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Ex vivo Confocal Microscopy


Ex vivo confocal microscopy (EVCM) is an emerging imaging technique that allows real-time microscopic examination of freshly excised tissue.

It has been proved to be a reliable diagnostic method with good histological correspondence for both normal and pathological skin[1] , with its applicability in other tissues also showing promising results in the scientific literature.

In dermatology, EVCM has been mainly studied and applied to the intraoperative analysis of the surgical margins of basal cell carcinoma (BCC) during micrographic Mohs surgery (MMS) with high overall sensitivity and specificity. It has been developed as an alternative to the optical microscopy examination of frozen sections stained with hematoxylin and eosin (H&E). Different from standard frozen sections, EVCM enables the evaluation of the resection margins immediately after excision without freezing the specimen, thereby reducing the investment of time by two-thirds and the need of technicians.

Many other possible applications in the dermatology field are under investigation, such as the study of other skin tumors, like squamous cell carcinoma (SCC) and melanoma. Its usefulness could also extend, in the future, to rapid histology at the bedside not only for skin cancer, but also for the diagnosis of inflammatory and infectious diseases. By quickly obtaining architectural and cytological details it can orient the clinical diagnosis before definitive histological examination. In these cases, EVCM's main advantage is that it eliminates the time for formalin-fixation and paraffin-embedding of specimens, which is needed for standard pathology, enabling a rapid examination of an entire skin sample with only the EVCM device. Importantly, EVCM does not alter the tissue and does not prevent subsequent definitive histopathological examination, which is still considered the gold standard.

It should be noted that fat tissue is usually modified by fixation during classical histology, whereas fat remains intact under EVCM.

 Its applicability extends to non-dermatological areas, with studies being conducted in EVCM rapid bedside evaluation of small tissue fragments of image guided core needle[2]  biopsies (CNB) or endoscopic biopsy specimens of breast, kidney, lung, liver, prostate and pancreas tissue with similar high overall sensitivity and specificity for differential diagnosis of malignant specimens.

In these cases, ex vivo optical imaging techniques are particularly appealing because they allow imaging of tissues in the fresh state, requiring very minimal tissue preparation, and do not cause any damage, distortion, or loss of tissue, unlike the other currently available techniques that can be used for immediate tissue assessment, such as frozen sections analysis, which is precisely not routinely recommended because precious tissue is lost in the cryostat when the frozen section is prepared for immediate histopathologic examination.

Touch preparation is another technique that is more commonly used currently for rapid evaluation of CNB specimens. However, this method does not allow accurate evaluation of the tumor cellularity, which is necessary for determining the adequacy of the CNB specimen for the basic diagnosis, as well as for other potential ancillary studies, such as immunohistochemistry and molecular testing. Also, the process of touching the tissue on the glass slide can result in distortion and loss of tissue from the CNB specimen, possibly harming the specimen for subsequent conventional histopathologic examination.

EVCM has been the focus of many studies involving margins assessment in lung cancer, conjunctival tumors, prostatectomy, brain tumors, urethral and ureteral margins during open radical cystectomy for bladder cancer.

How is it done?

In Sydney Melanoma Diagnostic Centre (SMDC) we have the first Ex Vivo Confocal machine in Australia and in the Southern Hemisphere: the 4th generation VivaScope 2500-G4.

The skin sample is mounted flat between two thick microscope glass slides attached together by small magnets. The skin sample is then settled on the microscope stage and the image is obtained.

The ex vivo confocal microscope works analyzing the reflectance of laser beams through the tissue. EVCM can analyze whole skin samples of up to 2 cm in diameter without the need to cut it into thin sections.

The images can be visualized in two different modes:

  • reflectance mode, where no staining is required and the images are in black and white as in in-vivo confocal microscopy.

  • fluorescence mode, when the entire surgical specimen is dipped in a solution of a fluorescent agent. The most used fluorescent agent is acridine orange that targets nuclei of the cells, facilitating the differentiation between keratinocytes, hair follicle epithelium, sebaceous and eccrine glands, fibroblasts and tumor cells relative to the surrounding tissue.

Interestingly, the same specimen can be observed both under the reflectance and fluorescence mode simultaneously and the combination of the two techniques can give complementary information on the architectural and cellular features and allow digital pseudo-coloring in pink and purple, simulating what we would see in regular Hematoxylin and eosin staining. 

Ex vivo Confocal Microscopy Images

The microscope produces horizontal images of 750 × 750 μm of the different layers of the skin up to a thickness of 200 μm. Single images are rapidly and automatically stitched together into a reconstructed mosaic image. The acquisition time for a single image of 750 × 750 μm is 0.68 seconds. These mosaic images can also be stacked upon each other, creating a cube that mimics the cutting through layers of the specimen as it's done in pathology when further assessment is necessary for more precise analysis.

As described previously, ex vivo optical imaging can enable visualization of tissues with minimal or no preparation and without destroying or losing tissue. Therefore, this technology has the potential for incorporation into surgical pathology practice including rapid bedside tissue qualification of specimens such as CNBs or endoscopic biopsies, intraoperative assessment of margins of surgical resections in multiple medical areas, or rapid examination of any small fragments that are sent by the surgeons for frozen section processing. Ex vivo optical imaging techniques can also be used to identify tissue representative of the lesion for sampling, genomic testing, or biobanking.

I would say at this level and many other tissues

not only needle but margins check; can you do a real paragraph on "other"

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