In vivo imaging of pigmented lesions in human skin was performed with a clinical multiphoton microscopy (MPM)- based tomograph (MPTflex, JenLab, Germany). Two-photon excited fluorescence was used for visualizing endogenous fluorophores such as NADH/FAD, keratin, melanin in the epidermal cells and elastin fibers in the dermis. Collagen fibers were imaged by second harmonic generation. Our study involved in vivo imaging of benign melanocytic nevi, atypical nevi and melanoma. The goal of this preliminary study was to identify in vivo the characteristic features and their frequency in pigmented lesions at different stages (benign, atypical and malignant) and to evaluate the ability of in vivo MPM to distinguish atypical nevi from melanoma. Comparison with histopathology was performed for the biopsied lesions. Benign melanocytic nevi were characterized by the presence of nevus cell nests at the epidermal-dermal junction. In atypical nevi, features such as lentiginous hyperplasia, acanthosis and architectural disorder were imaged. Cytological atypia was present in all the melanoma lesions imaged, showing the strongest correlation with malignancy. The MPM images demonstrated very good correlation with corresponding histological images, suggesting that MPM could be a promising tool for in vivo non-invasive pigmented lesion diagnosis, particularly distinguishing atypical nevi from melanoma. © 2013 SPIE.

beta-nicotinamide adenine dinucleotide (NADH)-alcohol dehydrogenase complex was compared to either UVA irradiation (364 nm; 50 mW cm-2; 0-60 min) or heat in order to investigate complex stability. Prior to irradiation, frequency-domain fluorescence lifetime measurements indicated the presence of two principal components having short (subnanosecond) and long (nanosecond) fluorescence lifetimes reflecting free and bound NADH respectively. UVA exposure resulted in decreased NADH fluorescence intensity concomitant with decreased absorption at 337 nm. However, UVA irradiation did not reduce the fractional contribution of the long-lived bound NADH. The photoinduced fluorescence decrease appeared to be caused by the formation of oxidized NAD+ and not on UVA-induced dissociation of the NADH-protein complex. Such dissociation, detected by a red-shifted fluorescence maximum and decreased fractional contribution of the nanosecond component, was observed when NADH-protein mixtures were heated.

Photostress has to be considered during optical micromanipulation of gametes. Ultraviolet light, including low-energy UVA (32-400 nm) radiation, as well as high-intensity near infrared (NIR) laser radiation may induce cell damage. A total number of 580 light-exposed sperm cells were studied in single-cell photostress experiments. Low-power (1.5 mW, 5.3 W/cm2) UVA exposure with 365 nm radiation of a standard mercury microscopy lamp to human spermatozoa resulted within 109 +/- 30 s in paralysis and within 310 +/- 110 s in cell death. Cytotoxic effects during cell manipulation with laser microbeams were found to be partly based on non-linear excitation phenomena, in particular two-photon absorption by endogenous cell chromophores. Two-photon absorption will be more intense in the case of pulsed laser microradiation, but occur also during micromanipulation with highly focused continuous wave (cw) microbeams used as laser tweezers ('optical traps'). In particular, short-wavelength NIR traps < 800 nm induce UVA-like biological effects (oxidative stress). For example, sperm trapping with 760 nm microbeams resulted in UVA-like autofluorescence modifications, paralysis within 35 +/- 20 s and cell death within 65 +/- 20 s. In contrast, laser microbeams at 800-1064 nm may act as relatively safe micromanipulation tools. In most optical traps multifrequency cw lasers are employed. Radiation of these lasers can magnify cytotoxic effects. Therefore, single-frequency laser operation should be preferred. In general, laser assisted cell micromanipulation requires a new understanding of microbeam-cell interaction, including aspects of non-linear optics.

The development of clinical multiphoton technologies has led to new, labelfree approaches for non-invasive, in vivo imaging of human skin. Recent studies have shown that multiphoton imaging can be used to assess a wide range of biological processes, including cancer, cellular metabolism, and the effects of skin treatments. The imaging devices MPTflex and its earlier version, DermaInspect, have been employed in a broad range of clinical applications spanning from characterizing and understanding keratinocyte metabolism to malignant melanoma detection and diagnosis, pigment biology, cosmetic treatments, and skin aging. The promising results indicate that in the near future, real-time non-invasive nonlinear “optical biopsies” can be performed at the bedside.

We constructed an advanced detection system for two-photon fluorescence microscopy that allows us to image in biological tissue and tissue phantoms up to the depth of a few mm with micron resolution. The innovation lies in the detection system which is much more sensitive to low level fluorescence signals than the fluorescence detection configuration used in conventional two-photon fluorescence microscopes. A wide area photocathode photomultiplier tube (PMT) was used to detect fluorescence photons directly from a wide (1 inch diameter) area of the turbid sample, as opposed to the photon collection by the microscope objective which can only collect light from a relatively small area of the sample. The optical path between the sample and the photocathode is refractive index matched to curtail losses at the boundaries due to reflections. The system has been successfully employed in the imaging of tissue phantoms simulating brain optical properties and in biological tissues, such as murine small intestine, colon, tumors, and other samples. The system has in-depth fluorescence lifetime imaging (FLIM) capabilities and is also highly suitable for SHG signal detection, such as collagen fibers and muscles, due to the intrinsically forward-directed propagation of SHG photons. © 2013 SPIE.

We use the phasor approach to fluorescence lifetime imaging and intrinsic biochemical fluorescence biomarkers in conjunction with image segmentation and the concept of cell phasor for deriving metabolic maps of cells and living tissues in vivo. In issues we identify and separate intrinsic fluorophores such as collagen, retinol, retinoic acid, porphyrin, flavins, free and bound nicotinamide adenine dinucleotide (NADH). Metabolic signatures of tissues are obtained by calculating the phasor fingerprint of single cells and by mapping the relative concentration of metabolites. This method detects small changes in metabolic signatures and redox states of cells. Phasor fingerprints of stem cells cluster according to their differentiation state in a living tissue such as the C. elegans germ line and the crypt base of small intestine and colon. Phasor FLIM provides a label-free and fit-free sensitive method to identify metabolic states of cells and to classify stem cells, normal differentiated cells and cancer cells both in vitro and in a live tissue. Our method could identify symmetric and asymmetric divisions, predict cell fate and identify pre-cancer stages in vivo. This method is a promising non-invasive optical tool for monitoring metabolic pathways during differentiation and carcinogenesis, for cell sorting and high throughput screening. © 2012 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).

Importance

Improvements in skin appearance resulting from treatment with fractionated picosecond-lasers have been noted, but optimizing the treatment efficacy depends on a thorough understanding of the specific skin response. The development of non-invasive laser imaging techniques in conjunction with laser therapy can potentially provide feedback for guidance and optimizing clinical outcome.

Objective

The purpose of this study was to demonstrate the capability of multiphoton microscopy (MPM), a high-resolution, label-free imaging technique, to characterize in vivo the skin response to a fractionated non-ablative picosecond-laser treatment.

Design, setting, and participants

Two areas on the arm of a volunteer were treated with a fractionated picosecond laser at the Dermatology Clinic, UC Irvine. The skin response to treatment was imaged in vivo with a clinical MPM-based tomograph at 3 hours and 24 hours after treatment and seven additional time points over a 4-week period.

Main outcomes and measures

MPM revealed micro-injuries present in the epidermis. Pigmented cells were particularly damaged in the process, suggesting that melanin is likely the main absorber for laser induced optical breakdown.

Results

Damaged individual cells were distinguished as early as 3 hours post pico-laser treatment with the 532 nm wavelength, and 24 hours post-treatment with both 532 and 1064 nm wavelengths. At later time points, clusters of cellular necrotic debris were imaged across the treated epidermis. After 24 hours of treatment, inflammatory cells were imaged in the proximity of epidermal micro-injuries. The epidermal injuries were exfoliated over a 4-week period.

Conclusions and relevance

This observational and descriptive pilot study demonstrates that in vivo MPM imaging can be used non-invasively to provide label-free contrast for describing changes in human skin following a fractionated non-ablative laser treatment. The results presented in this study represent the groundwork for future longitudinal investigations on an expanded number of subjects to understand the response to treatment in different skin types with different laser parameters, critical factors in optimizing treatment outcome. Lasers Surg. Med. 49:555-562, 2017. © 2017 Wiley Periodicals, Inc.

Importance

Basal cell carcinomas (BCCs) are diagnosed by clinical evaluation, which can include dermoscopic evaluation, biopsy, and histopathologic examination. Recent translation of multiphoton microscopy (MPM) to clinical practice raises the possibility of noninvasive, label-free in vivo imaging of BCCs that could reduce the time from consultation to treatment.

Objectives

To demonstrate the capability of MPM to image in vivo BCC lesions in human skin, and to evaluate if histopathologic criteria can be identified in MPM images.

Design, setting, and participants

Imaging in patients with BCC was performed at the University of California-Irvine Health Beckman Laser Institute & Medical Clinic, Irvine, between September 2012 and April 2014, with a clinical MPM-based tomograph. Ten BCC lesions were imaged in vivo in 9 patients prior to biopsy. The MPM images were compared with histopathologic findings.

Main outcomes and measures

MPM imaging identified in vivo and noninvasively the main histopathologic feature of BCC lesions: nests of basaloid cells showing palisading in the peripheral cell layer at the dermoepidermal junction and/or in the dermis.

Results

The main MPM feature associated with the BCC lesions involved nests of basaloid cells present in the papillary and reticular dermis. This feature correlated well with histopathologic examination. Other MPM features included elongated tumor cells in the epidermis aligned in 1 direction and parallel collagen and elastin bundles surrounding the tumors.

Conclusions and relevance

This study demonstrates, in a limited patient population, that noninvasive in vivo MPM imaging can provide label-free contrast that reveals several characteristic features of BCC lesions. Future studies are needed to validate the technique and correlate MPM performance with histopathologic examination.

Modifications of the porphyrin fluorescence during photodynamic treatment of ovarian cells, endometrial adenocarcinoma cells and rat uterus after administration of the porphyrin precursor δ-aminolevulinic acid and exposure with 630 nm laser radiation have been studied. The intracellular and intratissue fluorescence was excited with 407 nm radiation of a krypton ion laser and detected by spectral microscopic imaging using a modified microscope equipped with a slow-scan cooled camera and a liquid crystal filter. In particular, singlet oxygen induced effects due to photobleaching as well as formation of chlorin-type fluorescent photoproducts were detected at around 500, 630 and 670 nm. The photodynamically induced fluorescence modifications were found to vary between cell types. The major fluorescence peak of PP IX was photobleached 63% by a phototoxic fluence of about 20-40 J/cm2 in cells and within rat endometrium. Higher fluences were required to bleach the fluorescence at around 670 nm due to the formation of fluorescent photoproducts. No significant photoinduced modifications of autofluorescence occurred around 500 nm. © 2006 Elsevier GmbH. All rights reserved.