Abbreviations:SHG (second harmonic generation), THG (third harmonic generation), TPM (two-photon microscopy)
- •Recognize the newest techniques in biomedical research.
- •Describe how these techniques can be utilized and their limitations.
- •Describe the potential impact of these techniques.
- •Depth of light penetration
- •Reduced photobleaching and phototoxicity outside the focal plane
- •Optical sectioning and label-free visualization of autofluorescent molecules and structures
- •Potential phototoxicity in the focal plane after long-term imaging
- •Distorted z-resolution, in particular at higher depths levels
- •Potential thermal damage due to high-laser power pulses
- •High costs of instrument compared with confocal and conventional microscopy
- •High level of expertise required
The Basic Principles of TPM
|Skin Main Layer||Skin Sublayer||Cells in Each Layer||Endogenously Detectable Component||Spectral Positions (Excitation/Emission in nm)|
|Epidermis||Stratum corneum||5–6 layers of cornified dead cells||Keratin||(760–860/477–503)|
|Stratum lucidum||Dendritic epidermal T cells|
|NADPH (in living keratinocytes)||Free|
|Stratum granulosum||Bound to protein|
|Stratum spinosum||Melanin (eumelanin and pheomelanin)||440-420-475 (800/550)|
|Basal cell layer|
|Dermis||Dermal dendritic cells|
Dermal T cells
Other immune cells
Overview of Methodology (Sample Preparation and Filter Setup)
Experimental Dermatology Research and TPM
Clinical Dermatology and TPM
Conflict of Interest
Multiple Choice Questions
- 1.Which of the following is true about the basic principles of two-photon microscopy (TPM)?
- A.A two-photon microscope uses a pinhole.
- B.TPM requires collision of two low-energy photons almost simultaneously.
- C.UV spectra are used in TPM.
- D.Generation of second harmonic generation (SHG) requires absorption of two low-energy photons.
- 2.Which of the following is not an advantage of TPM?
- A.The depth of light penetration
- B.Less photobleaching outside the confocal volume
- C.Excellent z-resolution
- D.None of the above
- 3.TPM has been used in which of the following applications?
- A.Aging studies
- B.Inflammatory dermatoses
- C.Skin cancer diagnosis
- D.All of the above
- 4.Endogenous autofluorescent signals in the skin can be generated from which of the following?
- A.Elastin and collagen
- D.All of the above
- 5.Based on Figure 2b, using a mode-locked titanium-sapphire laser at 920 nm wavelength, which band-pass (BP) filter is most appropriate?
- A.Evans Blue with a BP filter of 505/30
- B.SHG with a BP filter of 641/25
- C.EGFP with a BP filter of 505/30
- D.None of the above
- Quiz and brief explanation of correct answers
- Teaching Slides
- Supplementary Table S1
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