Submicron resolution techniques: Multiphoton microscopy in skin disease

Non‐invasive optical examination plays a crucial role in various aspects of dermatology, such as diagnosis, management and research. Multiphoton microscopy uses a unique submicron technology to stimulate autofluorescence (AF), allowing for the observation of cellular structure, assessment of redox status and quantification of collagen fibres. This advanced imaging technique offers dermatologists novel insights into the skin's structure, positioning it as a promising ‘stethoscope’ for future development in the field. This review provides an overview of multiphoton microscopy's principles, technology and application in studying normal skin, tumour and inflammatory diseases, as well as collagen‐related and pigmentary diseases.


| INTRODUC TI ON
Skin disorders are highly prevalent and often cause significant distress for patients.The diagnosis of these skin diseases can be challenging due to their diverse and nonspecific clinical presentations, making it difficult for clinicians to diagnose.While histopathology of biopsied tissue is considered the gold standard for precise diagnosis, its invasive nature and the need for recovery a period make it impractical for screening purposes. 1However, advances in optical technology have provided new avenues for non-invasive skin examinations.The skin's large surface area and thin composition allow for effective light penetration, making it suitable for optical examinations. 2Non-invasive optical examination techniques, such as dermoscopy, reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) have improved the accuracy, specificity and sensitivity of clinical skin diagnosis.Multiphoton microscopy (MPM), also known as non-linear microscopy, has emerged as a promising tool for diagnosing skin diseases.One common form of MPM is two-photon microscopy (TPM) which uses two-photon excited fluorescence (TPEF) and second harmonic generation (SHG).Some current in vivo MPM systems (such as MPTfex-CARS JenLab GmbH) incorporate TPM with fluorescence lifetime analysis (FILM) to assess the metabolic state of keratinocytes. 3 Based on optical characteristics, MPM enables observation of the skin structure, collagen fibre characteristics and quantitative evaluation of cell metabolism and collagen content through special calculations.Therefore, MPM is suitable for diagnosing and evaluating the treatment outcomes of skin diseases.Furthermore, multiphoton technology, represented by the two-photon technology, has found extensive applications in biological and medical imaging, single-molecule detection and threedimensional (3D) imaging, demonstrating its vast potential for further development.

| BA S IC PRIN CIPLE S OF MPM TECHNI Q U E S
Maria Goppert Mayer first proposed the two-photon excitation theory in 1931, suggesting that fluorescent molecules simultaneously absorb two long-wavelength photons, causing them to transition to an excited state.The invention of the laser in 1960 allowed the two-photon effect to be verified and applied.In 1990, Denk and Webb invented the first two-photon fluorescence microscopy. 4Subsequently, the TPEF microscopic imaging technique was proposed and used after the 1980s. 5Biological tissues contain various fluorescent substances, such as chlorophyll, porphyrins, proteins, tryptophan, purines, pyrimidines, etc., which are suitable for excitation by photons.MPM is an imaging technique that excites non-linear optical effects, while single-photon imaging mainly excites linear optical imaging.Skin confocal microscopy (RCM) primarily used single-photon excitation for imaging.In single-photon excitation, the fluorescent material's nucleus absorbs single excitation photons, transitions from the ground state to the excited state, undergoes relaxation outside the nucleus, and then returns to the ground state, emitting an emission photon (emission photon).The energy of the fluorescent photon is slightly lower than that of the excitation photon due to energy consumption.MPM imaging technology mainly uses TPEF signal and SHG signal for imaging.Compared to single photons, TPEF predominantly involves the non-linear excitation of light.In this process, the fluorescent material absorbs two photons within a short time, undergoes relaxation and subsequently transitions to the same excited state, resulting in photon emission. 6,7r example, nicotinamide adenine dinucleotide (NADH) exhibits 450 nm fluorescence with single-photon excitation at 350 nm, whereas two-photon excitation produces 450 nm fluorescence using 700 nm excitation light.SHG fluorescence originates from noncentrosymmetric molecules, such as collagen and skeletal muscle.MPM offers several advantages, including the ability to obtain microenvironmental information about substances in a highly precise manner, distinguish substances that are indistinguishable by the two-photon technique and evaluate cellular metabolism. 8e specific advantages of MPM can be summarised as follows: (1) MPM enables unique observation of submicron structures, facilitating precise examination of individual cellular states within the dermal epidermis.Additionally, the unique SHG channel allows for the observation of collagen fibre distribution and structure. 1 forming cells becomes challenging when the excitation wavelength exceeds 800 nm. 7,12Studies have demonstrated that melanin in living skin exhibits optimal performance at 785 nm. 13 For melanoma, the ideal excitation wavelength for melanin is 800 nm. 14Collagen and elastin, which are key dermal components, can be excited at different wavelengths ranging from 760 to 840 nm.Typically, an 800nm wavelength is employed to visualise the fibrous structure of the dermis comprehensively. 7,9e human skin primarily comprises the epidermis, dermis and subcutaneous fat layer.The epidermis comprises the stratum corneum, granular layer, spinous layer, basal layer and dermal-epidermal junction (DEJ).The dermis is primarily composed of collagen fibres and elastic fibres.In our study, a portable skin cell microscope (TRANSCEND VIVOSCOPE, China) with an excitation fluorescence wavelength of 780 ± 10 nm was used to observe the morphology of each skin layer (Figure 1).The stratum corneum, characterised by keratin, exhibited non-nucleated polygonal keratinocytes appearing as irregular patches and bright spots.The granular layer located 10-20 μm deep, emitted fluorescence mainly from the cytoplasmic matrix.The cells in this layer were polygonal and flattened, with fluorescent granules and dark nuclei.At a depth of 20-40 μm, a honeycomb arrangement and a polygonal spinous layer were observed.The size of these cells gradually decreased with increasing depth.In this layer, the fluorescence originated primarily from NADH in the cytoplasm and mitochondria, while the nucleus did not exhibit fluorescence.In the basal layer, a distinctive ring of cells was visible.This layer displayed a central dermal papilla surrounded by a single layer of columnar cells, with a ring of basal cytochromes.TPEF (green) imaging highlighted elastic fibres with elongated reticular knots in the dermal reticular layer.SHG (red) imaging revealed thick strips or reticular patterns of collagen fibres.These findings are consistent with previous studies. 15

| THE APPLI C ATI ON OF MPM IN S K IN DISE A SE S 4.1 | Skin cancers
Early detection of skin tumours presents challenges, as they are prone to misdiagnosis.Various optical methods have been explored to improve the accuracy of non-invasive melanoma diagnosis. 16erefore, early non-invasive optical screening plays an increasingly significant role in skin tumour diagnosis.MPM allows the observation of cytological features of tumour tissue, and it further incorporates computational approaches to digitise AF and SHG values.By using algorithms and comparing these values to those of normal skin, MPM enables the calculation of peritumoural fibre changes.This comprehensive approach facilitates a more detailed characterisation of tumours using MPM.The characteristics of MPM in relation to common diseases are summarised in Table 1.
In a study conducted by Dimitrow et al., the sensitivity and specificity of TPM were assessed in patients with malignant melanoma (MM), in vivo on the skin (n = 8) and ex-vivo (n = 26) on pathological sections.The results demonstrated a sensitivity of 95% sensitivity and a specificity of 97%. 17MM is characterised by six main features, namely disorganised structure of the epidermis, blurred borders of keratinised cells, pleomorphic cells, polymorphic dendritic cells, large intercellular distances and ascending melanocytes and dendritic cells.The first four features are specific to MM.A decrease in fluorescence intensity can be observed in MM, which might be attributed to reduced cellular metabolic products in keratin-forming cells due to apoptosis or necrosis.However, MPM encounters challenges in distinguishing between dendritic cell types, such as melanocytes and Langerhans cells.Melanocytic nevus is characterised by nests of nevus cells at the true epidermal junction, sickle-shaped nevus cells exhibiting strong autofluorescence, uniform distributions of keratinocytes, well-defined borders and a scarcity of dendritic cells. 17 for disease progression and prognosis.This might be attributed to the role of metalloproteinases, which degrade type I collagen in the extracellular matrix (ECM) and promote the invasion and metastasis of cancer cells.Additionally, the d22 coefficient and anisotropy parameter were introduced to assess the collagen structure.The d22 coefficient represents the non-linear optical susceptibility, and it indicates the strongest signal when the fibres are aligned parallel to the polarisation direction of the laser beam.This index is lower in tumours.On the other hand, the anisotropy parameter represents the polarisation of the SHG signal, representing the collagen fibre orientation.Therefore, in tumour tissues, collagen fibres exhibit reduced density and a more straightened pattern, resulting in higher anisotropy parameter values compared with normal skin. 18MPM also enables qualitative and quantitative analysis of pigmented neoplasms, including pigmented nevi, dysplastic nevi and MM.The qualitative analysis focuses on comparing the tissue characteristics observed using MPM with those observed in pathological sections.In this study, a quantitative analysis was conducted using the multiphoton melanoma index (MMI), which included TPEF (F), SHG (S) and melanocyte morphology (D).The visualisation criteria with scores ranging from 0 to 3 were established for each parameter and the scores for the three parameters were summed to obtain the MMI (scores ranging from 0 to 9).According to this approach, common nevi typically score between 0 and 1, dysplastic nevi score between 1 and 4 and melanomas score between 5 and 8. Through statistical analysis, significant differences among the three diseases were observed, which could help diagnose atypical pigmented nevi in the future. 191][22] Based on these characteristics, Seidenari et al. conducted a study involving a large sample of 66 BCC cases and reported an overall sensitivity of 84.8% and specificity of 100% for MPM. 20RCM and multiphoton tomography (MPT) can visualise the nests and palisading edges of BCC cells.However, MPT offers higher resolution and enables the observation of nuclear diameter, nuclear/cytoplasm ratio and cell density of BCC. 23In addition to the characteristic manifestations of The study revealed a decreased IOD density value in BCC, along with a more consistent collagen orientation, decreased angle and increased fibre length. 24Sendín-Martín et al. also evaluated collagen fibres in BCC using FFT, CT-FIRE and CurveAlign methods.They compared BCC with normal skin, benign lesions and indolent BCC versus aggressive BCC.Generally, collagen distribution around BCC nests appeared more organised, and collagen orientation around indolent BCC subtypes (superficial and nodular) was more parallel. 25wever, MPM imaging has limited depth, making it less effective in observing nodular BCC.Therefore, MPM is suitable for superficial skin tumours or precancerous lesions.In squamous cell carcinoma in situ, MPM revealed features such as hyperkeratosis, Bowenoid dysplasia, speckled perinuclear and loss of cell polarity. 26For seborrheic keratosis, MPM exhibited central hyperfluorescent and peripheral hypofluorescent cells, which correspond to pathological papillomatous hyperplasia with hyperkeratosis. 27In actinic keratosis, MPM detected fluorescence features such as disorganised epidermal structure, heterogeneous cell morphology and fluorescence and increased cell space. 27Limited studies have focused on other precancerous lesions, and future studies could explore the relationship between MPM and other skin tumours such as Bowen's disease.
The SHG channel was used to observe collagen fibres and differentiate dermatofibrosarcoma (DFSP) from normal skin.In this study, the FFT method was used to extract the textural characteristics of collagen fibre bundles, while SHG fluorescence intensity was used to evaluate collagen density.The findings revealed that the SHG intensity in normal skin was 2.5 times higher than that in DFSP, indicating the disruption of collagen protofibrils and the loss of large collagen protofibrils in DFSP.The band-pass value was higher (p > 0.05) and the orientation index was marginally lower (p < 0.05) in DFSP compared with normal skin, suggesting a more scattered arrangement of collagen fibres and increased randomness in DFSP.Notable DFSP features include a blurred collagen texture, fibre thinning, reduced density and collagen dispersion. 28Therefore, MPM holds the potential for diagnosing DFSP based on collagen morphology and density. 29M enables the conversion of cell morphology heterogeneity in skin tumours, as well as variations in pigment content and cell arrangement morphology.By applying FFT, CT-FIRE, CurveAlign and other methods to analyse collagen and other indicators, the differences in collagen direction and density among different tumours can be quantified.This ability to discern dissimilarities allows for the distinction between various types of tumours.

| Inflammatory skin disease
Histopathology poses challenges in identifying inflammatory skin diseases.However, the development of non-invasive optical biopsy techniques has provided new diagnostic and follow-up approaches for inflammatory skin diseases. 30The following sections briefly summarise the applications of MPM in psoriasis, atopic dermatitis (AD) and localised scleroderma (LS).
In patients with psoriasis, MPM has been used for in vivo detection, revealing distinctive characteristics.These include punctate fluorescence in the stratum corneum, absence of the granular layer, increased nucleoplasmic ratio in the granular layer and widened intercellular spaces.Moreover, a prolonged dermal papilla layer (>100 μm) and enlarged psoriatic papillae, with twice the diameter of healthy skin (64.2 ± 22.6 μm and 28.8 ± 8.9 μm, respectively) were observed in the dermis.Psoriatic vessels were 83% larger than healthy vessels.Therefore, MPM enables the monitoring of changes in psoriasis before and after treatment by quantifying papilla length and vessel diameter. 31Furthermore, MPM examination allows for the observation of cells with nuclei in the psoriatic stratum corneum, a feature associated with accelerated epidermal cell turnover.However, due to hyperkeratosis and limited laser penetration, deeper cell layers might sometimes be challenging to visualise.Additionally, SHG appears at a greater depth (>100 μm) due to the elongation of the dermal papilla layer in psoriasis.These fluorescent characteristics are consistent with the histopathological findings. 32PM revealed distinct features of AD, including epidermal thickening, intercellular oedema and mitochondrial 'ring-like structures' around the nucleus, possibly due to increased NADH levels and are associated with inflammation. 32,33Combining MPM and immunohistochemistry, vascular thickening, curvature, and capillary loops were observed in erythematous and non-lesioned areas of AD tissue sections. 34In a study by Lu et al., three cases of LS were examined using MPM, revealing similar histopathological features such as a thinner epidermis, flattened epidermal protrusions, thickened collagen fibres parallel to the epidermis and reduced vascularity.Additionally, the study introduced the orientation ratio index of collagen bundles (ORICB) and SHG/TPEF index (STID) to quantitatively evaluate collagen and elastic fibres in the dermis and differentiate scleroderma skin from normal skin.The findings demonstrated higher ORICB in LS and higher STID in the deep dermis of LS compared with normal skin, indicating increased thickness and parallel alignment of collagen fibres in LS.Therefore, MPM facilitates rapid diagnosis of pathology sections without the time-consuming process of haematoxylin and eosin staining, thereby improving the efficacy of pathology diagnosis. 35In pemphigus vulgaris, MPM observations included medium-sized, uniform fluorescence of spinal layer cells, as well as loosening of the spinal layer cells and blister formation, aligning with the pathological structure. 27erall, MPM is less used in inflammatory skin diseases, which might be attributed to the challenges posed by hyperkeratosis and the resulting restricted laser penetration.Keratinocyte size, intensity of fluorescent substances in the cytoplasm, measurement of blood vessels and the observation of collagen using ORICB and STID are important parameters that help identify different inflammatory diseases.

| Ageing and scars
MPM also plays a crucial role in the study of skin ageing.The dermis, composed predominantly of collagen fibres and elastic fibres, Their study demonstrated the potential of guiding RF energy selection and evaluating treatment effect. 40 This suggests that SHG exhibits significant advantages in identifying collagen and allows for rapid diagnosis without the need for immunofluorescence staining on tissue sections. 46M offers unique advantages in the assessment of collagen dynamics in various processes such as wound healing, skin ageing, and scarring.Through the application of digital algorithms like SAAID, ImbrN, FFT and STI, MPM enables the observation of collagen directionality, collagen strength and density.These findings hold significant implications for future developments in anti-ageing treatments and the monitoring of scar treatment outcomes.2.
MPM and RCM possess characteristics with high (submicron and subcellular) resolution, allowing for the observation of skin cell features horizontally and vertically. 1,41RCM is widely used for its fast imaging, large field of view and ability to visualise blood vessels.
However, it has lower resolution compared with MPM and cannot distinguish between cell nuclei and cytoplasm. 53On the other hand, MPM leverages two-photon properties to assess cellular metabolism by fluorescence signals and identify collagen and elastic fibres.
However, MPM has certain limitations.First, the imaging size of 250 × 250 μm 2 might cause important information to be missed, affecting overall assessment.However, benchtop two-photon imaging generally has a larger range, making it more suitable to evaluate pathological sections.Second, the imaging depth was limited to SHG, similar to TPEF, is a form of non-linear optical imaging where two photons of the same frequency interact with an asymmetric medium, inducing the transition from the ground state to an imaginary state, resulting in the emission of photons with doubled frequencies and halved wavelengths.TPEF and SHG signals obtained from MPM are mutually corroborating and complementary, making TPEF and SHG composite imaging a promising new technique.In addition, the introduction of FILM enables the detection of fluorescence intensity decay in fluorescent substances.

( 2 )
Quantitative analysis of the redox state can be performed by analysing changes in fluorescence signals.For instance, variations in NADH fluorescence can indirectly assess mitochondrial metabolism and dynamics, providing insight into the occurrence of epidermal carcinogenesis. 9(3) MPM offers several advantages over RCM.The use of longer excitation wavelengths reduces light scattering and allows for deeper tissue penetration.Moreover, longer wavelengths, such as near-infrared light (700-1000 nm), induce less photodamage and phototoxicity to cells and tissues compared with RCM.This feature permits prolonged observation of living tissues.Additionally, MPM excitation and emission occur exclusively in the focal plane, minimising photobleaching outside of the focal plane. 10 | MPM IN S K IN IMAG ING Human skin contains abundant autofluorescent substances, including keratin, NADH, flavin adenine dinucleotide (FAD), melanin, collagen, elastin, porphyrins, tryptophan, flavin, cholecalciferol and lipofuscin.The presence of fluorescent materials has also been observed in ex vivo pathological sections.Long-wavelength infrared light excitation and deeper imaging depths enable MPM imaging without the need for fluorescent staining as it can elicit its fluorescent signal.The SHG signal originates from non-centrosymmetric molecules, such as collagen and myosin. 6,11Consequently, human keratin, cytoplasm, melanin and elastic fibres emit TPEF signals, while collagen fibres emit SHG signals.Conversely, the cell nucleus and vascular lumen do not emit fluorescent signals.The intensity of the fluorescence signal emitted after excitation of the fluorescent material is directly proportional to the amount of fluorescein present, forming the theoretical basis for the application of fluorescence imaging systems in biological research.MPM can distinguish the structures of different tissue layers within normal human skin tissue based on different excitation spectroscopy.Effective excitation of epidermal structures can be achieved at 760 nm, while fluorescence excitation of most keratin- Lentsch et al. observed that TPM provided a clearer visualisation of the epidermal junction and the location of MM nests compared with RCM images. 1 In a study conducted by Hompland et al., the density of collagen fibres was evaluated by introducing the pixel of SHG, which represents the percentage of the area containing the SHG signal.Based on the SHG observations, the collagen fibre density around MM was reduced compared with normal tissues, and collagen fibre density might serve as a predictive factor
BCC observed through MPM, Lin et al. introduced the concept of multiphoton fluorescence (MF) to the SHG index (MFSI) for quantifying the fluorescence of specific regions.The MFSI is calculated as (a − b)/(a + b), where (a) represents the MF pixel number and (b) represents the SHG pixel number.The quantitative analysis revealed that the SHG signal decreased and the AF signal increased within the tumour and tumour mesenchyme compared with the normal dermis.This might be attributed to increased collagenase activity in the tumour tissue and could potentially guide the determination of surgical margins in the future. 15,21Kiss et al. introduced four parameters to evaluate BCC, namely integrated optical density (IOD), fast Fourier transform (FFT), CT-FIRE and fibre alignment and orientation (CurveAlign).The IOD is derived from the intensity of TPEF signals (NADH and elastin) and SHG signals (collagen) using ImageJ software.A comprehensive assessment of collagen fibre characteristics, including collagen orientation index, fibre length, fibre angle and fibre alignment, was performed using FFT, CT-FIRE and CurveAlign.
produces SHG and TPEF signals, respectively.The SHG to AF ageing index of the dermis (SAAID) has been introduced.SAAID is calculated by comparing the autofluorescence (AF) pixel number (b) with the SHG pixel number (a): SAAID = (a − b)/(a + b).Skin ageing is characterised by a decrease in collagen fibre density and an increase in elastic fibre density, resulting in an increase in AF with age whereas and a decrease in SHG signals.MPM proves advantageous in studying skin ageing in vivo due to its deeper imaging penetration depth, reduced photobleaching, and lower phototoxicity. 36,37Wang et al. introduced an anisotropy ratio (a high ratio indicates fibre alignment and a low ratio indicates random orientation) to assess collagen orientation.Studies have indicated aged skin exhibits a high anisotropy ratio in elastic and collagen fibres, indicating a consistent fibre orientation. 38Pena et al. further emphasised the importance of combining three MPM parameters-SHG to TPEF, SAAID, and normalised imbrication (ImbrN)-for comprehensive ageing assessment.The 3D ImbrN index was used to assess the overlap of collagen and elastin fibres.Their findings indicated that ageing skin is characterised by a decrease in the SHG to TPEF ratio and 3D SAAID index, along with an increase in the 3D ImbrN index.39Real-time, non-invasive evaluation of treatment effects is currently lacking for monopolar radiofrequency (RF) therapy, which is used to delay skin ageing through dermal heating.Tsai et al. treated mice with varying RF energies and durations, and employed MPM for real-time, noninvasive observation of collagen changes following RF treatment.
Miyamoto et al. measured   the fluorophore NADH in epidermal keratinocytes of young and elderly women.The results revealed a significant decrease in NADH levels in aged skin keratinocytes, likely attributed to photoageing or environmental damage.Therefore, a change in NADH fluorescence intensity in the epidermis can indicate skin ageing.41MPM proves useful in assessing skin scars by examining elastic fibres (TPEF) and collagen fibres (SHG).Quantitative analysis of fibre density, collagen orientation and collagen density in scars is achieved through the introduction of indices such as SHG-to-TPEF index (STI), FFT, and collagen orientation index.42In MPM imaging, scars exhibit characteristics such as epidermal thinning, parallel arrangement of dermal collagen fibres, thin and disorganised collagen fibres and increased fragmentation of elastic fibres.While collagen levels are increased in the superficial and deep dermis, the superficial dermis experiences decreased elastin levels.43,44Furthermore, MPM observations revealed a decrease in collagen and elastin density with increasing age and scar course, which is consistent with the natural fading process of scars.45In a study conducted by Chen et al., SHG was employed to examine two cases of post-burn hyperplastic keloids using frozen and paraffin sections.The observations demonstrated elongated and wavy characteristics of collagen fibres, which correspond to the staining patterns of type I collagen antibodies.

4. 4 | 5 |
Application for other diseasesMPM plays a crucial role in evaluating wound healing processes.It has been demonstrated that the cellular metabolic rate can be assessed using NADH fluorescence lifetime imaging, which reveals increased fluorescence lifespan during the early stages of trauma.Additionally, MPM enables the assessment of collagen fibre changes through SHG imaging, particularly in late incision scar formation.47Furthermore, MPM offers valuable insights into the keratinocyte processes involved in wound healing metabolism, as indicated by optical redox ratio (FAD/[NADH+FAD]) and NADH fluorescence lifetime imaging, providing a quantitative marker for age-related delays in healing, as observed in young and aged mice.48MPM also proves useful in distinguishing between scarring and non-scarring alopecia.Scarring alopecia is characterised by inflammatory cells around hair follicles, reduced follicle diameter, and diminished sebaceous glands.Therefore, non-invasive MPM examination enables the assessment of changes in follicle size, sebaceous glands, and inflammatory cells to evaluate hair loss.49Additionally, MPM can identify early striae gravidarum, revealing distinctive characteristics of collagen bundles such as separation, disorganisation and a lack of organisation into bundles, along with an increase in elastic fibres.50Pigmentary diseases, including hyperpigmentation and hypopigmentation conditions, have gained significant attention in research.Lentsch et al. used MPM to evaluate melasma and observed that melanin in melasma predominantly localises in the cell membrane, while perilesional skin melanin concentration around the nucleus.The melasma area exhibited a significant increase in elastic fibres compared with perilesional skin.Additionally, dermal melanophages were more frequently observed in melasma.This study also introduced the melanin volume fraction as a quantitative measure of pigmentation, using an excitation wavelength of 880 nm to stimulate melanin.The results indicated significantly higher levels of epidermal melasma compared with perilesional skin (p = 0.03), while dermal/mixed melasma did not show a significant difference (p = 0.32).51Therefore, MPM could serve as a non-invasive diagnostic tool for melasma, enabling the monitoring of treatment outcomes.In another study,Shiu et al. combined   multiphoton microscopy and single-cell ribonucleic acid sequencing (scRNA-Seq) to uncover the pathogenesis of stable vitiligo.MPM imaging of normal skin revealed an increase in mitochondrial clustering in epidermal cells with increasing depth, whereas stable vitiligo lesions did not exhibit depth-dependent mitochondrial clustering.scRNA-Seq analysis and cell communication network analysis of keratinocytes revealed that stressed keratinocytes in stable vitiligo undergo a shift towards oxidative phosphorylation as an energy source, which might contribute to the limited therapeutic response in this condition.52Therefore, MPM combined with sequencing technology offers valuable insights into disease pathogenesis.COMPARISON WITH OTHER NON -INVA S IVE E X AMINATIONS MPM, RCM, OCT and UHFUS (ultra-high-frequency ultrasound) are imaging techniques that use optical and acoustic principles to enable real-time and dynamic in vivo imaging of the skin.Each of these tools possesses distinct characteristics that make them suitable for specific clinical applications.Therefore, it is crucial to have a comprehensive understanding of these characteristics to select the appropriate examination method.The features of different skin examination equipment are summarised in Table

6 |
<250 μm prevents the assessment of tumour depth and infiltration, necessitating the combination of skin ultrasound and OCT for future lesion evaluation.1 Third, MPM has slower imaging time and scanning speed compared with dermoscopy and RCM; however, this can be addressed in further design improvements.1 OCT and UHFUS primarily capture vertical images, enabling the determination of lesion boundaries, size and blood flow due to their deep penetration capabilities.OCT achieves a balance between resolution and penetration depth, while UHFUS provides deeper penetration at the expense of resolution.However, both techniques are unable to identify individual cell charcateristics.54A comprehensive evaluation of skin diseases requires the use of various examination equipment.In the case of BCC, RCM can be employed to identify the features of tumour cell nests, while MPM can assess tumour cell metabolism and dermal ECM properties.Furthermore, the combination of OCT and UHFUS helps determine the tumour margins and blood flow, thereby facilitating a comprehensive evaluation of BCC.Integrating these different examination tools is crucial for a comprehensive evaluation and holds promise for future advancements in skin assessment.CON CLUS ION This review focuses on the principles of two-photon excitation and the optical properties of the skin.The use of TPM in various skin diseases, including skin tumours, inflammatory skin diseases, collagen-related diseases and pigmentary diseases, has been highlighted.MPM's submicron technology enables the non-invasive detection of skin conditions and offers immense potential for enhancing human-computer interaction and reducing motion artefacts.With continued advancements in equipment, there is an opportunity to further explore the application of MPM in in vivo skin disease diagnosis and treatment assessment.MPM serves as a valuable tool for non-invasive, real-time detection and evaluation of treatment outcomes.

TA B L E 2
Characteristics of different skin examination equipment.