Laser Treatment Monitoring with Reflectance Confocal Microscopy

Laser treatments have become popular in Dermatology. In parallel to technologic development enabling the availability of different laser wavelengths, non-invasive skin imaging techniques, such as reflectance confocal microscopy (RCM), have been used to explore morphologic and qualitative skin characteristics. Specifically, RCM can be applied to cosmetically sensitive skin areas such as the face, without the need for skin biopsies. For these reasons, apart from its current use in skin cancer diagnosis, our systematic review reveals how RCM can be employed in the field of laser treatment monitoring, being particularly suitable for the evaluation of variations in epidermis and dermis, and pigmentary and vascular characteristics of the skin. This systematic review article aims to provide an overview on current applications of RCM laser treatment monitoring, while describing RCM features identified for different applications. Studies on human subjects treated with laser treatments, monitored with RCM, were included in the current systematic review. Five groups of treatments were identified and described: skin rejuvenation, scar tissue, pigmentary disorders, vascular disorders and other. Interestingly, RCM can assist treatments with lasers targeting all chromophores in the skin and exploiting laser induced optical breakdown. Treatment monitoring encompasses assessment at baseline and examination of changes after treatment, therefore revealing details in morphologic alterations underlying different skin conditions and mechanisms of actions of laser therapy, as well as objectify results after treatment.


Introduction
Over the past few decades, non-invasive treatments are increasingly requested in Dermatology [1][2][3][4]. Among these, laser therapy became popular due to technologic development leading both to the availability of different light wavelengths targeting different chromophores and to protocols reducing the downtime of treatment, according to patients' requests [3][4][5][6].
In this scenario, in vivo reflectance confocal microscopy (RCM) has emerged as a non-invasive technique enabling horizontal visualization at different layers of the skin with good contrast and high resolution, providing cytologic and architectural details [7][8][9].
It has been proven as an excellent add-on tool for diagnostic purposes in Dermatology as well as for the analysis of healthy skin, since RCM provides an optical "histological" biopsy of the living tissue in a totally non-invasive manner, therefore avoiding scars, which is pivotal for aesthetic areas such as the face [10]. Specifically, RCM has been mainly employed in Cosmetic Dermatology to examine presence of regular/irregular keratinocytes at epidermis, collagen morphology and eventual elastosis at dermal level and pigmentary characteristics of the skin [8,10,11]. Importantly, these parameters have been recently standardized according to semi-quantitative and qualitative scales in order to improve recognition, reliability and reproducibility of evaluations [10]. Another advantage of noninvasive RCM is its ability to enable repeated examination of a given skin area, facilitating dynamic evaluation of skin changes, such as those that occur during treatment monitoring. RCM has been used to assess the effectiveness of various laser therapies in treating a variety of skin conditions, including acne, rosacea, and post-inflammatory hyperpigmentation. By offering non-invasive, real-time evaluation of skin changes, RCM can assist clinicians in customizing laser treatments to meet the individual needs of patients, optimizing treatment outcomes [10,12].
To summarize, RCM has become an essential tool in the field of dermatology, revolutionizing the approach of clinicians towards non-invasive skin treatments. In addition to its diagnostic and treatment monitoring applications, RCM has also been used to assess the efficacy of laser therapy in various skin conditions, including skin rejuventation, scars, pigmentatry and vascular disorders. By providing non-invasive, real-time evaluation of skin changes, RCM can assist clinicians in tailoring laser treatments to meet the unique needs of individual patients, resulting in optimal treatment outcomes.
Currently, an overview about RCM in laser treatment monitoring is lacking. Therefore, we systematically review literature on the topic in order to summarize fields of application and to identify pre-and post-treatment RCM features that can be assessed in laser treatment monitoring.

Materials and Methods
Studies conducted on human subjects involving laser treatments for skin conditions monitored with RCM were screened.
Electronic databases were systematically searched and included MEDLINE (PubMed), Web of Science and Cochrane library databases. Search strategy adopted was similar across the databases and developed using the following keyword: "laser" AND "reflectance confocal microscopy". Our search included studies from inception to October 2022.
Two authors independently screened the abstracts for inclusion and exclusion criteria (SG and SA). In case of doubt or discordance, a third opinion was obtained (CL).
Studies were excluded based on the following criteria: • language other than English • in vitro or animal studies • not involving laser treatments • concerning tumors and not used to understand underlying mechanisms of laser therapies • studies without specific RCM features described before and after treatment • studies involving less than 8 patients From each of the included studies, the following data were extracted: first author, year of publication, indication, number of patients and numbers of female patients, age, skin type/ethnicity, study type, laser type, RCM criteria at baseline and variations posttreatment, follow up timing, efficacy and safety.

Study Selection
A total of 142 records were screened after duplicate and preliminary screening removal. Based on title/abstract screening, 101 studies were excluded. Forty-one full texts were thus assessed for eligibility and 20 studies were included in the qualitative synthesis, Figure 1.
A total of 142 records were screened after duplicate and preliminary screen removal. Based on title/abstract screening, 101 studies were excluded. Forty-one full t were thus assessed for eligibility and 20 studies were included in the qualitative synth Figure 1.
A summary of RCM features is presented in Table 1; an overview of studies included is reported in Table 2.

Epidermal layer
Regular honeycombed [14,18,19,23] Polygonal keratinocytes with homogeneous size and shape; cell border is well outlined and preserved Mottled pigmentation [14,16,17] Clustered bright keratinocytes in context of honeycombed pattern Spongiosis [18] Honeycombed pattern where the outline of the epidermal cells and the intercellular junctions appeared brighter and larger compared with normal skin Exocytosis [18] Bright particles corresponding to lymphocytes Fine scales [23] Highly reflective round-to-polygonal areas Micro-thermal zone or micro-injuries-ablative zone [14,18,19,31] empty spaces or black micro-holes Micro-thermal zone -thermal modified zone [31] areas with a whitish ring exhibiting high reflectance Extracellular deposits of melanin [23] bright round-to-polygonal areas and aggregated granules were observed throughout the epidermis

Dermo-epidermal junction
Polycyclic papillary contours [14,17] Bulbous projections and cords, with variably convoluted arrangement Edged dermal papillae [23] Dark round-to-oval structures surrounded by a rim of bright monomorphic cells Sebaceous glands [31] Roundish to oval-shaped annular structures, corresponding to glandular parenchyma and centered by hair follicles

Dermis
Thin reticular collagen [16,21] Bright thin fibrillar structures forming delicate weblike pattern; this structure can be detected around follicular openings Coarse collagen [14,16,18,20] Coarse filamentous thick structures with tendency to be packed; weblike pattern is still observable but with larger and irregularly spaced meshes Huddle collagen [14] Large hyporefractive blotches of amorphous and hyporeflective material; individual collagen fibers are no longer visible Curled bright fibers [14] Highly refractive thick and short undulated fibers, sometimes forming compact masses when severe solar elastosis is present "Neat-wall" [21] distortion of the normal DEJ, visible only in mosaic images, similar to a welldemarcated wall separating regular areas of junction, which are formed by round papillae and fibrillar or reticular collagen

Skin Rejuvenation
A total of 5 studies explored the role of RCM in laser monitoring for skin rejuvenation [13][14][15][16][17], Table 2. Most of the studies reported the use of lasers targeting water into the skin, including fractional CO 2 laser and non-ablative fractional erbium laser while one study encompassed 1064 nm fractional PSL use, exploiting laser induced optical breakdown (LIOB). A total of 35 patients were treated for skin rejuvenation of the face while 18 for the neck. Mean follow up of patients was 1 to 4 months. Interestingly, RCM treatment monitoring enabled the visualization of variations supporting clinical improvement observed after laser treatments.       There were also melanophages in the papillary dermis. In surrounding normal skin, there was less melanin in the epidermis and DEJ than there was in the melasma lesions. The pigment intensity of each skin layer (spinous layer, basal layer, and papillary dermis) was investigated 1 and 24 h after treatment.
After PAL treatment, there was a decrease in melanin-induced reflectance in the spinous layer and basal layer. In contrast, treatment with QS Nd:YAG led to slight or non-significant improvement in the spinous layer and aggravated findings in the basal layer.
After the treatments, a decrease of melanin-induced reflectance was shown in melasma lesion and surrounding normal skin. However, there was more improvement in the surrounding normal skin than in the melasma lesions.
After 1 h of PAL or QS Nd:YAG treatment, 37.5% of subjects in both groups demonstrated melanocyte activation in the basal layer.
One and two subjects showed melanocyte activation (dendritic) 24  with Vivacam, follicular plugs and hyperpigmented rings were observed in 6 (75%) and 4 (50%) of the melasma lesions from eight subjects, respectively (Figure 1). These hyperpigmented rings on dermoscopy were observed as hyper-reflectant rings on RCM in 4 (50%) of the melasma lesions  The ablative zone of the stratum spinosum showed a progressive reduction at 14-day after treatment and complete resolution at day-28. The ablative zone was smaller at the basal layer as compared to thte stratum spinosum. Thermally modified zones, surrounding the ablative zones, were observed at different time periods. From day3 to day14, the thermally modified zone was visualized as a highly refractile ring. At day28, even after epidermal regeneration was completed, a thermally modified zone was observable in some subjects.
Bright and fine fibers corresponding to newly formed collagen throughout the papillary dermis were also visible. Two months after laser application, a fine and bright collagen network was consistently observed.  Early RCM parameters after laser treatment included the onset of micro-holes or microcolumns within the honeycombed pattern, corresponding to the micro-ablation induced by the fractional lasers, and dendritic cells at epidermal level, related to post-treatment inflammation, 1 to 6 weeks after treatment [14,15]. About 1 to 4 months after treatment, a clinical reduction of hyperpigmentation associated to aging (photoaging) was found to correspond to the reduction of both epidermal and dermo-epidermal junction (DEJ) RCM features of pigmentation, represented by mottled pigmentation or polycyclic papillary contours, respectively. Importantly, an increased number of dermal papillae and appearance of long straight fibers presenting a parallel alignment, or reticular collagen, was observed [13][14][15][16][17]. Interestingly, this last RCM feature corresponded to a clinical improvement of wrinkles/rhytids [16,17].

Scar Tissue
A total of six studies concerning treatment monitoring with RCM after laser treatments of scar tissue were included. Two studies involved acne scars, other two atrophic and hypertrophic surgical scars and the remnant two striae distensae. Similarly to skin rejuvenation, laser sources included CO 2 fractional laser and non-ablative resurfacing lasers as well as PSL. RCM features included honeycombed or cobblestone pattern in epidermis, epidermal thickness, dermal papillae at DEJ and thin reticulated fibers or coarse collagen at dermal level [15,[18][19][20][21][22], Table 2.
In detail, immediately after fractional CO 2 laser or PSL to treat acne or surgical scars, black micro-holes surrounded by well-defined or fringed borders within the surrounding tissue could be observed [15,[18][19][20][21][22]. Following the first week after treatment, a progressive repair of skin layers was observed. Three to 6 months post-treatment, thin reticulated collagen fibers arranged to form a net, with variable brightness of the collagen fibers, were observed [15,[18][19][20]. Interestingly, this arrangement has been related to collagen remodeling [14].
Interestingly, both surgical scars and striae distensae showed parallel collagen fibers, mainly orthogonal to major axis of scar tissue. These parallel collagen fibers were significantly reduced after CO 2 laser and PSL for striae distensae [21,22]. Interestingly, a new confocal feature, the "neat wall", was described at baseline in striae distensae [21], Figure 2. This parameter corresponds to a distortion of the normal DEJ, visible only in RCM mosaic images; it resembles a well-demarcated wall separating regular areas of DEJ, which are formed by round papillae and fibrillar or reticular collagen (definition tab RCM). This parameter was reduced in patients receiving more than 4 CO 2 laser sessions, as compared to those receiving ≤4 sessions at 4-week post-treatment [21].

Pigmentary Disorders
Due to the ability of RCM to visualize pigmentation at different layers, RCM has been used as a treatment monitoring tool in different pigmentary disorders: solar lentigines, café au lait macules (CALMs), infraorbital dark circles, melasma [23][24][25][26][27], Table 2. Intuitively, the main laser source employed was Q-switched and a comparison with this laser source and PSL was available for one study.
In solar lentigines, edged dermal papillae and polycyclic papillary contours were observed at baseline. Immediately after Q-switched treatment, dark structureless areas could be observed throughout the epidermis and dermal papillae were hyporefractive. After 10 days, at the DEJ, non-edged dermal papillae were observed, in 9 out of 12 cases containing a few melanophages. Bright reflective rims surrounding the dermal papillae were no longer observed at the DEJ [23].
For CALMs, length and density of papillae were estimated. Interestingly, CALMs with irregular borders showed lower length and density of papillae as compared to those with smooth borders and better response to laser treatment [24].
Two studies were performed concerning the treatment of infraorbital dark circles in a total of 60 women of which half treated with Q-switched ruby and Q-switched Nd:YAG laser. At baseline, greater melanin deposition in the upper dermis of the dark circle area was observed, as compared to the cheekbone skin (control), but no significant difference in epidermal pigmentation. After 8 sessions, about 70% of subjects showed over 50% improvement of pigmentation, while the control area did not show any variations [25,26].

Pigmentary Disorders
Due to the ability of RCM to visualize pigmentation at different layers, RCM has been used as a treatment monitoring tool in different pigmentary disorders: solar lentigines, café au lait macules (CALMs), infraorbital dark circles, melasma [23][24][25][26][27], Table 2. Intuitively, the main laser source employed was Q-switched and a comparison with this laser source and PSL was available for one study.
In solar lentigines, edged dermal papillae and polycyclic papillary contours were observed at baseline. Immediately after Q-switched treatment, dark structureless areas could be observed throughout the epidermis and dermal papillae were hyporefractive. After 10 days, at the DEJ, non-edged dermal papillae were observed, in 9 out of 12 cases containing a few melanophages. Bright reflective rims surrounding the dermal papillae were no longer observed at the DEJ [23].
For CALMs, length and density of papillae were estimated. Interestingly, CALMs with irregular borders showed lower length and density of papillae as compared to those with smooth borders and better response to laser treatment [24].
Two studies were performed concerning the treatment of infraorbital dark circles in a total of 60 women of which half treated with Q-switched ruby and Q-switched Nd:YAG laser. At baseline, greater melanin deposition in the upper dermis of the dark circle area was observed, as compared to the cheekbone skin (control), but no significant difference in epidermal pigmentation. After 8 sessions, about 70% of subjects showed over 50% improvement of pigmentation, while the control area did not show any variations [25,26].
Differently from what observed for dark circles, melasma patients can show pigmentation located at different layers. Jo et al. compared the effects of PSL and Qswitched laser on melasma. After treatment, either an increase of activated melanocytes at basal layer of epidermis or an increased amount of melanophages were observed at upper dermis 24 h after treatment [27]. Interestingly, the presence of dendritic-shaped cells after treatment has been associated with a relapse of melasma 3 months after multiple sessions of Q-switched laser treatment [28]. Figure 3 shows the presence of superficial pigmentation represented by mottled pigmentation at epidermal level at baseline and the disappearance of the pigmentation after Q-switched laser treatment. Differently from what observed for dark circles, melasma patients can show pigmentation located at different layers. Jo et al. compared the effects of PSL and Q-switched laser on melasma. After treatment, either an increase of activated melanocytes at basal layer of epidermis or an increased amount of melanophages were observed at upper dermis 24 h after treatment [27]. Interestingly, the presence of dendritic-shaped cells after treatment has been associated with a relapse of melasma 3 months after multiple sessions of Q-switched laser treatment [28]. Figure 3 shows the presence of superficial pigmentation represented by mottled pigmentation at epidermal level at baseline and the disappearance of the pigmentation after Q-switched laser treatment.

Vascular Disorders
Treatment monitoring of port-wine stains (PWS) with RCM has been reported in two papers [29,30], Table 2. PWS are a type of vascular malformation that affect the skin and can cause significant psychological distress. Treatment options include pulsed dye laser (PDL) therapy, which targets the abnormal blood vessels to reduce their appearance. Based on different clinical response and RCM analysis of blood flow, RCM features associated to resistance to treatment for facial PWS were identified, being represented by high blood flow, large diameter, deep location [30]. Interestingly, based on different depths of vessels, different pulse durations of laser are related to different reduction of diameter vessels while no influence has been observed for density [29].
Overall, RCM is a valuable tool for monitoring the vascular response to laser therapy and for characterizing the microvascular changes associated with various vascular disorders. Further research is needed to fully understand the potential applications of RCM in the field of vascular medicine.

Vascular Disorders
Treatment monitoring of port-wine stains (PWS) with RCM has been reported in two papers [29,30], Table 2. PWS are a type of vascular malformation that affect the skin and can cause significant psychological distress. Treatment options include pulsed dye laser (PDL) therapy, which targets the abnormal blood vessels to reduce their appearance. Based on different clinical response and RCM analysis of blood flow, RCM features associated to resistance to treatment for facial PWS were identified, being represented by high blood flow, large diameter, deep location [30]. Interestingly, based on different depths of vessels, different pulse durations of laser are related to different reduction of diameter vessels while no influence has been observed for density [29].
Overall, RCM is a valuable tool for monitoring the vascular response to laser therapy and for characterizing the microvascular changes associated with various vascular disorders. Further research is needed to fully understand the potential applications of RCM in the field of vascular medicine.

Other Applications
Laser treatment monitoring with RCM has also been explored in other fields [31,32], Table 2. Wound healing has been studied after CO2 laser on healthy skin and results observed were in line with previous findings related to applications for skin resurfacing and scar tissue treatment [31].
Additionally, RCM has been employed to monitor sebaceous hyperplasia treated with dye laser. At baseline, lesions appeared like dilated sebaceous duct surrounded by dilated vessels [32].

Other Applications
Laser treatment monitoring with RCM has also been explored in other fields [31,32], Table 2. Wound healing has been studied after CO 2 laser on healthy skin and results observed were in line with previous findings related to applications for skin resurfacing and scar tissue treatment [31].
Additionally, RCM has been employed to monitor sebaceous hyperplasia treated with dye laser. At baseline, lesions appeared like dilated sebaceous duct surrounded by dilated vessels [32].

Discussion
Nowadays, laser treatment monitoring can be performed with non-invasive skin imaging techniques, such as RCM [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. RCM is an advanced technique enabling the visualization of the skin at a quasi-histological resolution [7,9]. It has been mainly applied to the field of skin cancers [33,34] but has expanding applications in Cosmetic Dermatology [1,35]. As a matter of fact, the possibility to visualize pigmentation at different layers, collagen and vessels in a non-invasive manner, makes RCM particularly suitable to monitor laser treatments. Accordingly, when laser treatments are applied on cosmetically sensitive areas such as the face or are employed to treat aesthetic conditions, it would be preferable to avoid scarring as a consequence of skin biopsies [36,37].
Our results highlight that RCM can be applied to monitor skin rejuvenation treatments, scar tissue, pigmentation and vessels at different layers after laser therapy.
Specifically, RCM applied to skin rejuvenation and scar tissue treatment procedures revealed the advantage to monitor of skin healing process after laser therapy and to objectify variations of epidermis, DEJ and collagen or elastosis in the dermis. Additionally, RCM enables the visualization of micro-holes or microcolumns within the epidermal pattern, that have been associated to fractional laser treatment [14,15]. Similar findings were observed with histology [13].
Considering the high reflectivity of melanin, RCM is particularly useful to study pigmention distribution in different skin layers in different pigmentary disorders [38], overcoming the limitations related to other non-invasive tools, such as Wood's lamp and dermoscopy which cannot locate the pigment precisely [39]. Interestingly, both pigment depth and the presence of dendritic-shaped cells in the epidermis have been associated with poor response to laser treatments, therefore highlighting the prognostic impact of RCM [27,28].
Our study reveals that specific terminology employed for healthy skin analysis can also be applied to define variations occurring after laser approach for skin rejuvenation, scar tissue and pigmentation [8,10,14,37].
Lastly, for vascular disorders, RCM has been employed to monitor the changes in blood vessels following laser treatments for port-wine stains. The ability to assess the depth, diameter, and density of blood vessels non-invasively may help optimize treatment parameters and improve overall treatment outcomes [29,30].
Currently, there are no studies comparing RCM with other techniques in laser treatment monitoring but some authors reported the alternative or complementary use of other techniques such as digital photography with automated features count and 3D imaging. Histologic studies, representing the reference for skin analysis, are mainly employed on ex vivo samples or in vivo in areas other than the face or on scars on the face in order to minimize visible scarring in aesthetic sites [40][41][42]. Therefore, many non-invasive techniques have been applied to the field of laser monitoring, apart from RCM, such as dermoscopy, digital photography with automatic features count (VISIA) and 3D assessment [3,[43][44][45][46][47]. These techniques enable the visualization of pigment and vessels but, differently from RCM, they cannot be employed for the analysis of dermal features such as collagen characteristics and analyses per each skin layer.
In conclusion, RCM is a non-invasive technique that has shown promise in monitoring a variety of laser treatments in dermatology providing insights into the observation of variations associated with clinical improvement. RCM features identified in our systematic review can be applied to future studies to objectively evaluate changes in the skin and improve our understanding of the mechanisms of action of lasers. By facilitating noninvasive, real-time evaluation of skin changes, RCM can assist clinicians in tailoring laser treatments to meet the unique needs of individual patients, ultimately leading to optimal treatment outcomes.