Fluorescent fibre-optic confocal characterization of in vivo epidermal changes in atopic eczema.

BACKGROUND/AIMS: Fibre-optic confocal imaging (FOCI) allows non-invasive visualization of live skin in vivo. A contrast agent, a fluorophore, is injected into the dermis. FOCI images are horizontal optical sections with cellular resolution. The aim was to study in vivo epidermal changes and the cellular structure of keratinocytes in moderate to severe atopic eczema (AE). METHODS: Eight patients with AE with active lesions on the forearms were studied and compared to a control group of six healthy individuals, and two cases of AE without activity. Fluorescein sodium was used as fluorophore. A hand-held fibre-optic laser scanner (Stratum® ) was used. The study included morphometric analyses. RESULTS: The confocal in vivo images identified characteristic features of epidermis and keratinocytes in active AE vs healthy skin controls. FOCI could non-invasively image acanthosis, spongiosis, and parakeratosis in AE. Epidermal oedema and micro-vesicles were visualized. Morphometry based on FOCI demonstrated 14% increased width of keratinocytes of atopic skin vs healthy controls. The epidermal structures and organization in distinctive cell layers were deviant as a result of the disease. CONCLUSIONS: Fibre-optic confocal imaging can visualize essential epidermal structures of atopic eczema directly in vivo, in real-time, and with cellular resolution thus without disturbing the natural state of the skin. FOCI is primarily a research tool, but with a potential to become used in the clinic for non-invasive microscopic diagnosis of AE and monitoring of effect of therapies.

Fluorescent fibre-optic confocal imaging of lesional and non-lesional psoriatic skin compared with normal skin in vivo.

BACKGROUND/AIMS: Fibre-optic confocal imaging (FOCI) allows non-invasive visualization of live skin in vivo. A contrast agent, a fluorophore, is injected in the dermis. FOCI images are optical sections from a horizontal (en face) view. The aim was to study epidermis and the cellular structure of keratinocytes of psoriatic plaques and adjacent non-lesional with healthy skin as a reference. METHODS: Twelve patients with stable plaque psoriasis were studied and compared with a control group of eight healthy individuals. Fluorescein sodium was used as fluorophore. A hand held fibre-optic laser scanner (Stratum(®); Optiscan Pty., Melbourne, Australia) was used. The study included morphometric analyses. RESULTS: The confocal in vivo images demonstrated characteristic features of epidermis and keratinocytes in lesional and non-lesional skin vs. healthy skin. Morphometry based on FOCI demonstrated an approximately 30% increased width of keratinocytes of psoriatic skin vs. healthy control, and the number of keratinocytes per viewing field was reduced. FOCI allowed non-invasive visualization of cell nuclei and parakeratosis of psoriatic epidermis. The horizontal width of dermal papillae of psoriatic skin was increased by approximately 50% as compared with healthy skin, and the flow of erythrocytes in the papillar vessels could be observed in real-time. CONCLUSION: FOCI can directly visualize essential epidermal structures of plaque psoriasis in vivo, in real-time and with cellular resolution without the need of taking biopsies and thus without disturbing the natural state of the skin. FOCI is a versatile future tool for non-invasive microscopic diagnosis and therapy follow-up of psoriasis.

Fluorescence confocal laser scanning microscopy for in vivo imaging of epidermal reactions to two experimental irritants.

BACKGROUND: Fibre-optic fluorescence confocal laser scanning microscopy (CLSM) is a novel non-invasive technique for in vivo imaging of skin. The cellular structure of the epidermis can be studied. A fluorophore, e.g. fluorescein sodium, is introduced by an intradermal injection or applied to the skin surface before scanning. Images are horizontal optical sections parallel to the skin surface. Fluorescence CLSM has hitherto not been applied to experimental contact dermatitis. OBJECTIVE: The aim was to study the applicability of fluorescence CLSM for in situ imaging of irritant contact dermatitis reactions caused by established model irritants, e.g. sodium lauryl sulphate (SLS) and pelargonic acid (PA). METHODS: Twelve healthy individuals volunteered. The flexor aspect of the right and the left forearm was exposed to SLS in water and PA in isopropanol and occluded under Finn Chambers for 24 h. The reactions were rated clinically and, following epicutaneous and intra-dermal application of fluorescein sodium, studied by fluorescence CLSM, magnification x 1000. RESULTS: Both irritants disturbed the epidermal intercellular borders, which became blurred, thickened and variably altered. This was interpreted as being a result of chemical damage to cellular membranes. Cell borders might show a double contour as a result of inter-cellular oedema. PA might increase the size of individual keratinocytes interpreted as a result of intra-cellular disturbance with oedema. SLS-exposed sites showed clusters of keratinocytes with visible nuclei in the outer layers of the epidermis, e.g. a parakeratotic shift supposed to be due to increased cell proliferation elicited by SLS. The isopropanol vehicle and PA did not interfere with the CLSM imaging technique or the experimental procedures. SLS, being a detergent, however, modified the physico-chemical properties of the skin surface and both disturbed epicutaneous labelling with the flurophore and immersion oil coupling between the skin surface and the optical system. Thus, SLS was technically more difficult to study by CLSM than PA. CONCLUSIONS: This preliminary study demonstrated the applicability of fluorescence CLSM for a detailed study of experimental skin irritants in vivo. Essential findings were disturbed and widened cell borders, swelling of keratinocytes by PA and induction of a parakeratotic shift by SLS with clusters of keratinocytes holding nuclei in the epidermis. Fluorescence CLSM offers a unique opportunity to study the inter- and intracellular water compartments directly in the epidermis in situ and an opportunity to visualize cell proliferation manifested as parakeratosis. Fibre-optic fluorescence CLSM of irritant reactions is, however, technically more complicated than reflectance CLSM and may not be applicable to any irritant. SLS applied epicutaneously interacted with the skin surface and coupling to the microscope and was thus found to be more difficult to study technically than PA. PA dissolved in isopropanol is for technical reasons, and with SLS as alternative, considered the preferred model irritant.

In vivo imaging of intradermal tattoos by confocal scanning laser microscopy.

BACKGROUND/AIMS: In vivo confocal laser scanning microscopy (CLSM) is a new method for high-resolution imaging of intact skin in situ. Horizontal mapping of the outer skin is provided (magnification x 1000). OBJECTIVES: Tattooing is popular all over the world; however, tattooed skin has not been studied in using CLSM. RESULTS: Tattoos in two volunteers were studied using the Vivascope1500 of Lucid Inc. Subepidermal massive deposits of dense, clustered pigment granules up to about 3 mum in size corresponding to black tattoos, and more scarce and diffuse deposits, corresponding to red, blue and green tattoos, were observed. Diffuse pigment granules tended to accumulate in the outer dermis underneath the level of the basement membrane zone. CONCLUSIONS: Dermal pigments from tattoos can be imaged in vivo using CLSM. This application of CLSM has an important future potential for pre-evaluation of tattoos before laser removal, predicting good or poor outcome of laser removal.

Fluorescence fibre-optic confocal microscopy of skin in vivo: microscope and fluorophores.

BACKGROUND/AIMS: Fibre-optic confocal imaging in vivo is a new approach in the assessment of human skin. The objective is to describe a novel instrument and its operation and use in combination with fluorophores. METHODS: The Stratum is a fibre-optic fluorescence confocal microscope especially developed for the study of skin and mucous membranes. The system is flexible and any body site can be studied with a hand-held scanner. The light source is a 488 nm argon ion laser. Horizontal (en face) images of the epidermis and outer dermis are produced with cellular resolution. Magnification is approximately 1000 x . Fluorescein sodium is routinely used as fluorophore (intradermal injection or application to the skin surface). This fluorophore is safe for human use in vivo, but other substances (rhodamine B, Acridine Orange, green fluorescent protein, curcumin) have also been studied. RESULTS: The instrument produces sharp images of epidermal cell layers from the epidermal surface to the sub-papillary dermis, with sub-cellular resolution. The scanner is flexible in use. The technique of intradermal fluorophore injection requires some skill. CONCLUSIONS: We consider this fibre-optic instrument a potentially important tool in skin research for non-invasive optical biopsy of primarily the epidermis. Present use is focussed on research applications, where the fluorophore distribution in the skin may illustrate morphological changes in the epidermis.