Two-photon microscopy (TPM), a high-resolution three-dimensional imaging technology based on nonlinear optical effects, has demonstrated significant application potential in the field of cosmetic efficacy evaluation in recent years. Its core advantages lie in its non-invasive, in vivo, in situ, label-free real-time imaging capability, enabling deep observation of dynamic changes in cells and the extracellular matrix within skin tissue. This provides a scientific basis for evaluating the safety and efficacy of cosmetics. Below are its specific applications and technical characteristics:

I. Technical Principle and Advantages

1. Deep Imaging Capability                                                                                        

TPM employs long-wavelength lasers (typically 780-1300 nm) for excitation, minimizing tissue scattering and absorption. Its penetration depth (>200 µm) surpasses that of single-photon confocal microscopy (~100 µm), though less than three-photon microscopy (~500 µm), providing clear visualization of skin structures from the epidermis down to the superficial dermis.

For example, it can visualize the distribution of substances like keratin, collagen fibers, elastic fibers, melanin, as well as autofluorescence signals from metabolic markers like NAD(P)H/FAD.

Figure 1. Stratum Corneum: Typically display an irregular contour with sharp margins, where dark grooves and bright lines are readily observable

Figure 2. Stratum Granulosum: Consists of slightly flattened keratinocytes. Cellular and nuclear structures are clearly observable, enabling assessment of their regular arrangement and absence of voids or defects

Figure 3. Stratum Spinosum: Composed of polygonal keratinocytes. A honeycomb-like, tightly packed arrangement of polygonal cells is observable. Compared to the stratum granulosum, the cell size in the stratum spinosum is significantly reduced

Figure 4. Stratum Basale: Basal cell pigment forms rings around dermal papillae in 3D scans. Collagen fibers occupy the ring interiors, and the encircling cell layer comprises the stratum basal

Figure 5. Dermal Elastic Fibers: Located beneath the stratum basale, these fibers exhibit a reticular (network-like) configuration. If follicular voids are encountered during image acquisition at this depth, reacquisition is required

Figure 6. Dermal Collagen Fibers: Exhibit a textured, banded, or reticular morphology. If follicular voids appear during scanning of this layer, reacquisition is required.

(Source of the above TPM images: CIRS Testing)

2. High Resolution and Multimodal Imaging

• Lateral resolution ≤ 0.8 µm, capable of resolving individual cells and extracellular matrix morphology (e.g., collagen fiber arrangement).
• Combines two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) techniques to detect metabolic substances (e.g., NAD(P)H) and collagen fibers separately, enabling multi-parameter quantitative analysis.

3. Non-invasiveness and Real-time Dynamic Monitoring

Requires no tissue sectioning or staining, circumventing structural damage typical of conventional histological processing. Supports repeated imaging of identical sites, facilitating longitudinal evaluation of cosmetic effects.

Figure 7. a) Traditional Microscopy: Requires sectioning + staining (Image source: Internet)

Figure 7. b) Two-Photon Microscopy: Dual-channel natural horizontal optical sectioning imaging (Image source: CIRS Testing)

Note: Dual-channel refers to: Autofluorescence channel - Keratin (stratum corneum), NAD(P)H & FAD (living keratinocytes), Elastic fibers (reticular dermis), Melanin (stratum basale); SHG channel - Collagen fibers (reticular dermis).

II. Specific Applications in Cosmetic Efficacy Evaluation

1. Skin Barrier and Moisturizing Efficacy Evaluation

• Evaluates the reparative effects of moisturizing products by monitoring stratum corneum thickness, intercellular lipid arrangement, and water distribution. For example, comparing changes in keratin fluorescence intensity at the skin surface before and after product use quantifies moisturizing effects.

Figure 8. a) After 28 days of product use, the surface morphology of the stratum corneum becomes smoother (visible reduction in voids, keratinocyte arrangement becomes more uniform)

Figure 8. b) After 28 days of product use, the upper epidermal layer gradually thickens (dashed line indicates the dermal-epidermal junction).

 (Source of above TPM images: CIRS Testing)

2. Microneedling Wound Detection

• Uses TPM to detect microneedle wounds on arm skin after microneedling procedures in medical aesthetics.

Figure 9. Clear irregular wounds exist in the stratum corneum, and corresponding signal voids appear in the reticular fiber layer, indicating the microneedles penetrated the stratum basale

(Source: CIRS Testing)

3. Anti-aging and Firming Efficacy Research

• Collagen Fiber Imaging: The SHG channel visually displays changes in collagen fiber density and arrangement, evaluating the efficacy of anti-wrinkle products.

Figure 10. SHG Channel Collagen Fibers --- Planar Image

Figure 11. SHG Channel Collagen Fibers --- 3D Image

(Source: CIRS Testing)

Note:

• Collagen fibers are functional proteins, relatively coarse, distributed in bundles 1-20 µm in diameter, densely packed, and interwoven into a network.
• TPM scanning depth for collagen fibers is typically 40 µm or deeper.
• Collagen fibers (red) can be observed in the SHG channel. Planar images primarily show a textured, banded, or reticular structure. 3D images, due to the stacking of collagen fibers at different depths, mainly present as a 3D network or fluffy cloud-like structure.

Figure 12. Monitoring Epidermal Thickness Changes

Note: Changes over follow-up period: After 28 days of product use, upper epidermal thickness significantly improved by 8.45%. 0 µm indicates the stratum corneum surface; 0-40 µm is the epidermal layer; 50-60 µm (dashed line) is the dermal-epidermal junction, where red collagen fibers appear, considered the shallowest dermis, and green cellular structures disappear, considered the deepest epidermis; 60-120 µm is the superficial dermis, red signals are collagen fibers, green signals are elastic fibers

Figure 13. Fluorescence Channel & SHG Channel --- 3D Image

(Source: CIRS Testing)

• Metabolic Activity Analysis: The NAD(P)H/FAD fluorescence intensity ratio reflects cellular metabolic status, indirectly evaluating antioxidant and anti-aging efficacy.

4. Safety Evaluation

• Observes phototoxicity or inflammatory reactions of cosmetic ingredients on skin cells (e.g., abnormal cell morphology, abnormally enhanced fluorescence signals).

III. Application Directions

Cosmetic Efficacy Evaluation:

• • Development of human efficacy methods for cosmetics, such as research into skin fiber morphology testing methods based on two-photon excitation technology.
  • Application in population-based skin baseline research. Literature documents TPM analysis of changes in dermal fiber network morphology characteristics with age, defining the dermal morphology scoring system MDMS (MLT-based Dermis Morphology Score).
  • In vivo evaluation of skin aging, defining the dermal aging index SAAID (SHG-to-AF aging index of dermis).
  • Research and exploration of cosmetic ingredient mechanisms of action, such as efficacy verification of collagen-stimulating products.

Medical Aesthetic Efficacy Evaluation:

• • Efficacy evaluation of laser skin treatments.
  • Detection of human cell activity.
  • Visualization assessment of human skin collagen fibers.
  • Visualization assessment of human skin elastic fibers.

If you need any assistance or have any questions, please get in touch with us via cosmetic-ck@cirs-group.com.