The gold standard for diagnosis in dermatology is histopathologic examination of tissue. Although cutaneous biopsies are associated with a relatively low morbidity, there is always a risk for infection, bleeding, and scarring. Biopsies also destroy the site of interest and the dynamic changes over time cannot be observed. Moreover, it consumes time! The search to overcome the drawbacks of the traditional skin biopsy and to provide high resolution images has led to the development of non-invasive techniques such as dermoscopy and in-vivo confocal microscopy.

Confocal scanning laser microscopy(CSLM), often referred as reflectance confocal microscopy, is a novel non-invasive imaging technique that enables the identification of cells and tissues with nearly histologic resolution. The confocal microscope was originally designed by Marvin Minsky (1955), which has further undergone decades of improvisation in structure. With the presently available ones, it is possible to image the skin in vivo, in real time and display on a monitor the 3D morphology and dynamic events. Skin can also be imaged by a confocal microscope in freshly biopsied (in vitro) specimens without processing and staining that is required for routine histopathology. The maximum depth of visualization by CSLM is 200-350µm below the stratum corneum covering the epidermis, papillary dermis and superficial reticular dermis. It allows a horizontal scanning of the imaged tissue, with the advantage of exploring a larger field of view compared with vertical sectioning.

In conventional microscopy, a tissue section is placed on the microscope stage and the entire field of the specimen is simultaneously illuminated by light and visualized. The illumination of other parts of the sample surrounding the point of interest, results in a “background noise,” which compromises the quality of the image.

However, in CSLM, a beam of light (generated by a laser medium) is focused on a small spot inside the tissue through the microscope objective via a dichroic mirror. The mirror is capable of reflecting the light as well as allowing it to pass through. The same objective gathers the reflected light coming back from the tissue, and is projected on a screen. Confocal microscopy overcomes the problem of “background noise” using a small pinhole aperture in a screen that allows only the light emitting from the desired focal spot to pass through. Any light outside of the focal plane (the scattered light) is blocked by the screen. In optical terms, the pinhole is placed in a conjugate focal plane as the tissue specimen (hence the designation “confocal”). A sensitive light detector, such as a photomultiplier tube, on the other side of the pinhole is used to detect the confocal light. (See figure) This technique allows the specimen to be imaged one “point” at a time. Because the sample is not actually sectioned, it is possible to image a “stack” of virtual, confocal image planes that can later be used to make tomographic images, similar to the reconstructions of magnetic resonance imaging or computed tomography scanners in medicine.

Applications of CSLM in dermatology:

• Microscopic analysis of skin structures (including hairs and nails) and their composition at different anatomic sites.
• Detection of premalignant changes in pigmented skin conditions.
• Identification of fungal hyphae and Sarcoptes scabiei mite.
• Monitoring number and activity of inflammatory cells in diseases like psoriasis, lichen planus etc.
• Visualizing intradermal tattoo pigments for laser treatment.
• Defining margins in Mohs surgery.

Limitations of confocal microscopy include the depth of imaging within thick samples and cost compared with conventional microscopes. Nevertheless, technologies for microscopy are promising and are still being improved.