Anti‐Stokes fluorescence from endogenously formed protoporphyrin IX – Implications for clinical multiphoton diagnostics

Multiphoton imaging based on two‐photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two‐photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one‐photon anti‐Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Label‐free spectroscopic tissue characterization using fluorescence excitation‐scanning spectral imaging

Spectral imaging approaches provide new possibilities for measuring and discriminating fluorescent molecules in living cells and tissues. These approaches often employ tunable filters and robust image processing algorithms to identify many fluorescent labels in a single image set. Here, we present results from a novel spectral imaging technology that scans the fluorescence excitation spectrum, demonstrating that excitation‐scanning hyperspectral image data can discriminate among tissue types and estimate the molecular composition of tissues. This approach allows fast, accurate quantification of many fluorescent species from multivariate image data without the need of exogenous labels or dyes. We evaluated the ability of the excitation‐scanning approach to identify endogenous fluorescence signatures in multiple unlabeled tissue types. Signatures were screened using multi‐pass principal component analysis. Endmember extraction techniques revealed conserved autofluorescent signatures across multiple tissue types. We further examined the ability to detect known molecular signatures by constructing spectral libraries of common endogenous fluorophores and applying multiple spectral analysis techniques on test images from lung, liver and kidney. Spectral deconvolution revealed structure‐specific morphologic contrast generated from pure molecule signatures. These results demonstrate that excitation‐scanning spectral imaging, coupled with spectral imaging processing techniques, provides an approach for discriminating among tissue types and assessing the molecular composition of tissues. Additionally, excitation scanning offers the ability to rapidly screen molecular markers across a range of tissues without using fluorescent labels. This approach lays the groundwork for translation of excitation‐scanning technologies to clinical imaging platforms.     

Video‐assisted parathyroid gland mapping with autofocusing

Preservation of the parathyroid gland (PTG) in neck endocrine surgery is important for regulating the amount of calcium in the blood and within the bones. Localization of the PTG has been attempted using various methods such as ultrasound, sestamibi, computerized tomography, magnetic resonance imaging and indocyanine green fluorescence imaging. These methods cannot be used during surgery, have high sensitivity or have PTG specificity. However, autofluorescence technique has shown high sensitivity and does not require exogenous contrast. In this study, a new optical system was designed and developed into a clinical system. The system enabled easier and faster focusing on the surgical area and high‐resolution video imaging while maintaining a clear image. The system was located above the head of the surgeon. The surgeon was able to see the real‐time autofluorescent image on the monitor next to the operating table at any time to locate the PTG. The PTG buried in the adipose tissue and connective tissue was located easily and accurately. The clinical trial conducted in this study consisted of 56 parathyroid cases in 26 patients. For the statistical results, the sensitivity and accuracy in this redesigned autofluorescent imaging system were 98.1% and 96.4%, respectively.      

Quantitative and rapid estimations of human sub‐surface skin mass using ultra‐high‐resolution spectral domain optical coherence tomography

Non‐invasive and quantitative estimations for the delineation of sub‐surface tumor margins could greatly aid in the early detection and monitoring of the morphological appearances of tumor growth, ensure complete tumor excision without the unnecessary sacrifice of healthy tissue, and facilitate post‐operative follow‐up for recurrence. In this study, a high‐speed, non‐invasive, and ultra‐high‐resolution spectral domain optical coherence tomography (UHR‐SDOCT) imaging platform was developed for the quantitative measurement of human sub‐surface skin mass. With a proposed robust, semi‐automatic analysis, the system can rapidly quantify lesion area and shape regularity by an en‐face‐oriented algorithm. Various sizes of nylon sutures embedded in pork skin were used first as a phantom to verify the accuracy of our algorithm, and then in vivo, feasibility was proven using benign human angiomas and pigmented nevi. Clinically, this is the first step towards an automated skin lesion measurement system.

In vivo optical coherence tomography (OCT) image of angioma (A). Thin red arrows point to a blood vessel (BV).     

Collagen peptides modulate the metabolism of extracellular matrix by human dermal fibroblasts derived from sun‐protected and sun‐exposed body sites

Clinical data published in recent years have demonstrated positive effects of collagen hydrolysate (CH) on skin aging clinical signs. CH use as food supplement has a long history; however, few studies have addressed the underlying purpose of CH on the cellular and molecular biology of skin cells that could elucidate clinical improvement findings. Wide diversity of characteristics has been reported for dermal fibroblasts derived from different body sites and it is unknown whether collagen peptides could modulate differently cells from chronological aged and photoaged skin areas. This study investigated the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from chronological aged (sun‐protected) and photoaged (sun‐exposed) body sites. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of dermal matrix precursor and main protein, procollagen I and collagen I, respectively. These effects were confirmed in the human dermal equivalent model. The increase in collagen content in the cultures was attributed to stimulation of biosynthesis and decreased collagen I metabolism through inhibition of metalloproteinase activity (MMP) 1 and 2. Modulation of CH in dermal metabolism did not differ between cells derived from sun‐protected and sun‐exposed areas, although lower concentrations of CH seemed to be enough to stimulate sun‐exposed‐derived HDFs, suggesting more pronounced effect in these cells. This study contributes to understanding the biological effects of CH on skin cells and viability of its use as a functional ingredient in food supplements.     

High‐intensity‐focused ultrasound and phase‐sensitive optical coherence tomography for high resolution surface acoustic wave elastography

Elastography has the ability of quantitatively evaluating the mechanical properties of soft tissue; thus it is helpful for diagnosis and treatment monitoring of many diseases, for example, skin diseases. Surface acoustic waves (SAWs) have been proven to be a non‐invasive, non‐destructive method for accurate characterization of tissue elastic properties. Current SAW elastography using high‐energy laser pulse or mechanical shaker still have some problems. In order to improve SAW elastography in medical application, a new technique was proposed in this paper, which combines high‐intensity‐focused ultrasound as a SAWs impulse inducer and phase‐sensitive optical coherence tomography as a SAWs detector. A 2% agar‐agar phantom and ex‐vivo porcine skin were tested. The data were processed by a new algorithm based on the Fourier analysis. The results show that the proposed method has the capability of quantifying the elastic properties of soft tissue‐mimicking materials. The lateral resolution of the elastogram has been significantly improved and the different layers in heterogeneous material could also been distinguished. Our improved technique of SAW elastography has a large potential to be widely applied in clinical use for skin disease diagnosis and treatment monitoring.      

Stem cell recovering effect of copper‐free GHK in skin

The peptide Gly‐His‐Lys (GHK) is a naturally occurring copper(II)‐chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper‐GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper‐GHK promotes the survival of basal stem cells in the skin. However, the effects of copper‐free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and β1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK‐treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper‐free GHK showed similar effects with copper‐GHK. Thus, it can be said that copper‐free GHK can be used in industry to obtain the effects of copper‐GHK in vivo. Further study is necessary to explore the relationship between copper‐free GHK and copper‐GHK. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Migration‐ and Proliferation‐Promoting Activities in Human Keratinocytes of Zea mays Flower Absolute and Its Chemical Composition

Zea mays L. (ZM) has cytotoxic and anti‐inflammatory activities, but its biological activities such as skin regeneration and wound healing in human skin have not been reported. In the present study, we tested the effects of ZM flower (ZMF) absolute on proliferation and migration of human keratinocytes (HaCaTs) and identified its components by using gas chromatography/mass spectrometry (GC/MS) analysis. GC/MS analysis revealed that the ZMF absolute contained 13 constituents, and it increased HaCaT proliferation and migration. The ZMF absolute enhanced the phosphorylation levels of serine/threonine‐specific protein kinase (Akt), p38 mitogen‐activated protein kinase (MAPK), and extracellular signal‐regulated kinase1/2 in HaCaTs. In addition, the absolute induced an increase in sprout outgrowth of HaCaTs. The present study reports for the first time that ZMF absolute may promote skin wound healing and/or skin regeneration by stimulating proliferative and migratory activities in dermal keratinocytes through the Akt/MAPK pathway. Therefore, ZMF absolute may be a promising natural material for the use in skin regeneration and/or wound healing applications.     

Interactions of donor sources and media influence the histo‐morphological quality of full‐thickness skin models

Human artificial skin models are increasingly employed as non‐animal test platforms for research and medical purposes. However, the overall histopathological quality of such models may vary significantly. Therefore, the effects of manufacturing protocols and donor sources on the quality of skin models built‐up from fibroblasts and keratinocytes derived from juvenile foreskins is studied. Histo‐morphological parameters such as epidermal thickness, number of epidermal cell layers, dermal thickness, dermo‐epidermal adhesion and absence of cellular nuclei in the corneal layer are obtained and scored accordingly. In total, 144 full‐thickness skin models derived from 16 different donors, built‐up in triplicates using three different culture conditions were successfully generated. In univariate analysis both media and donor age affected the quality of skin models significantly. Both parameters remained statistically significant in multivariate analyses. Performing general linear model analyses we could show that individual medium‐donor‐interactions influence the quality. These observations suggest that the optimal choice of media may differ from donor to donor and coincides with findings where significant inter‐individual variations of growth rates in keratinocytes and fibroblasts have been described. Thus, the consideration of individual medium‐donor‐interactions may improve the overall quality of human organ models thereby forming a reproducible test platform for sophisticated clinical research.     

双光子显微镜在生物医学中的应用及其进展

光学显微镜的发展历史是一段不断提高显微镜的分辨率和对比度的历史。双光子显微镜是近30年来非线性显微镜的研究发展的代表。它在分辨率上与共聚焦显微镜相当,但在成像的层析穿透深度上有显著提高,并且大大减少了光毒性与光漂白。由于生物细胞组织中富有各种自家荧光源,因此双光子显微镜被广泛应用于皮肤组织甚至癌组织以及细胞的成像。基于共聚焦扫描显微镜的双光子显微镜可以很容易的与二次谐波显微镜组合,对皮肤组织中的重要成分胶原纤维进行成像。双光子显微镜还可以结合其他非线性光学现象对组织以及细胞进行成像,显示其强大的生命力。将来随着携带方便且廉价的双光子显微镜的出现,双光子显微镜有望在临床医学上发挥其有效的作用。     

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

A compact high‐speed full‐field optical coherence microscope for high‐resolution in vivo skin imaging

A compact high‐speed full‐field optical coherence microscope has been developed for high‐resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm³ and a weight of 210 g. Full‐field illumination with low‐coherence light is achieved with a high‐brightness broadband light‐emitting diode. High‐speed full‐field detection is achieved by using part of the image sensor of a high‐dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 μm × 1.2 μm (axial × transverse), over a field of view of 245 × 245 μm². Images of human skin, revealing in‐depth cellular‐level structures, were obtained in vivo and in real‐time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm², but at a reduced depth.      

TLR4‐mediated skin carcinogenesis is dependent on immune and radioresistant cells

Skin cancers are the most commonly diagnosed cancers. Understanding what are the factors contributing to skin tumour development can be instrumental to identify preventive therapies. The myeloid differentiation primary response gene (MyD)88, the downstream adaptor protein of most Toll‐like receptors (TLR), has been shown to be involved in several mouse tumourigenesis models. We show here that TLR4, but not TLR2 or TLR9, is upstream of MyD88 in skin tumourigenesis. TLR4 triggering is not dependent on lipopolysaccharide associated to skin‐colonizing bacteria, but on the high mobility group box‐1 protein (HMGB1), an endogenous ligand of TLR4. HMGB1 is released by necrotic keratinocytes and is required for the recruitment of inflammatory cells and for the initiation of inflammation. The expression of TLR4 on both bone marrow‐derived and radioresistant cells is necessary for carcinogenesis. Consistently, a human tissue microarray analysis showed that melanoma and colon cancer display an over‐expression of TLR4 and its downstream adaptor protein MyD88 within tumours. Together, our results suggest that the initial release of HMGB1 triggers a TLR4‐dependent inflammatory response that leads to tumour development.     

Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently‐labeled cells in highly autofluorescent tissue

Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP‐expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single‐band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Oncoprotein BMI‐1 induces the malignant transformation of HaCaT cells

BMI‐1 (B‐cell‐specific Moloney murine leukemia virus integration site 1), a novel oncogene, has attracted much attention in recent years for its involvement in the initiation of a variety of tumors. Recent evidence showed that BMI‐1 was highly expressed in neoplastic skin lesions. However, whether dysregulated BMI‐1 expression is causal for the transformation of skin cells remains unknown. In this study, we stably expressed BMI‐1 in a human keratinocyte cell line, HaCaT. The expression of wild‐type BMI‐1 induced the malignant transformation of HaCaT cells in vitro. More importantly, we found that expression of BMI‐1 promoted formation of squamous cell carcinomas in vivo. Furthermore, we showed that BMI‐1 expression led to the downregulation of tumore suppressors, such as p16INK4a and p14ARF, cell adhesion molecules, such as E‐Cadherin, and differentiation related factor, such as KRT6. Therefore, our findings demonstrated that dysregulated BMI‐1 could indeed lead to keratinocytes transformation and tumorigenesis, potentially through promoting cell cycle progression and increasing cell mobility. J. Cell. Biochem. 106: 16–24, 2009. © 2008 Wiley‐Liss, Inc.     

Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited autofluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

UVA influenced the SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in human skin primary fibroblasts

Both SIRT1 and UVA radiation are involved in cellular damage processes such as apoptosis, senescence and ageing. MicroRNAs (miRNAs) have been reported to be closely related to UV radiation, as well as to SIRT1. In this study, we investigated the connections among SIRT1, UVA and miRNA in human skin primary fibroblasts. Our results showed that UVA altered the protein level of SIRT1 in a time point–dependent manner. Using miRNA microarray, bioinformatics analysis, we found that knocking down SIRT1 could cause up‐regulation of miR‐27a‐5p and the latter could down‐regulate SMAD2, and these results were verified by qRT‐PCR or Western blot. Furthermore, UVA radiation (5 J/cm²), knocking down SIRT1 or overexpression of miR‐27a‐5p led to increased expression of MMP1, and decreased expressions of COL1 and BCL2. We also found additive impacts on MMP1, COL1 and BCL2 under the combination of UVA radiation + Sirtinol (SIRT1 inhibitor), or UVA radiation + miR‐27a‐5p mimic. SIRT1 activator resveratrol could reverse damage changes caused by UVA radiation. Besides, absent of SIRT1 or overexpression of miR‐27a‐5p increased cell apoptosis and induced cell arrest in G2/M phase. Taken together, these results demonstrated that UVA could influence a novel SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in skin primary fibroblasts, and may provide potential therapeutic targets for UVA‐induced skin damage.     

Application of the Isoenzyme Profile in the Development of Cell‐Based Devices

To detect cell cross‐contamination and verify the origin of the cells of an artificial organ, the sensitive isoenzyme assay was chosen to monitor the quality test of cell‐based devices. Authoritative cell evaluation of artificial skin products has been established in this study. Human and porcine cell suspensions with total cell counts of between 1×10⁵ and 4×10⁶ were individually tested to determine the activity of isoenzymes. Human fibroblast, mixed with 1% to 100% of porcine fibroblast, could be significantly distinguished in the isoenzyme assay. Based on the glucose‐6‐phosphophate‐dehydrogenase analysis, the human fibroblast tested in this study belonged to the B type human cells. Lactate dehydrogenase (LD), malate dehydrogenase (MD) and mannose phosphate isomerase isoenzyme (MPI) activities obviously revealed that a different pattern corresponds to the percentage of human and porcine cell mixtures. The discriminatory limit of MPI, LD and MD activity can reach up to 1% of sensitivity of the isoenzyme analysis. This sensitive isoenzyme analysis method allows us to routinely test cellular biomaterials whether interspecies cell line cross‐contamination has occurred in the development of artificial organs.