Research letter: A new fluorescent three- dimensional and deep- imaging technique for histological identification of individual tumor cells in extramammary Paget's disease

EMPD tumor border histologically. Compared to their method, our new technique requires only a LUCID/PK cocktail and TPM; there is no requirement for immunohistochemical staining or sample processing, such as making paraffin blocks or slices. Our procedure also has s higher resolution and is very easy to perform in any lab-oratory where TPM is available. Furthermore, confocal laser microscopy can detect the signal in tissue ( < 100 μ m thick). Further experiments are underway to establish the non- invasive “mapping biopsy” technique.


Research letter: A new fluorescent three-dimensional and deep-imaging technique for histological identification of individual tumor cells in extramammary Paget's disease
In extramammary Paget's disease (EMPD), Paget cells are sometimes detected outside the clinical border (subclinical extension) with a skipping pattern. Therefore, "mapping biopsy" is performed to evaluate the clinical border before complete resection. To increase the proportion of Paget cells in biopsy specimens, it is necessary to increase the number of biopsies. However, this is associated with increased pain and discomfort to the patient. The tumor border decision is also a critical factor for prognosis. If we could detect the EMPD tumor cells from the skin surface directly without cutting the patient skin, we could check numerous points where the mapping biopsy is necessary for detecting the tumor border, and it may bring a good prognosis with a skin surgery.
Recently, we have established a new fluorescent threedimensional deep-imaging technique using two-photon excitation fluorescence microscopy for skin lesions. 1,2 The new technique is very good at describing the skin tissue structure in the epidermis and dermis, for example, acrosyringium, individual keratinocytes, and nerve fibre. 1 Hence, to resolve the abovementioned issue, we evaluated whether a new fluorescent three-dimensional deep-imaging technique using two-photon excitation fluorescence microscopy for skin lesions is applicable for mapping biopsy in EMPD. EMPD samples resected during surgery were used in this study. Briefly, skin tissue samples were collected via 2-mm disposal biopsy punches from six areas around the main EMPD lesion. After fixation with 4% paraformaldehyde in PBS for 30 min, the samples were processed for propidium-iodide staining, and optically cleared using the transparency-enhancing ilLUmination of Cleared organs to IDentify target molecules method (LUCID), 3 and newly established push-pull pyrene probe bearing a ketone acceptor group (PK), which is a solvatochromic dye. 4 The samples were evaluated by two-photon excitation fluorescent microscopy (TPM), as described previously. 1  Two-photon microscopy is a very useful application of multiphoton microscopy in dermatological studies. 5 Conventional skin tissue slices have autoimmunofluorescence and the basic structure of the epidermis, which can reveal the dermis and skin appendages.
However, the resolution of this procedure is inferior to conventional HE staining. Therefore, a new technique using TPM with immunohistochemistry was developed that can reveal the detailed structure of skin appendages. 6 Two-photon microscopy with immunohistochemical analysis has already been applied to EMPD, 7

K E Y WO R DS
extramammary Paget disease, human skin, mapping biopsy, solvatochromic probe, two-photon excitation microscopy

ACK N O WLE D G E M ENTS
The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textc heck.com/certi ficat e/RycH7j.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflict of interest to declare. Video S1. EMPD lesion in the epidermis with three-dimensional observation.

FU N D I N G I N FO R M ATI O N
Video S2. EMPD lesion scanned from the surface to the dermis.