Detection of oral squamous cell carcinoma with ex vivo fluorescence confocal microscopy: Sensitivity and specificity compared to histopathology

Real‐time microscopic imaging of freshly excised tissue enables a rapid bedside‐pathology. A possible application of interest is the detection of oral squamous cell carcinomas (OSCCs). The aim of this study was to analyze the sensitivity and specificity of ex vivo fluorescence confocal microscopy (FCM) for OSCCs and to compare confocal images visually and qualitatively with gold standard histopathology. Two hundred eighty ex vivo FCM images were prospectively collected and evaluated immediately after excision. Every confocal image was blindly assessed for the presence or absence of malignancy by two clinicians and one pathologist. The results were compared with conventional histopathology with hematoxylin and eosin staining. OSCCs were detected with a very high sensitivity of 0.991, specificity of 0.9527, positive predictive value of 0.9322 and negative predictive value of 0.9938. The results demonstrate the potential of ex vivo FCM in fresh tissue for rapid real‐time surgical pathology.     

Identification of ex‐vivo confocal laser scanning microscopic features of melanocytic lesions and their histological correlates

Ex‐vivo confocal laser scanning microscopy (CLSM) offers rapid tissue examination. Current literature shows promising results in the evaluation of non‐melanoma skin cancer but little is known about presentation of melanocytic lesions (ML). This study evaluates ML with ex‐vivo CLSM in comparison to histology and offers an overview of ex‐vivo CLSM characteristics. 31 ML were stained with acridine orange or fluorescein and examined using ex‐vivo CLSM (Vivascope2500^®; Lucid Inc; Rochester NY) in reflectance and fluorescence mode. Confocal images were correlated to histopathology. Benign and malignant features of the ML were listed and results were presented. Sensitivity and specificity were calculated using contingency tables. The ML included junctional, compound, dermal, Spitz and dysplastic nevi, as well as various melanoma subtypes. The correlation of the confocal findings with histopathology allowed the identification of different types of ML and differentiation of benign and malignant features. The study offers an overview of confocal characteristics of ML in comparison to histology. Ex‐vivo CLSM does not reproduce the typical in‐vivo horizontal mosaics but rather reflects the vertical histological presentation. Not all typical in‐vivo patterns are detectable here. These findings may help to evaluate the ex‐vivo CLSM as an adjunctive tool in the immediate intraoperative diagnosis of ML.

Superficial spreading malignant melanoma. Histopathology (H&E stain; 200×) correlated to the reflectance (RM; 830 nm) and fluorescence mode (FM; 488 nm) in the ex‐vivo CLSM (Vivablock^® by VivaScan^®, acridine orange).     

Ex vivo confocal laser scanning microscopy: An innovative method for direct immunofluorescence of cutaneous vasculitis

Ex vivo confocal laser scanning microscopy (ex vivo CLSM) offers an innovative diagnostic approach through vertical scanning of skin samples with a resolution close to conventional histology. In addition, it enables fluorescence detection in tissues. We aimed to assess the applicability of ex vivo CLSM in the detection of vascular immune complexes in cutaneous vasculitis and to compare its diagnostic accuracy with direct immunofluorescence (DIF) microscopy. Eighty‐two sections of 49 vasculitis patients with relevant DIF microscopy findings were examined using ex vivo CLSM following staining with fluorescent‐labeled IgG, IgM, IgA, C3 and fibrinogen antibodies. DIF microscopy showed immunoreactivity of vessels with IgG, IgM, IgA, C3 and Fibrinogen in 2.0%, 49.9%, 12.2%, 59.2% and 44.9% of the patients, respectively. Ex vivo CLSM detected positive vessels with the same antibodies in 2.0%, 38.8%, 8.2%, 42.9% and 36.7% of the patients, respectively. The detection rate of positive superficial dermal vessels was significantly higher in DIF microscopy as compared to ex vivo CLSM (P < .05). Whereas, ex vivo CLSM identified positive deep dermal vessels more frequently compared to DIF microscopy. In conclusion, ex vivo CLSM could identify specific binding of the antibodies in vessels and showed a comparable performance to conventional DIF microscopy in diagnosing vasculitis.     

Direct observation of spectral differences between normal and basal cell carcinoma (BCC) tissues using confocal Raman microscopy

Raman spectroscopy has strong potential for providing noninvasive dermatological diagnosis of skin cancer. In this study, confocal Raman microscopy was applied to the dermatological diagnosis for one of the most common skin cancers, basal cell carcinoma (BCC). BCC tissues were obtained from 10 BCC patients using a routine biopsy and used for confocal Raman measurements. Autofluorescence signals from tissues, which interfere with the Raman signals, were greatly reduced using a confocal slit adjustment. Distinct Raman band differences between normal and BCC tissues for the amide I mode and the PO2- symmetric stretching mode showed that this technique has strong potential for use as a dermatological diagnostic tool without the need for statistical treatment of spectral data. It was also possible to precisely differentiate BCC tissue from surrounding noncancerous tissue using the confocal Raman depth profiling technique. We propose that confocal Raman microscopy provides a novel method for dermatological diagnosis since direct observations of spectral differences between normal and BCC tissues are possible.     

In vivo nonlinear optical imaging to monitor early microscopic changes in a murine cutaneous squamous cell carcinoma model

Early detection of cutaneous squamous cell carcinoma (cSCC) can enable timely therapeutic and preventive interventions for patients. In this study, in vivo nonlinear optical imaging (NLOI) based on two‐photon excitation fluorescence (TPEF) and second harmonic generation (SHG), was used to non‐invasively detect microscopic changes occurring in murine skin treated topically with 7,12‐dimethylbenz(a)anthracene (DMBA). The optical microscopic findings and the measured TPEF‐SHG index show that NLOI was able to clearly detect early cytostructural changes in DMBA treated skin that appeared clinically normal. This suggests that in vivo NLOI could be a non‐invasive tool to monitor early signs of cSCC.

In vivo axial NLOI scans of normal murine skin (upper left), murine skin with preclinical hyperplasia (upper right), early clinical murine skin lesion (lower left) and late or advanced murine skin lesion (lower right).     

Simultaneous immunofluorescence and histology in pemphigus vulgaris using ex vivo confocal laser scanning microscopy

Ex vivo confocal laser scanning microscopy (ex vivo CLSM) provides rapid, high-resolution imaging and immunofluorescence examinations of the excised tissues. We aimed to evaluate the applicability of ex vivo CLSM in histomorphological and direct immunofluorescence (DIF) examination of pemphigus vulgaris (PV). 20 PV sections were stained with fluorescent-labeled anti-IgG and anti-C3 using various dilutions and incubation periods. Subsequently, the determined ideal staining protocol was applied on 20 additional PV and 20 control sections. Ex vivo CLSM identified intraepidermal blisters and acantholytic cells in 80% and 60% of PV patients, respectively. The sensitivity of ex vivo CLSM in detecting intraepidermal fluorescence was 90% both with IgG and C3. The specificity of staining for IgG and C3 was 70% and 90%, respectively. Histomorphological and immunofluorescence features of PV could be detected within the same ex vivo CSLM session showing a comparable performance to conventional histopathology and DIF microscopy.     

Ex-vivo fluorescence confocal microscopy with digital staining for characterizing basal cell carcinoma on frozen sections: A comparison with histology

Ex-vivo fluorescence confocal microscopy (FCM) has been used on fresh tissue, but there is little experience on frozen sections. We evaluated the applicability of FCM on frozen sections of basal cell carcinomas (BCCs), stained with acridine orange and digitally colored to simulate hematoxylin and eosin (H&E) dyes. We compared our diagnostic accuracy in detecting and subtyping BCCs with FCM to our gold standard (H&E stained frozen sections used in 3D horizontal micrographic surgery). Fourty-six primary BCCs were analyzed for free margins as well as histological subtype with all FCM modes and conventional H&E staining. Adnexa, artifacts and diagnostic confidence were evaluated. Free margins were identified with a sensitivity and specificity of 92% and 91%. Concordance for tumor subtype was 88%. FCM may be used on both fresh tissue and frozen samples, although with reduced performance and different artifacts. The device is useful for the intraoperative diagnosis, subtyping and margin-mapping of BCCs.      

Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.     

人食管癌EC9706单克隆的分离培养及其生物学特性的研究

用限制性稀释法对食管癌EC9706细胞系进行单细胞克隆分离,建立了3个亚系,分别命名为EC1、EC2和EC3。细胞遗传学分析显示,EC1仍为混合克隆,EC2和EC3的染色体众数分别为117条和62条。对EC2和EC3的生物学特性进行比较,生长曲线、平皿集落形成能力实验、流式细胞分析和细胞运动能力实验均表明,两者的恶性程度有显著差别,EC2较低,EC3较高。成功分离的2个食管癌单克隆细胞亚系,可为食管癌发生发展的分子机制研究、基因治疗研究、耐药机制研究以及抗肿瘤药物筛选提供实验材料。     

Shedding light on fibered confocal fluorescence microscopy: Applications in biomedical imaging and therapies

Discoveries of major importance in life sciences and preclinical research are linked to the invention of microscopes that enable imaging of cells and their microstructures. Imaging technologies involving in vivo procedures using fluorescent dyes that permit labelling of cells have been developed over the last two decades. Fibered confocal fluorescence microscopy (FCFM) is an imaging technology equipped with fiber‐optic probes to deliver light to organs and tissues of live animals. This enables not only in vivo detection of fluorescent signals and visualization of cells, but also the study of dynamic processes, such cell proliferation, apoptosis and angiogenesis, under physiological and pathological conditions. This will allow the diagnosis of diseased organs and tissues and the evaluation of the efficacy of new therapies in animal models of human diseases. The aim of this report is to shed light on FCFM and its potential medical applications and discusses some factors that compromise the reliability and reproducibility of monitoring biological processes by FCFM. This report also highlights the issues concerning animal experimentation and welfare, and the contributions of FCFM to the 3Rs principals, replacement, reduction and refinement.      

Integrated photoacoustic and hyperspectral dual‐modality microscopy for co‐imaging of melanoma and cutaneous squamous cell carcinoma in vivo

Skin carcinoma such as melanoma (MM) and cutaneous squamous cell carcinoma (cSCC) are considered as the highest mortality and the most aggressive skin cancers in dermatology. In view that early diagnosis and treatment can greatly improve the survival rate and life quality of the patients, developing noninvasive and effective evaluation methods is of great significance for the detection and identification of early stage cutaneous cancers. In this article, we propose a hybrid photoacoustic and hyperspectral dual‐modality microscopy to evaluate and differentiate skin carcinoma by structural and multiphysiological parameters. The proposed system's imaging abilities are verified by mimic phantoms and normal mice experiments. Furthermore, in vivo characterization and evaluation results of MM and cSCC mice are obtained successfully, which prove this novel method could be used as a reliable and useful method for skin cancer detection in early stages.     

Perfectly registered multiphoton and reflectance confocal video rate imaging of in vivo human skin

We present a multimodal in vivo skin imaging instrument that is capable of simultaneously acquiring multiphoton and reflectance confocal images at up to 27 frames per second with 256 × 256 pixel resolution without the use of exogenous contrast agents. A single femtosecond laser excitation source is used for all channels ensuring perfect image registration between the two‐photon fluorescence (TPF), second harmonic generation (SHG), and reflectance confocal microscopy (RCM) images. Images and videos acquired with the system show that the three imaging channels provide complementary information in in vivo human skin measurements. In the epidermis, cell boundaries are clearly seen in the RCM channel, while cytoplasm is better seen in the TPF imaging channel, whereas in the dermis, SHG and TPF channels show collagen bundles and elastin fibers, respectively. The demonstrated fast imaging speed and multimodal imaging capabilities of this MPM/RCM instrument are essential features for future clinical application of this technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Potential of contrast agents to enhance in vivo confocal microscopy and optical coherence tomography in dermatology: A review

Distinction between normal skin and pathology can be a diagnostic challenge. This systematic review summarizes how various contrast agents, either topically delivered or injected into the skin, affect distinction between skin disease and normal skin when imaged by optical coherence tomography (OCT) and confocal microscopy (CM). A systematic review of in vivo OCT and CM studies using exogenous contrast agents on healthy human skin or skin disease was performed. In total, nine CM studies and one OCT study were eligible. Four contrast agents aluminum chloride (AlCl) n = 2, indocyanine green (ICG) n = 3, sodium fluorescein n = 3 and acetic acid n = 1 applied to CM in variety of skin diseases. ICG, acetic acid and AlCl showed promise to increase contrast of tumor nests in keratinocyte carcinomas. Fluorescein and ICG enhanced contrast of keratinocytes and adnexal structures. In OCT of healthy skin gold nanoshells, increased contrast of natural skin openings. Contrast agents may improve delineation and diagnosis of skin cancers; ICG, acetic acid and AlCl have potential in CM and gold nanoshells facilitate visualization of adnexal skin structures in OCT. However, as utility of bedside optical imaging increases, further studies with robust methodological quality are necessary to implement contrast agents into routine dermatological practice.      

Focal adhesion complex proteins in epidermis and squamous cell carcinoma

Focal adhesions (FAs) are large, integrin-containing, multi-protein assemblies spanning the plasma membrane that link the cellular cytoskeleton to surrounding extracellular matrix. They play critical roles in adhesion and cell signaling and are major regulators of epithelial homeostasis, tissue response to injury, and tumorigenesis. Most integrin subunits and their associated FA proteins are expressed in skin, and murine genetic models have provided insight into the functional roles of FAs in normal and neoplastic epidermis. Here, we discuss the roles of these proteins in normal epidermal proliferation, adhesion, wound healing, and cancer. While many downstream signaling mechanisms remain unclear, the critically important roles of FAs are highlighted by the development of therapeutics targeting FAs for human cancer.     

Ex vivo imaging of T cells in murine lymph node slices with widefield and confocal microscopes

Naïve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay ¹, originally set up by neurobiologists and transposed recently to murine thymus ². In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.