ++ |
Polarization | +++ | ++ | |
Coherency (C) | + | +++ | |
Fibers waviness | Straightness parameter (Ps) | + | ++ |
Fibers thickness and distance | Plot profile | + | + |
Parameters | Meaning | Interpretation in the Image |
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Mean | Average value | The average value of gray tones |
Standard Deviation | The standard deviation of the gray values used to generate the mean gray value | Contrast |
Integrated Density | Product of the image’s area and mean gray value | Lightness/darkness |
Skewness | It quantifies how symmetrical the distribution is relative to the mean value | The imbalance between the extent of areas (or number of pixels) that are darker or brighter than the mean |
Kurtosis | It quantifies whether the shape of the data distribution matches the Gaussian distribution | The spread of gray tones around the mean value |
Parameters | Meaning | Interpretation in the Image |
---|---|---|
Inverse difference moment (IDM) | Quantifies the local similarities present in the image | Homogeneity |
Energy | Probabilities of different gray levels in the image | Uniformity |
Inertia | The similarity in gray levels between neighboring pixels | Contrast |
Entropy | Measure the lack of spatial organization inside the image | Randomness |
Correlation | Dependence of gray levels between two pixels separated by a certain distance | Regularity in repetition patterns |
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Borile, G.; Sandrin, D.; Filippi, A.; Anderson, K.I.; Romanato, F. Label-Free Multiphoton Microscopy: Much More Than Fancy Images. Int. J. Mol. Sci. 2021, 22, 2657. https://doi.org/10.3390/ijms22052657
Borile G, Sandrin D, Filippi A, Anderson KI, Romanato F. Label-Free Multiphoton Microscopy: Much More Than Fancy Images. International Journal of Molecular Sciences. 2021; 22(5):2657. https://doi.org/10.3390/ijms22052657
Chicago/Turabian StyleBorile, Giulia, Deborah Sandrin, Andrea Filippi, Kurt I. Anderson, and Filippo Romanato. 2021. "Label-Free Multiphoton Microscopy: Much More Than Fancy Images" International Journal of Molecular Sciences 22, no. 5: 2657. https://doi.org/10.3390/ijms22052657
APA StyleBorile, G., Sandrin, D., Filippi, A., Anderson, K. I., & Romanato, F. (2021). Label-Free Multiphoton Microscopy: Much More Than Fancy Images. International Journal of Molecular Sciences, 22(5), 2657. https://doi.org/10.3390/ijms22052657