Label-free virtual-histology of skin tissues using a Photoacoustic Remote Sensing (PARS) microscope is explored. The PARS captures optical absorption contrast facilitating label-free imaging of numerous biomolecules. Combined with AI-based image processing, PARS provides virtual histology images label-free directly from unprocessed tissues. The label-free contrast means several virtual stains may be applied to each tissue section. Subsequent results are explored in unprocessed human skin with basal cell carcinoma, and squamous cell carcinoma. Clinical comparison studies indicate there is similar diagnostic quality between the PARS and gold-standard histology preparations. This represents a significant milestone in the development of a clinically ready microscope for label-free skin cancer histology.

Ex vivo confocal microscopy (EVCM) images tissues rapidly at near histological resolution without the need for histological processing. Other health institutes can benefit by sending their tissues to confocal experts in formalin, a readily available media that preserves tissue integrity. Fresh tissues imaged with an EVCM device at various timepoints after being put in formalin exhibited easier tissue flattening, improved visualization of the epidermis, reduced tissue movement (due to fat fixation), improved image contrast, and lack of photobleaching (due to dye fixation). Normal skin structures and tumors were readily identified at all TPs by an expert in real-time.

We developed a rapid tissue clearing technique combined with two-photon fluorescence microscopy (cFTPM) to image melanoma in situ biopsy volumetrically. cFTPM can provide rapid volumetric melanoma in situ biopsy images with the potential to provide same-day melanoma biopsy turnaround time with full volumetric images. The overall time span of our process, including tissue clearing and imaging, is faster than conventional histology processing.

A non-invasive means to determine lesion depth (I.e. Breslow Thickness) and an indirect, lesion-specific assessment of cellular morphology could substantially improve the efficacy of melanoma screening. Such applications would be of great benefit in aiding diagnosis, reducing the number of unnecessary biopsies, and improving treatment options for patients. Using Spatial Frequency Domain Imaging (SFDI), we have developed a spectral method to isolate depth-specific scattering properties and hence, differentiate pigmented lesion volumes from underlying tissue by its structural morphology. This method was evaluated on solid phantoms that emulate the optical properties of suspect pigmented lesions over multiple thicknesses.

Accurately classifying burn severity is crucial to inform proper treatment. Here, we quantitatively compare the efficacy of machine learning (ML) burn classification algorithms using multispectral imaging versus conventional digital color imaging data as inputs. We imaged 80 porcine burns that underwent biopsy and histology for ground truth categorization into “skin graft needed” versus “no graft needed” groups. The accuracy of our ML algorithm with a transfer learning architecture was 97.5% for the multispectral model, 57.5% for the digital-color model, and 57.5% for the multispectral+digital-color model. This result strongly supports the use of multispectral imaging over digital-color imaging for burn classification.

Confocal Raman spectroscopy (CRS) has been used for noninvasive biochemical analysis of biological tissue in vivo. Reflectance confocal microscopy (RCM) has been integrated with CRS as visual guidance to facilitate simultaneous square-shaped region or point of interest micro-Raman measurement. However, these methods do not allow the acquisition of representative Raman spectra from and only from the area that covers the target micro-structure that has various morphologies. To solve this problem, we developed a method to acquire Raman spectra from a region of interest that has an arbitrary shape under simultaneous RCM imaging guidance using a single laser source.

Auretek is the first of its kind portable Confocal Raman Spectroscopy (CRS) system equipped with fiber based handheld probe, that cascades dual-wavelength lasers. With its flexible probe, Auretek can acquire CRS data of skin from various parts of the human body with depth profiling. A clinical study was conducted to investigate the effects of ceramide-based moisturizer (CBM) on skin physiology and biochemistry. Quantitative skin component characterization in different epidermal layers was done with spectral unmixing analysis of the CRS data. Even though the analysis revealed a significant increase in ceramide content on the side of CBM application, the increase was in the layers within the stratum corneum.

Inflammatory skin diseases affect a large proportion of the population, for instance the prevalence of eczema is 20%. However, objective disease measurements to assess severity are lacking for this common inflammatory skin condition. In this study, we assess the use of a non-invasive optical method to characterize different biomarkers in eczema patients. Raman spectroscopy which measures molecular bond vibrations was used to assess different disease characteristics including keratin, ceramide, protein and water content. Eczema patients showed a lower amount of lipid content (eg. ceramides), and asymmetric protein and bound water. These findings will be further explored to correlate with disease severity.

This project proposes an alternate method to DIN/ISO parameters for analyzing the effects of intrinsic and extrinsic aging on skin morphology. This method improves the efficacy of assessing aging by evaluating the surface and subsurface of the skin. The volumetric data obtained using OCT was processed using a machine learning approach to obtain DEJ segmentation, which was further segmented to obtain the morphological information of the micro-plateaus. The results showed that the flattening of DEJ due to aging is accompanied by changes in the size and shape of micro-plateaus in the DEJ.

In this study, a portable and ultrahigh-resolution OCT (UHR-OCT) system is developed, and the axial and transverse resolutions are approximately 2 and 5 m, respectively. Although picosecond lasers are widely used for clinical treatment, it is difficult to predict and observe the treatment outcome. The developed UHR-OCT system is implemented to observe the photodamage induced by the picosecond laser and the laser-induced optical breakdown (LIOB) can be observed. Furthermore, the recovery process is studied and compared by UHR-OCT. The results indicate that the UHR-OCT system can clearly identify the skin structures, and the induced photodamage can be quantitatively evaluated.

Crisaborole 2% ointment is a non-steroidal treatment for mild-moderate atopic dermatitis (AD) and may have a better safety profile than topical corticosteroids (TCS), which produce pronounced epidermal atrophy. In this study we compared crisaborole to betamethasone valerate (BMV) in terms of changes to dermal collagen, using PS-OCT to quantify dermal birefringence. 37 subjects were enrolled in this study, which involved daily application of BMV & crisaborole. Results showed BMV produced a significant increase in dermal birefringence, whereas crisaborole showed no significant change. PS-OCT may thus have an important role in the safety assessment of non-steroidal alternatives to TCS.

Cutaneous vascular malformations (CVM), such as port wine stain (PWS), often appear in highly visible parts of patient and cause emotional and social problems. In this work, we utilized our novel anti-vascular technique, termed photo-mediated ultrasound therapy (PUT), which combines nanosecond duration laser pulses synchronized with ultrasound bursts to treat blood vessels. The feasibility of PUT in the treatment of cutaneous microvessels was evaluated in a chicken wattle model in vivo. The treatment results showed perfusion stop in microvessels up to 1.5mm deep in chicken wattle, as demonstrated by both the OCT angiography and the color change in skin images after the treatment. The treatment effect and safety such as epidermal tissue damage and inflammation were also evaluated by H&E histology.

Pneumatic control devices were constructed to apply negative pressure on targeted human skin areas to increase or decrease blood flow for improving laser therapy. The effect of pneumatic control on blood flow in a cutaneous skin region was evaluated using optical coherence tomography-angiography (OCT-A). Dynamic changes in blood flow under the application of negative pressure to the dorsal hand and forearm skin were confirmed in acquired OCT-A images. We also observed the device size-dependent difference in blood flow change. Results suggest the feasibility of bi-directional blood flow control using a pneumatic control device.

Large area fractional laser treatment (LAFLT) is a new fat removal and weight loss concept, which applies microscopic thermal injuries to large parts of the skin. These injuries induce a controlled metabolic response which temporarily increases the energy expenditure (EE), thereby reducing overall weight. In a mouse model, we investigated the dosimetry settings to induce this metabolic reaction. Therefore, mice were housed in metabolic cages and their body composition was analyzed. Results showed a significant EE increase (31% - 91%, p < 0.05) over a 6-day period which seems to suggest that an enhanced metabolic state can be achieved.

Despite the effectiveness of using fractional picosecond 1064-nm Nd:YAG lasers for facial rejuvenation or resurfacing, the underlying mechanisms of the dermal remodeling with this nonablative treatment are not fully understood. We perform harmonic generation and reflectance confocal microscopy for the in vivo observation of laser-induced optical breakdown. We have found that the induced vacuoles extend to the papillary dermis using second harmonic generation imaging, which is different from the histopathology examinations and previous studies using optical coherence tomography and reflectance confocal microscopy. Our finding suggests that the fractional picosecond laser may induce collagen degeneration through suboptimal thermal effects.

This paper presents a hand-held single-mode fiber-based polarization-sensitive optical coherence tomography (PS-OCT) system with a single input polarization state. One drawback of this system is that the polarization becomes arbitrary, which can be solved by placing a polarization controller in the sample arm. A calibration target with an azimuthally varying optic axis integrated with the handheld probe will provide an absolute orientation axis measurement. This significantly reduces cost, time and availability of PS-OCT for clinical applications. With the home-built PS-OCT, we investigate the reorganization of collagen and blood vessel in a wound on the dorsal side of the human hand.

Intrinsic aging is associated with metabolic dysregulation and altered tissue organization. Using label-free multiphoton microscopy, skin was non-invasively imaged in vivo in young and aged mice and optical biomarkers of metabolism and altered collagen organization were compared. Aged skin demonstrated decreased NADH autofluorescence intensity, higher mitochondrial fractal dimensions, and longer mean NADH fluorescence lifetimes compared to young skin. Moreover, aged dermal collagen exhibited greater randomness of fiber organization. These findings demonstrate that multiphoton microscopy is a viable technique to monitor multiple structural and biochemical changes associated with the aging process.

This conference presentation was prepared for the Photonics in Dermatology and Plastic Surgery 2023 conference at SPIE BiOS, SPIE Photonics West 2023.

Detection of hemoglobin (tHb), water (tH2O), and microvascular perfusion dynamics is of great importance as these indicate early signs of tissue physiological changes related to diseases. We have developed a physiological model based on tissue absorption and scattering properties measured by a customized spatial frequency domain imaging (SFDI) system. We performed an in-vivo investigation to evaluate the imager’s ability to characterize dermal response under a noxious heating protocol. In this initial study, we found that noxious heating induced changes in extravascular water content, hemoglobin, and tissue morphology that can be interpreted as vascular perfusion and dilation, inflammation, and edema.

Here we propose a novel method to automatically delineate and quantify DEJ zone morphometrics in human skin on a three dimensional (3D) basis in vivo. Quantitative characterization of the DEJ in terms of 3D interdigitation (I), arithmetic mean roughness (Sa), and root mean square roughness (Sq) were calculated. These DEJ features as a function of age were analyzed. The results show age-dependent morphological differences.

This conference presentation was prepared for the Photonics in Dermatology and Plastic Surgery 2023 conference at SPIE BiOS, SPIE Photonics West 2023.

Monitoring hair follicle development is important to interpret hair pathophysiology and follicular-related diseases such as alopecia, folliculitis, acne, and keratosis pilaris. We introduce label-free visualization of hair follicles based on optical coherence microscopy (OCM) and quantitative analysis of the structure using OCM imaging data. The hair follicle regeneration of mice was monitored using our home-built serial OCM. Through this process, we obtained the volumetric anatomy and functional feature of the hair follicle. We also quantified the hair follicle structure including hair root, bulb, papilla, and follicle wall based on OCM data. Our result provides insight into the hair cycle investigation.

Polarization-sensitive optical coherence tomography (PS-OCT) is a promising tool for non-invasive skin imaging, with its capability for depth-resolved, high-resolution, fast, and polarization-sensitive imaging. Thirty volunteers are recruited for skin imaging on the palm, arm, forehead, and eye corner. A segmentation algorithm based on intensity images will segment the epidermis and dermis layers and also stratum corneum if applicable. Multi-contrast images, including phase retardation, local birefringence, and degree of polarization uniformity, will be obtained from different skin layers. Possible relationships between the optical properties and skin features including layered structure, collagen organization, melanin concertation, and skin roughness will be investigated.

This conference presentation was prepared for the Photonics in Dermatology and Plastic Surgery 2023 conference at SPIE BiOS, SPIE Photonics West 2023.