Quick overview of possibilities offered by reflectance and fluorescence confocal

From dermoscopedia

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 Author(s): Amélie Boespflug, Luc Thomas
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Reflectance confocal microscopy (RCM) is a non-invasive, mainly in vivo technique, that permits a visualization at the cellular level of the skin until a vertical depth of 0,5 mm. Because it is an expensive, time-consuming tool, it is only available in a few university hospitals worldwide but its indications in ento-dermatology, onco-dermatology and inflammatory skin diseases are increasing.


Conventional microscopy produces 2D images and is limited as it can only “see” as far into the specimen as the light can penetrate[1]. It is used to analyze histological samples after fixation and staining.


Confocal microscopy produces 3D images and enables the reconstruction of three-dimensional structures from sets of images obtained at different depths (a process known as optical sectioning) within a thick object. Confocal microscopy "sees" images one depth level at a time. [2]

Technical aspects[edit]

Confocal microscopy uses optical sectioning to generate virtual horizontal sectioning of the examined tissue with a micrometric definition and reconstruct 3D images with colors ranging from white to black on a vertical distance of 0,5mm. Highly reflective material, mainly melanin, will appear white, whereas unreflective material, mainly water, will appear black.

Clinical applications[edit]

Infectious diseases[edit]

Confocal microscopy can be used to diagnose and monitor treatment efficacity in fungal diseases like onychomycosis [3] and tinea nigra [4] and can also be used for the diagnosis of parasites like scabies [5] and Demodex Follicularum [6]. Viruses cannot directly be seen but the cytopathic effect can be identified.

Onco-dermatology[edit]

Confocal microscopy can be used to help the diagnosis of sub-clinical actinic keratosis and monitor the response to treatment [7]. Confocal microscopy can increase the accuracy of dermoscopy in the diagnosis of lentigo melanoma and help delimit the margins before surgery[8]. It has also been studied to help delimit the surgical margins of basal cell carcinomas[9] and pigmented squamous cell carcinomas [10].

Nails[edit]

In vivo Reflective confocal microscopy

Because it is impossible to visualize structures through the nail plate, nail RCM is mainly used for the per-operative examination of subungual structures after plate avulsion[11] except in anecdotal uses for the diagnosis and treatment of onychomycoses [12]


In vivo reflectance confocal microscopy on the nail is mainly used in the differential diagnosis of melanocytic tumors during surgical procedures with an exploration of the nail bed and matrix. It requires, after nail plate avulsion under local anesthesia (trap door technique, lateral avulsion or complete nail plate desinsertion) in order to visualize the entire surface of the matrix from the distal part of the lunula to the proximal matricial cul-de-sac, a sterile isolation of the surgical field by a translucent sterile wrap. The direct examination of subungual structures by the handheld detector is possible though sterile oil immersion of its optical. Different depth of examination images are grabbed during the live examination of the structures. The main advantage of this technique is to offer in the majority of preoperatively doubtful cases of melanonychia striata an extemporaneous diagnosis of malignancy then allowing to offer wide excision of the nail unit within an unique surgical session reducing dramatically the disability time when compared to the biopsy-then-excise two step classical melanoma management procedure [13][14][15].


If the principal interest of RCM to diagnose nail tumors is in melanoma, other extemporaneous diagnosis of SCC, onychomatricoma, glomus cell tumor and neuroma can be made [16].


Ex vivo confocal microscopy

Not all skin tumors contain enough reflective material to allow a reflectance confocal microscopy diagnosis. In this case exogenous fluorescent dyes can be used to enhance contrast and to allow better visualization of microstructures. This is the purpose of florescence confocal microscopy, unfortunately for the moment no sufficiently safe trans-epidermically bioavailable fluorescent dye is available. This technique can only be used ex-vivo. Ex vivo confocal microscope are bi-modal and offer reflectance mode imaging as well as fluorescence mode image at different wavelengths. The use of acridine orange, that stains the nuclei of the cells allow the production of nice micrometric level histopathological images. However, in contrast with reflectance microscopy no virtual sectioning is possible since acridine orange only decorates the surface of the tissue specimen in contact with the dye.


The freshly excised tumor is bisected into two parts then put in contact with the dye and after rinsing out of the dye the two bisected parts of the tumor is placed, section up, on a glass slide directly mounted towards the optical of the device. Image (up to 2cm x 2 cm) is obtained by point by point scanning of the surface of the specimen. A complete unique image is obtained with 5 to 15 minutes depending on the size of the examination field. The device offers the possibility to precisely re-target specific areas of the lesion for further high power examination. The main advantage of this technique is to offer a good alternative to classical frozen-section extemporaneous examination of tumors with a complete conservation of all surgically excised tissue (yet bisected into two parts) for further classical paraffin-embedded tissue processing for pathological examination.


Confocal microscopy is a non invasive technique that is currently in development in dermatology. It has mostly been studied to help the diagnosis of cancerous lesions and help establish the best surgical margins, but its application in inflammatory (psoriasis, pytiriasis rubra pilaire) and genetic (brooke Speigler, Galli Galli, Hailey-Hailey) dermatology is also being evaluated.



References

  1. https://en.wikipedia.org/wiki/Optical_microscope
  2. https://en.wikipedia.org/wiki/Confocal_microscopy
  3. Pharaon M, Gari-Toussaint M, Khemis A, Zorzi K, Petit L, Martel P, Baran R, Ortonne JP, Passeron T, Lacour JP, Bahadoran P. Diagnosis and treatment monitoring of toenail onychomycosis by reflectance confocal microscopy: prospective cohort study in 58 patients. J Am Acad Dermatol. 2014 Jul;71(1):56-61.
  4. Veasey JV, Avila RB de, Ferreira MAM de O, Lazzarini R. Reflectance confocal microscopy of tinea nigra: comparing images with dermoscopy and mycological examination results. Anais Brasileiros de Dermatologia 2017;92:568–9.
  5. Cinotti E, Labeille B, Cambazard F, Perrot JL. Reflectance confocal microscopy in infectious diseases. Giornale Italiano Di Dermatologia E Venereologia : Organo Ufficiale, Societa Italiana Di Dermatologia E Sifilografia 2015;150:575–83.
  6. Pampín A, Floristán U, Gamo R, Ascanio ML, López-Estebaranz JL. Facial spinulosis caused by Demodex folliculorum : diagnostic assessment by means of reflectance confocal microscopy. Clinical and Experimental Dermatology 2017;42:348–50.
  7. Ulrich M, Reinhold U, Falqués M, Rodriguez Azeredo R, Stockfleth E. Use of reflectance confocal microscopy to evaluate 5-fluorouracil 0.5% /salicylic acid 10% in the field-directed treatment of sub-clinical lesions of actinic keratosis: sub-analysis of a Phase III, randomised, double-blind, vehicle-controlled trial. Journal of the European Academy of Dermatology and Venereology 2017.
  8. Hibler BP, Yélamos O, Cordova M, Sierra H, Rajadhyaksha M, Nehal KS, et al. Handheld reflectance confocal microscopy to aid in the management of complex facial lentigo maligna. Cutis 2017;99:346–52.
  9. Iftimia N, Yélamos O, Chen C-SJ, Maguluri G, Cordova MA, Sahu A, et al. Handheld optical coherence tomography–reflectance confocal microscopy probe for detection of basal cell carcinoma and delineation of margins. Journal of Biomedical Optics 2017;22:76006.
  10. Shahriari N, Grant-Kels JM, Rabinovitz HS, Oliviero M, Scope A. Reflectance Confocal Microscopy Criteria of Pigmented Squamous Cell Carcinoma In Situ. The American Journal of Dermatopathology 2017:1.
  11. Debarbieux S, Hospod V, Depaepe L, Balme B, Poulalhon N, Thomas L. Perioperative confocal microscopy of the nail matrix in the management of in situ or minimally invasive subungual melanomas. Br J Dermatol. 2012 Oct;167(4):828-36.
  12. Hongcharu W, Dwyer P, Gonzalez S, Anderson RR. Confirmation of onychomycosis by in vivo confocal microscopy. J Am Acad Dermatol. 2000 Feb;42(2 Pt 1):214-6.
  13. Hongcharu W, Dwyer P, Gonzalez S, Anderson RR. Confirmation of onychomycosis by in vivo confocal microscopy. J Am Acad Dermatol. 2000 Feb;42(2 Pt 1):214-6.
  14. Fattouh K, Debarbieux S, Depaepe L, Amini-Adle M, Balme B, Thomas L. Routine use of perioperative in vivo reflectance confocal microscopy of the nail matrix in melanonychia striata: about 30 cases. Br J Dermatol. 2016 Oct 3.
  15. Fernandes Massa A, Debarbieux S, Depaepe L, Dalle S, Balme B, Thomas L. Pigmented squamous cell carcinoma of the nail bed presenting as a melanonychia striata: diagnosis by perioperative reflectance confocal microscopy. Br J Dermatol. 2013 Jul;169(1):198-9.
  16. Debarbieux S, Gaspar R, Depaepe L, Dalle S, Balme B, Thomas L. Intraoperative diagnosis of nonpigmented nail tumours with ex vivo fluorescence confocal microscopy: 10 cases. Br J Dermatol. 2015 Apr;172(4):1037-44.