Feature characterization of scarring and non-scarring types of alopecia by multiphoton microscopy.

OBJECTIVES: Non-invasive visualization of hair follicles is important for proper diagnosis and management of alopecia; however, histological assessment remains the gold standard. Laser imaging technologies have made possible noninvasive in vivo evaluation of skin and hair follicle. The aim of this study was to evaluate the ability of multiphoton microscopy (MPM) to non-invasively identify morphological features that can distinguish scarring from non-scarring alopecia. METHODS: MPM images were obtained from areas on the scalp affected by alopecia. Investigators blinded to the diagnosis analyzed hair follicle and shaft sizes. Patients were recruited and imaged at the UC Irvine Health Medical Center and the University of California, Irvine Beckman Laser Institute. Patients with androgenetic alopecia (AGA) and alopecia areata (AA), and scarring alopecia, in particular frontal fibrosing alopecia (FFA) were recruited and imaged from July 2016 to July 2017. RESULTS: We imaged 5 normal scalp subjects and 12 patients affected by non-scarring (7 subjects) and scarring (5 subjects) alopecia. In normal and non-scarring alopecia patients, MPM identified presence of sebaceous glands associated with hair follicles. MPM images of scarring alopecia were characterized by the presence of inflammatory cells surrounding hair follicles. Measurements of hair follicle diameter sizes were found to be significantly smaller in scarring alopecia patients compared to normal (P < 0.001) and compared to non-scarring alopecia patients (P = 0.046); non-scarring hair follicles were also significantly smaller than normal hair follicles (P = 0.043). CONCLUSIONS: This study shows that MPM imaging can non-invasively identify morphological features that distinguish scarring from non-scarring alopecia. Further studies are needed to validate this technique and evaluate its potential to be used as an aid for guiding treatment. Lasers Surg. Med. 51:95-103, 2019. © 2018 Wiley Periodicals, Inc.

From morphology to biochemical state - intravital multiphoton fluorescence lifetime imaging of inflamed human skin.

The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients' bedsides. These 'optical biopsies' generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

Imaging the Different Mechanisms of Prostate Cancer Cell-killing by Tumor-targeting Salmonella typhimurium A1-R.

BACKGROUND/AIMS: We previously developed a genetically-modified bacterial strain of Salmonella typhimurium, auxotrophic for leucine and arginine, which also expresses green fluorescent protein (GFP), termed S. typhimurium A1-R. S. typhimurium A1-R was found to be effective against metastatic human prostate, breast, pancreatic, cervical and ovarian cancer, as well as osteosarcoma, fibrosarcoma and glioma, in clinically relevant nude mouse models. MATERIALS AND METHODS: To understand the tumor cell-killing mechanism of S. typhimurium A1-R-GFP, we studied the interaction of S. typhimurium A1-R-GFP with three different prostate cancer cell lines in vitro. S. typhimurium-GFP invasion, proliferation, and means of killing in three different human prostate cancer cell lines were visualized by confocal fluorescence microscopy with the Olympus FV1000. RESULTS: We found that S. typhimurium A1-R-induced cancer-cell death had different mechanisms in different prostate cancer cell lines, occurring through apoptosis and necrosis in the PC-3 prostate cancer cell line, and by cell bursting in the LNCaP and DU-145 prostate cancer cell lines. The time required for S. typhimurium A1-R-GFP to kill the majority of cancer cells varied from line to line, ranging from 2 hours to 48 hours. CONCLUSION: Understanding the various mechanisms of cancer-cell killing by S. typhimurium A1-R will be important for its use as a general therapeutic for cancer.

The bulge area is the major hair follicle source of nestin-expressing pluripotent stem cells which can repair the spinal cord compared to the dermal papilla.

Nestin has been shown to be expressed in the hair follicle, both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be pluripotent and be able to form neurons and other non-follicle cell types. The nestin-expressing pluripotent stem cells from the DP have been termed skin precursor or SKP cells. The objective of the present study was to determine the major source of nestin-expressing pluripotent stem cells in the hair follicle and to compare the ability of the nestin-expressing pluripotent stem cells from the BA and DP to repair spinal cord injury. Transgenic mice in which the nestin promoter drives GFP (ND-GFP) were used in order to observe nestin expression in the BA and DP. Nestin-expressing DP cells were found in early and middle anagen. The BA had nestin expression throughout the hair cycle and to a greater extent than the DP. The cells from both regions had very long processes extending from them as shown by two-photon confocal microscopy. Nestin-expressing stem cells from both areas differentiated into neuronal cells at high frequency in vitro. Both nestin-expressing DP and BA cells differentiated into neuronal and glial cells after transplantation to the injured spinal cord and enhanced injury repair and locomotor recovery within four weeks. Nestin-expressing pluripotent stem cells from both the BA and DP have potential for spinal cord regeneration, with the BA being the greater and more constant source.

In-vivo intratissue ablation by nanojoule near-infrared femtosecond laser pulses.

Non-invasive intratissue ablation was performed in the cornea of living rabbits by using 80 MHz near-infrared intense nanojoule femtosecond laser pulses. The intratissue surgical effect was induced by multiphoton absorption at a wavelength of 800 nm and was ascertained by histological examination. Highly precise intratissue ablation was obtained with no detrimental effects to the overlying or underlying layers. Activated keratocytes in the laser-treated corneas were detected with two-photon imaging postoperatively. Intratissue femtosecond laser ablation thus has potential as a effective technique in refractive surgery for the treatment of visual disorders.

Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers.

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm2. The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 x 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman's layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.

Intratissue surgery with 80 MHz nanojoule femtosecond laser pulses in the near infrared.

The use of 1 nanojoule near infrared 80 MHz femtosecond laser pulses for highly precise intratissue processing, in particular for intraocular refractive surgery, was evaluated. Destructive optical breakdown at TW/cm2 light intensities in a subfemtoliter intrastromal volume was obtained by diffraction-limited focussing with an 40x objective (N.A. 1.3) and beam scanning 50 to 140 microm below the epithelial surface. Using the same system at GW/cm2 intensities two-photon excited autofluorescence imaging was used to determine the target of interest and to visualize intraocular laser effects. Histological examination of laser-exposed porcine eyes reveal a minimum cut size below 1 microm without destructive effects to surrounding tissues.