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.     

Identification of ex‐vivo confocal scanning microscopic features and their histological correlates in human skin

Ex‐vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. The first generation of ex‐vivo devices have already shown promising results in the ex‐vivo evaluation of basal cell carcinoma compared to Mohs surgery. Nevertheless, for the diagnostics of pathological skin lesions the knowledge of normal skin features is essential. Therefore we examined 50 samples of healthy skin from various donor sites including head and neck (n = 25), trunk (n = 10), upper (n = 10) and lower extremities (n = 5) using a new generation ex‐vivo CLSM device offering three different laser wavelengths and compared the findings to the corresponding histological sections. In correlation with the histopathology we identified different layers of the epidermis, differentiated keratinocytes from melanocytes and described in detail skin appendages including hair follicle, sebaceous and sweat glands. Furthermore, structures of the dermis and subcutis were illustrated. Additionally, artefacts and pitfalls occurring with the use of ex‐vivo CLSM have been documented. The study offers an overview of the main ex‐vivo CLSM skin characteristics in comparison to the standard histological examination and helps to recognize and avoid common artefacts.

Anatomy of a hair follicle in the reflectance mode (RM) CLSM, fluorescence mode (FM) CLSM and in a routine hematoxylin‐eosin stained histological section (H).     

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).     

Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin

We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.     

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.     

Sensitivity and efficiency of four immunohistochemical methods as defined by staining of artificial sections

Direct (DIF) and indirect (IIF) immunofluorescence, indirect immunoperoxidase conjugate (IPC) and unlabelled antibody peroxidase antiperoxidase (PAP) staining was performed on sections of artificial substrate containing different concentrations of human immunoglobulin (Ig)A or IgG. Detection sensitivity, in terms of the lowest amount of discernible antigen, was evaluated by direct microscopy and by microphotometry. Staining efficiency (signal-to-noise ratio) was evaluated by microphotometry. Only minor differences in antigen detection sensitivity were found when IPC and PAP were compared with DIF and IIF under appropriate conditions. The sensitivity of DIF was only marginally improved by raised conjugate concentration and prolonged incubation time. Microphotometry of DIF on ethanol-fixed IgA substrate revealed that the staining intensity increased proportionally with the antigen concentration whereas on formaldehyde-fixed substrate a progressive masking of the antigen was indicated which, however, could be overcome by applying raised conjugate concentration and prolonged incubation time. Such antigenic self masking was of relatively little importance to IPC and PAP staining, probably because of the inherent amplification in these methods. An additional masking effect due to extraneous protein was revealed by DIF when ethanol-fixed sections had been soaked in bovine serum albumin and postfixed with formaldehyde; unmasking was achieved by proteolytic treatment of the sections.     

Defining the role of complement in experimental pemphigus vulgaris in mice

Parenteral passive transfer of human pemphigus vulgaris IgG (PV IgG) into neonatal mice reproduces the cutaneous disease. We used this model to study the role of complement in the development of acantholysis in three steps. Peptic F(ab')2 fragments were prepared from PV IgG and were injected into seven newborn mice, and all animals developed acantholytic skin blisters without local complement activation, as shown by direct immunofluorescence. These fragments were reduced and alkylated to produce Fab' fragments with equivalent in vitro binding activity. The monovalent fragments were given in an identical fashion to five littermates but failed to produce disease even though they were bound in the epidermis in vivo. Intact PV IgG was injected in 20 genetically C5-deficient neonates (B10-D2-OSN strain) and 20 control neonates (B10-D2-NSN, normal complementemic). Extensive blistering, with a positive Nikolsky sign, was produced in all 40 animals. PV IgG was given to 34 BALB/c neonates that were complement depleted by pretreatment with cobra venom factor (CoF) for 24 hr, and to 38 untreated neonates from the same litters. There was no difference in the disease produced after CoF treatment in animals that received high doses of PV IgG (5 to 15 mg/g/day). In animals receiving 2.5 mg PV IgG/g/day, blister formation was delayed and the final extent of the cutaneous lesions was less in CoF-treated mice (n = 12) than in normal complementemic controls (n = 12, p less than 0.02). These results show that complement activation is not an essential mechanism in PV IgG-induced acantholysis in vivo, but it does have an amplifying effect on the development of cutaneous lesions under certain conditions, and lesions can be induced in vivo by bivalent F(ab')2 fragments of PV IgG, but not by the monovalent Fab' fragments, suggesting that cross-linking of the cell surface antigen is an initiating signal in acantholysis.     

Sensitivity and efficiency of four immunohistochemical methods as defined by staining of artificial sections

Summary Direct (DIF) and indirect (IIF) immunofluorescence, indirect immunoperoxidase conjugate (IPC) and unlabelled antibody peroxidase antiperoxidase (PAP) staining was performed on sections of artificial substrate containing different concentrations of human immunoglobulin (Ig)A or IgG. Detection sensitivity, in terms of the lowest amount of discernible antigen, was evaluated by direct microscopy and by microphotometry. Staining efficiency (signal-to-noise ratio) was evaluated by microphotometry. Only minor differences in antigen detection sensitivity were found when IPC and PAP were compared with DIF and IIF under appropriate conditions. The sensitivity of DIF was only marignally improved by raised conjugate concentration and prolonged incubation time. Microphotometry of DIF on ethanol-fixed IgA substrate revealed that the staining intensity increased proportionally with the antigen concentration whereas on formaldehyde-fixed substrate a progressive masking of the antigen was indicated which, however, could be overcome by applying raised conjugate concentration and prolonged incubation time. Such antigenic self masking was of relatively little importance to IPC and PAP staining, probably because of the inherent amplification in these methods. An additional masking effect due to extraneous protein was revealed by DIF when ethanol-fixed sections had been soaked in bovine serum albumin and postfixed with formaldehyde; unmasking was achieved by proteolytic treatment of the sections.This work was supported by the Norwegian Cancer Society, the Norwegian Research Council for Science and the Humanities, and Anders Jahres Foundation     

Exercise-heat acclimation in humans alters baseline levels and ex vivo heat inducibility of HSP72 and HSP90 in peripheral blood mononuclear cells

The induction of cellular acquired thermal tolerance (ATT) during heat acclimation (HA) in humans is not well described. This study determined whether exercise-HA modifies the human heat shock protein (HSP)72 and HSP90 responses and whether changes are correlated with physiological adaptations to HA. Using a 10-day HA protocol comprising daily exercise (treadmill walking) in a hot environment (T(a) = 49 degrees C, 20% RH), we analyzed baseline and ex vivo heat-induced expression of HSP72 and HSP90 in peripheral blood mononuclear cells (PBMCs) isolated prior to exercise from eight subjects on day 1 and 10 of the HA protocol. Classical physiological responses to HA were observed, including significantly reduced heart rate and core body temperature, and significantly increased sweating rate. Baseline levels of HSP72 and HSP90 were significantly increased following acclimation by 17.7 +/- 6.1% and 21.1 +/- 6.5%, respectively. Ex vivo induction of HSP72 in PBMCs exposed to heat shock (43 degrees C) was blunted on day 10 compared with day 1. A correlation was identified (r(2) = 0.89) between changes in core temperature elevation and ex vivo HSP90 responses to heat shock between days 1 and 10, indicating that volunteers demonstrating the greatest physiological HA tended to exhibit the greatest blunting of ex vivo HSP induction in response to heat shock. In summary, 1) exercise-HA resulted in increased baseline levels of HSP72 and HSP90, 2) ex vivo heat inducibility of HSP72 was blunted after HA, and 3) volunteers demonstrating the greatest physiological HA tended to exhibit the greatest blunting of ex vivo HSP induction in response to heat shock. These data demonstrate that physiological adaptations in humans undergoing HA are accompanied by both increases in baseline levels and changes in regulation of cytoprotective HSPs.     

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness,cell‐specific multiphoton fluorescence brain imaging

We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.     

Transcranial photobiomodulation attenuates pentylenetetrazole‐induced status epilepticus in peripubertal rats

Convulsive status epilepticus is the most common neurological emergency in children. Transcranial photobiomodulation (tPBM) reverses elevated rodent neurotransmitters after status epilepticus (SE) yet whether tPBM can attenuate seizure behaviors remains unknown. Here, we applied near‐infrared laser at wavelength 808 nm transcranially to peripubertal Sprague‐Dawley rats prior to pentylenetetrazole (PTZ) injection. Hematoxylin‐eosin, immunofluorescence (IF) staining with anti‐parvalbumin (PV) and terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay after IF staining was performed. Behaviorally, tPBM attenuated the mean seizure score and reduced the incidence of SE and mortality. Histochemically, tPBM reduced dark neurons in the cortex, hippocampus, thalamus and hypothalamus, lessened the apoptotic ratio of parvalbumin‐positive interneurons (PV‐INs) and alleviated the aberrant extent of PV‐positive unstained somata of PCs in the hippocampus. Conclusively, tPBM attenuated PTZ‐induced seizures, SE and mortality in peripubertal rats and reduced PTZ‐induced neuronal injury, apoptosis of PV‐INs and preserved PV positive perisomatic inhibitory network in the hippocampus.     

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.      

In vivo non-contact measurement of human iris elasticity by optical coherence elastography

Quantifying the mechanical properties of the iris can offer valuable insights into the pathophysiology of primary angle closure glaucoma. However, current techniques for iris elastography remain ex vivo with limited clinical applications. This article describes a proposition for a non-contact and non-invasive air-puff optical coherence elastography (OCE) system that can evaluate iris elasticity in vivo. Ten eyes recruited from seven subjects underwent OCE imaging acquisition under three different illumination conditions. The Young's modulus of each eye was detected and shown to be inversely proportional to the iris length, indicating a relationship between mechanical properties and morphology of the iris. With its noninvasive and high-resolution features, this air-puff system shows great potential for applications in clinical ophthalmology.     

Dexamethasone inhibits plasminogen activator activity in experimental pemphigus in vivo but does not block acantholysis

In vitro studies have suggested that autoantibody-stimulated increases in epidermal plasminogen activator (PA) may be an important pathogenetic mechanism in pemphigus vulgaris (PV). We measured PA in murine epidermis after i.p. injection of normal human IgG (NH IgG) and PV IgG, with and without exposure to dexamethasone (DEX). BALB/c neonates received i.p. injections of saline control or DEX (20 mg/kg). Twenty-four hours later, they received a second injection of saline or DEX and a single dose of NH or PV IgG (20 mg/gm body weight). After 24 hr, epidermis was obtained and was sequentially extracted in 0.14 M NaCl, pH 6.8, and 0.5% Triton X-100 in 0.1 M Tris, pH 8.1. Epidermal PA was assayed in the Triton-Tris supernatant by a two-stage colorimetric reaction and was expressed as milliPloug units per milligram of protein (mPu/A280). PA in animals injected with NH IgG was 0.21 +/- 0.11 mPu/A280 (n = 8). Epidermal PA was increased in animals with cutaneous lesions of pemphigus to 0.42 +/- 0.29 (n = 15). Treatment with DEX decreased PA levels in both animals receiving NH IgG and PV IgG by 80%, to 0.04 +/- 0.05 (n = 15) and 0.09 +/- 0.07 (n = 7), respectively. Despite the decreased PA activity, all animals in the PV IgG and the PV IgG-plus-DEX group had identical and extensive cutaneous disease, and lesions developed at the same time points. This finding shows that PV autoantibodies can stimulate increases in epidermal PA, but reduction of PA by corticosteroids does not inhibit acantholysis in vivo. There is no clear correlation between PA and disease activity in the murine model of pemphigus.     

Localisation of fungal hyphae in wood using immunofluorescence labelling and confocal laser scanning microscopy

This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood.     

Microlesion healing dynamics in in vivo porcine skin after treatment with 1064 nm picosecond-domain Nd:YAG laser

The fractionated picosecond laser produces microscopic lesions in the epidermis and dermis due to laser-induced optical breakdown (LIOB). There have been multiple histological reports, but the present literature lacks detailed in vivo studies after treatment with high-power laser systems. Our study aimed to characterize the healing patterns of microlesions induced with 150 ps duration 1064 nm MLA-type picosecond laser. The induced picosecond laser-tissue reactions with pulse energy of 50–250 mJ and different treatment modes were observed in in vivo porcine skin model over 10 days after the laser procedure. A macroscopic evaluation was combined with microscopic histological analysis to observe the healing dynamics of laser-induced microlesions. Superficial, intraepidermal cavitation bubbles were induced using microbeam fluence of 4–20 J/cm². Skin irritation scores positively correlated with pulse energy and dose. Our findings demonstrate that dose and pulse energy had a direct impact on epidermal thickness and lesions healing dynamics.     

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8+ T cells with detection by ELISPOT and HLA-multimer staining

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R ² = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.     

Multicomposite super‐resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions

Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.