Abstract
Conventional histopathological techniques, such as hematoxylin and eosin staining, are limited to 4–5 μm-thick tissue sections, restricting visualization to two-dimensional planes. Moreover, acquisition of three-dimensional horizontal images from the skin surface remains challenging, hindering precise assessment of tumor margins in skin lesions. This challenge is particularly pronounced in extramammary Paget’s disease (EMPD), in which diffuse epidermal tumor cell spread complicates accurate evaluation of lesion extent. We hypothesized that combining horizontal sectioning with identification of individual tumor cells would enhance the determination of surgical margins. In this study, we developed a deep-imaging technique utilizing fluorescent solvatochromic dyes (LipiORDER® and HistoBright®) and two-photon microscopy to achieve high-resolution tumor margin visualization in EMPD. This technique enables identification of tumor cells in frozen and paraffin-embedded tissue blocks, as well as in live skin tissue under physiological conditions. Our novel approach holds substantial promise for improving the precision of surgical-margin assessment in EMPD and other cutaneous malignancies.
I. Introduction
Hematoxylin and eosin (H&E) staining has long been the standard for histological evaluation of skin sections, helping to advance understanding of dermatological disease morphology and lesion localization substantially. This technique has facilitated diagnosis of skin lesions and determination of excision margins for malignant tumors. However, conventional observation methods are restricted to tissue sections approximately 4–5 μm thick [7], making it difficult to visualize an excised specimen fully [1, 12].
Additionally, conventional H&E staining does not permit three-dimensional (3D) visualization of tissues, particularly horizontal sections from the skin surface (Z-axis data). This limitation hinders stereoscopic understanding of complex skin structures. In contrast, two-photon microscopy (TPM) combined with fluorescent dyes offers a high-resolution, deep-imaging technique capable of visualizing structures from the skin surface to subcutaneous adipose tissue in a single scan. This approach also enables 3D imaging of blood vessels, nerves, and skin appendages, providing a more detailed representation of skin architecture [9].
Extramammary Paget’s disease (EMPD) exemplifies the limitations of conventional diagnostic methods in accurately assessing lesion extent. EMPD lesions, often biopsied or excised before surgical treatment, are typically processed as frozen sections or paraffin blocks and evaluated using H&E staining. Despite these efforts, local recurrence is frequently observed [6], implying that conventional pathological evaluations do not always ensure complete removal of lesions. This uncertainty complicates the delineation of boundaries between EMPD lesions and surrounding normal skin, presenting substantial challenges for dermatologists.
EMPD is characterized by the presence of Paget cells and is typically classified as an intraepithelial cutaneous disease. These malignant cells spread horizontally within the epidermis, impeding precise assessment of lesion extent [3]. We hypothesized that developing a method for observing individual Paget cells in a 3D horizontal section from the skin surface would have significant clinical implications, particularly in guiding the extent of surgical resection.
This study investigated the potential of TPM and fluorescent dyes as alternatives to conventional H&E staining for the assessment of EMPD morphology and lesion boundaries. Specifically, the solvatochromic dyes LipiORDER® and HistoBright® were utilized to develop two approaches. The first involved imaging frozen or paraffin-embedded tissue blocks, while the second introduced a novel method for direct imaging of excised EMPD specimens. These techniques represent considerable advances in evaluating EMPD tumor margins and hold promise for improving surgical outcomes.
II. Materials and Methods
Ethics
This study was conducted in accordance with the principles of the Declaration of Helsinki. Leftover clinical samples were used with approval from the Institutional Ethics Committee of Ehime University School of Medicine (approval number: 18022009).
Tissue sample preparation for H&E staining
Excised specimens were collected from four patients diagnosed with EMPD. The EMPD tissues embedded in paraffin blocks were sectioned into coronal slices at a thickness of 5 μm and stained using the conventional H&E staining method.
Tissue sample preparation in frozen or paraffin blocks
Excised specimens were collected from four patients diagnosed with EMPD. After the removal of EMPD skin lesions, tissue blocks (2 × 2 mm) were excised from each patient, encompassing the epidermis, dermis, and adipose tissue. The samples were fixed in freshly prepared 4% (v/v) paraformaldehyde in phosphate buffer (pH 7.4) for 24–48 hr at 4°C. One block was embedded in frozen medium; another block was embedded in paraffin. Normal control samples, obtained from residual skin during the excision of skin tumors, were processed using the same embedding methods as the EMPD samples.
Optical clearance using transparency-enhancing technology (OCTET) and solvatochromic pyrene probe staining in frozen or paraffin blocks
Both frozen and paraffin blocks were optically cleared using the LUCID method to enable observation within the skin tissue [8, 9]. The solvatochromic dyes LipiORDER® and HistoBright® (Funakoshi Co., Ltd., Tokyo, Japan) were utilized as pyrene probes [2, 14]. A 10-μM pyrene probe solution was prepared in LUCID to create a master mix solution (MMS), which was stored at room temperature. Tissue sections were initially stained with Hoechst dye in phosphate-buffered saline (PBS) overnight at room temperature. After several washes with PBS, the sections were immersed in the MMS for 76 hr at room temperature.
Tissue sample preparation in postoperative resection specimens (living tissue)
Excised specimens were obtained from four patients diagnosed with EMPD. After lesion removal, 2 × 2-mm tissue blocks, including the epidermis, dermis, and adipose tissue, were excised and placed in Dulbecco’s Modified Eagle Medium (D-MEM). Normal control samples, obtained as residual skin from tumor-excision procedures, were processed using the same method and placed in D-MEM.
Solvatochromic pyrene probe staining in postoperative resection specimens (living tissue)
A 10-μM solution of LipiORDER® and HistoBright® was prepared in D-MEM. Tissue blocks were immersed in the pyrene probe solution without LUCID and incubated for 2 hr with 5% CO2.
Image acquisition
Tissue sections were imaged using light microscopy and TPM (AX R MP and A1 R MP, Nikon, Japan with Alcor 920-2, SPARK LASERS, France, and InSight DS+, Spectra Physics, USA) with a ×20 APO LWD 20XC NA1.0 lens and a ×25 APO LWD 25XC NA1.10 water-immersion lens (AX R MP and A1 R MP). TPM imaging was performed using excitation lasers at 920 nm (ALCOR) and 960 nm (InSight DS+). Second harmonic generation (SHG) signals from collagen fibers were captured by splitting the signals with dichroic mirrors at 488 nm and shot pass filter at 460 nm in the case of 920 nm excitation, and dichroic mirror at 495 nm was used in the 960 nm excitation. Fluorescence signals from the pyrene probe and Hoechst dye were detected at 500–550 nm and 563–593 nm, respectively, using a GaAsP-type photomultiplier module. Image stacks were acquired from the skin surface, corresponding to an area of 0.71 × 0.71 mm2 (2,048 × 2,048 pixels, 0.35 μm/pixel) of normal and EMPD tissues.
Image analysis
Images were processed using Nikon NIS-Elements version 5.21 software (Nikon, Japan). To enhance image clarity, all images were applied median 3 × 3 filtering and advanced denoising. All samples were evaluated along the vertical axis, covering the entire depth of the sections or tissue blocks. Maximum intensity projection images were saved as a single file in tagged image format (TIF, 8-bit).
Determination of the ease of identifying Paget cells (brightness difference value [BDV])
We assessed the optimal tissue conditions and fluorescent dyes for enhancing the identification of Paget cells. The ease of morphological identification of Paget cells was determined by comparing the brightness difference between the cell membrane and the cytoplasm in images saved in TIF format.
RGB (R: red, G: green, B: blue) is a method of representing colors by combining the three primary colors of light. For each Paget cell, the RGB values of the cell membrane and cytoplasm were measured at four points using PhotoDirector 365® (CyberLink, Ltd., New Taipei City, Taiwan). The brightness value was calculated using the formula 0.3 × R + 0.59 × G + 0.11 × B [4]. This formula is designed to align with human brightness perception by combining RGB values. An area with higher brightness values become more perceivable. The BDV was defined as the difference between the average brightness values of the cell membrane and the cytoplasm.
The eight Paget cells were examined under each condition (frozen/LipiORDER®, frozen/HistoBright®, paraffin/LipiORDER®, and paraffin/HistoBright®) to minimize variability, and the average values were calculated. Statistical analyses were performed using GraphPad Prism Software (version 10.4.0). Additionally, we hypothesized that the ease of identifying Paget cells would be associated with the difference in BDVs between Paget cells and keratinocytes, considering the presence of Paget cells within keratinocyte layers. To test this hypothesis, the BDVs of keratinocytes were measured, and the differences in BDVs between Paget cells and keratinocytes were calculated for each condition.
III. Results
We observed frozen and paraffin-embedded EMPD tissues using fluorescent dyes (LipiORDER® or HistoBright®) and TPM. The results of these observations are summarized below. To optimize identification of Paget cells, we evaluated various combinations of tissue preparation methods and fluorescent dyes by calculating BDVs, which quantify luminance differences between the cell membrane and cytoplasm. Additionally, we examined Paget cells in EMPD tissue immediately after excision, without freezing or paraffin-embedding processes. Our experimental setup was intended to carefully replicate the in vivo state of tumor tissue. This approach offers considerable potential to enhance clinical understanding and improve diagnostics for EMPD by enabling observation of Paget cells under conditions that closely approximate their natural state.
H&E staining
HE staining showed multiple Paget’s cells within the epidermis; Paget’s cells had large, pale cytoplasm and eccentric, spindle-shaped nuclei (Supplementary Fig. S1).
Imaging results for frozen blocks
LipiORDER® staining
Both normal and EMPD tissues in frozen blocks were stained with LipiORDER® in combination with Hoechst/LUCID (Hoechst/LUCID/LipiORDER®) and imaged at various magnifications (Fig. 1). In normal tissues, keratinocytes exhibited homogeneous green fluorescence, forming a uniform “paving stone” pattern in the superficial epidermis and at the dermo-epidermal junction. Individual keratinocytes appeared uniform in size; they displayed finely rounded nuclei positioned centrally within the cytoplasm.
In EMPD tissues, invading Paget cells were observed in both the superficial epidermis and at the dermo-epidermal junction (Supplementary Fig. S2, S3, video). These Paget cells were easily distinguishable due to their dark, clear cytoplasm and the brighter, more distinct green fluorescence along their cell membranes relative to surrounding keratinocytes. This contrast enabled clear identification of Paget cells within the tissue architecture. Additionally, these Paget cells exhibited a blue, eccentric nucleus and infiltrated the hair follicle (Supplementary Fig. S4).
HistoBright® staining
Staining and imaging using HistoBright® in combination with Hoechst/LUCID (Hoechst/LUCID/HistoBright®) were also performed on both normal and EMPD tissues in frozen blocks (Fig. 2). Normal keratinocytes displayed homogeneous bright-yellow to brownish-yellow fluorescence. These cells formed a uniform “paving stone” pattern in the superficial epidermis. At the dermo-epidermal junction, this pattern diminished regularly due to the absence of staining in dermal papillae. Individual keratinocytes appeared uniform in size; their nuclei were visualized as bluish-green fluorescence within the cytoplasm.
In EMPD tissues, invading Paget cells were detected in both the superficial epidermis and at the dermo-epidermal junction (Supplementary Fig. S3, video). Paget cells exhibited slightly darker cytoplasms, and their nuclei were poorly visible within the cytoplasm. The tumor cell membranes appeared marginally thicker and displayed brighter yellow fluorescence compared with surrounding keratinocytes. Based on subjective assessments of visibility, considering the brightness of the cell membrane and the relative darkness of the cytoplasm, frozen/LipiORDER® was considered superior to frozen/HistoBright® for identifying Paget cells.
BDV comparison between LipiORDER® and HistoBright® staining
To evaluate the ease of identifying Paget cells objectively, we performed BDV measurements. For frozen/LipiORDER®, the mean BDVs of Paget cells were 44.94 cd/m2 (range: 40.00–48.25 cd/m2) in the superficial epidermis and 41.97 cd/m2 (range: 38.25–48.00 cd/m2) at the dermo-epidermal junction (Fig. 3). For frozen/HistoBright®, the mean BDVs were 28.25 cd/m2 (range: 14.50–36.50 cd/m2) in the superficial epidermis and 28.75 cd/m2 (range: 23.25–39.75 cd/m2) at the dermo-epidermal junction.
The higher BDVs observed with frozen/LipiORDER® relative to frozen/HistoBright® confirmed that LipiORDER® provided greater contrast between the cell membrane and cytoplasm of Paget cells. This objective evaluation aligned with our subjective observations. Furthermore, differences in BDVs between Paget cells and keratinocytes were more pronounced with frozen/LipiORDER®, further supporting its superior performance.
Based on these findings, we concluded that frozen/LipiORDER® is more effective than frozen/HistoBright® for observing Paget cells in EMPD tissues, making it the preferred method for detailed imaging and analysis.
Imaging results for paraffin blocks
LipiORDER® staining
Both normal and EMPD tissues embedded in paraffin were stained with the Hoechst/LUCID/LipiORDER® mixture and observed at various magnifications (Fig. 4). Normal keratinocytes displayed homogeneous green fluorescence and formed a uniform “paving stone” pattern in the superficial epidermis and at the dermo-epidermal junction. Individual keratinocytes appeared uniform in size, with nuclei stained blue.
In EMPD tissues, invading Paget cells were observed in the superficial epidermis and at the dermo-epidermal junction (Supplementary Fig. S3, video). However, their visibility was lower than with LipiORDER® staining in frozen blocks, hindering identification.
HistoBright® staining
For comparison, the Hoechst/LUCID/HistoBright® mixture was applied to both normal and EMPD tissues embedded in paraffin (Fig. 5). Normal keratinocytes exhibited homogeneous bright-yellow to brownish-yellow fluorescence and formed a uniform “paving stone” pattern in the superficial epidermis. This pattern diminished at the dermo-epidermal junction due to the absence of staining in dermal papillae. Individual keratinocytes appeared uniform in size, with nuclei stained bright blue.
In EMPD tissues, Paget cells were observed in the superficial epidermis and at the dermo-epidermal junction (Supplementary Fig. S3, video). Paget cells in the superficial epidermis displayed bright blue nuclei, whereas those at the dermo-epidermal junction showed less distinct nuclear staining. The cytoplasm of Paget cells appeared darker; their cell membranes were slightly thicker and brighter yellow relative to surrounding keratinocytes. Additionally, these Paget cells infiltrated into the epidermis around sweat ducts (Supplementary Fig. S5).
Based on subjective assessment, identification of Paget cells in paraffin-embedded tissues was more effective using LipiORDER® when evaluating cytoplasmic contrast. However, HistoBright® performed better than LipiORDER® in terms of membrane brightness.
BDV comparison between LipiORDER® and HistoBright® staining
The mean BDVs of Paget cells stained with paraffin/LipiORDER® were 26.16 cd/m2 (range: 23.00–30.25 cd/m2) in the superficial epidermis and 21.31 cd/m2 (range: 13.50–30.25 cd/m2) at the dermo-epidermal junction (Fig. 3). For paraffin/HistoBright®, the mean BDVs were 21.97 cd/m2 (range: 17.00–27.50 cd/m2) in the superficial epidermis and 20.47 cd/m2 (range: 15.00–27.00 cd/m2) at the dermo-epidermal junction.
The BDV comparison between paraffin/LipiORDER® and paraffin/HistoBright® did not reveal clear superiority for identifying Paget cells. Similarly, when comparing BDVs between Paget cells and keratinocytes, no significant difference was observed between the two staining methods.
Considering the results and comparing frozen and paraffin-embedded EMPD tissues, we concluded that the frozen/LipiORDER® combination was the optimal method for identifying Paget cells.
Imaging results for EMPD tissue in postoperative resection specimens
After evaluating various combinations of fixation methods and fluorescent dyes, we determined that the frozen-LipiORDER® combination provided the greatest ease in identifying Paget cells. We hypothesize that freezing produced superior results compared with paraffin embedding because it preserves tissue closer to its native state and avoids potential alterations introduced by processes such as deparaffinization.
To observe tissue in a condition as close to in vivo as possible (living tissue), we examined excised EMPD tissue using LipiORDER® immediately after resection. The fluorescent staining procedure (D-MEM/LipiORDER®) was applied to both living-normal tissue and living-EMPD tissue (Fig. 6). This method revealed green-stained keratinocytes that were uniform in size. Invading Paget cells were detected in the superficial epidermis and at the dermo-epidermal junction (Supplementary Fig. S6, video).
Paget cells exhibited green cytoplasm and appeared larger than surrounding keratinocytes. However, the central regions of the tissues were not adequately stained green, resulting in insufficient visualization in these areas. In contrast, when the fluorescent staining procedure (D-MEM/HistoBright®) was applied to the tissue, keratinocytes and Paget cells were not observed (data not shown).
IV. Discussion
EMPD tissues were successfully observed using TPM and fluorescent dyes, marking the first instance of evaluating optimal methods for imaging skin tissue embedded in frozen or paraffin blocks using high-quality fluorescence imaging techniques. Additionally, horizontal lesion areas in fresh EMPD tissue were successfully identified immediately after excision using LipiORDER® fluorescent dyes with TPM. This novel technique provides a rapid method for analyzing fresh EMPD tissue without requiring embedding in frozen or paraffin blocks. Moreover, it enables evaluation of preoperative tumor tissue in a state closely approximating in vivo conditions.
In the observation of horizontal sections, conventional methods necessitate preparing numerous thin sections to achieve comprehensive visualization of the entire epidermis. In addition, multiple procedures, such as H&E staining and dehydration, are required to observe each of the thin sections, whereas our technique does not require such procedures. This method enabled the evaluation of individually distributed EMPD cells throughout the entire epidermal layer. Thus, it is very useful for assessing the extent of EMPD lesions, as EMPD is a characteristic progression that progresses through individual intraepidermal infiltrates. Although we did not continuously observe the transition zone from the lesion to the normal area or analyze large tissue samples at this time, further observation for this area would be expected to confirm tumor formation in the tissue. In particular, the lesion margins may appear as depigmented macules, making it difficult to determine the extent of the lesion macroscopically. This method allows for the identification of individual EMPD cells and may be superior to visual observation in accurately identifying the affected area.
EMPD is a rare neoplasm that typically arises in areas with a high density of apocrine glands, such as the genitalia, perianal region, and axillae. Paget cells, identifiable by their large size and abundant clear cytoplasm in H&E staining, often exhibit a distinctive “shotgun pattern” within the epidermis. Although histopathology generally allows straightforward identification of these tumor cells, the clinical presentation of EMPD skin lesions is highly variable. Lesions may appear as erosions, scaling, erythema, or other nonspecific features, hindering accurate detection and delineation of the exact margins of tumor cell involvement based on macroscopic examination alone. For this reason, “mapping biopsies” are often performed before surgical treatment to delineate lesion extent preoperatively [5]. Multiple punch biopsies are taken around the tumor lesion to determine the surgical border. However, false-negative mapping results can lead to incomplete resection. This highlights the need for numerous biopsy samples to achieve diagnostic accuracy that can guide surgical treatment.
Generally, biopsied or excised lesions are fixed in frozen or paraffin blocks and evaluated via H&E staining. However, recurrence has been observed at resection margins, implying that current methods present challenges in histologically understanding EMPD and determining the full extent of lesions [6, 11, 15].
Paget cells are scattered throughout the epidermal layer [13]. Therefore, horizontal imaging of tumor cells along the Z-axis and a 3D representation of the epidermis are necessary to evaluate individual Paget cells. Conventional H&E-stained sections require numerous slides to construct a 3D image. To overcome this limitation and improve visualization of individual EMPD lesion cells, we used TPM. This approach enables deep-tissue evaluation of the entire epidermal layer and constructs 3D images, offering a more detailed representation of tumor spread within these structures.
When imaging tissues with TPM, we used solvatochromic dyes, specifically pyrene probes (LipiORDER® and HistoBright®). These pyrene probes are highly photostable and bright, making them ideal for staining lipid components of skin tissue, cell membranes, peripheral nerve sheaths, and sebaceous glands [2, 9]. Both LipiORDER® and HistoBright® are commercially available dyes. Previously, we reported successful observation of Paget cells in 4% paraformaldehyde-fixed EMPD tissues using OCTET, LipiORDER®, and TPM [10]. For this study, we clarified frozen and paraffin-embedded tissue blocks using the LUCID method. Hoechst/LipiORDER® or Hoechst/HistoBright® mixtures were then applied, and the tissues were imaged using TPM. Both LipiORDER® and HistoBright® enabled deep-tissue imaging and clearly visualized keratinocytes and Paget cells. The LipiORDER® staining mixture showed these cells in green tones, whereas the HistoBright® mixture rendered them in yellow tones.
In summary, the optimal tissue condition and fluorescent dye for identifying Paget cells in this experiment was frozen/LipiORDER®. The reduced clarity in identifying Paget cells with paraffin/LipiORDER® relative to frozen/LipiORDER® may be attributable to the processes involved in paraffin embedding, deparaffinization, and the superior preservation of tissue in a state closer to living tissue. These factors prompted us to explore the possibility of observing Paget cells in conditions approximating living tissue, without the use of 4% paraformaldehyde in phosphate buffer, freezing, or paraffin embedding.
We evaluated whether this technique could identify Paget cells under physiological conditions. EMPD samples excised during surgery were placed in a medium containing LipiORDER® and incubated for 2 hr before observation with TPM. Our research group previously reported a method involving 4% paraformaldehyde fixation and LUCID treatment for excised specimens [10]. However, we found that Paget cells can also be observed without the use of fixatives or clearing agents. This approach offers simpler and faster evaluation compared with preparation of H&E-based pathological specimens, which involves multiple steps such as slicing and staining.
The fluorescent dye LipiORDER® was particularly suitable for this method. In frozen-LipiORDER, the cytoplasm of Paget cells appeared black, whereas in living-tissue-LipiORDER, it appeared green. This difference implies that the polarity of cytoplasmic fluids varies depending on tissue conditions. Although the resolution of skin structures, cell membranes, and EMPD lesions in living tissue-LipiORDER was slightly lower than with frozen-LipiORDER, this method enabled generation of Z-axis data and 3D images.
In conclusion, because individual Paget cells spread epidermotropically, conventional H&E methods face limitations in delineating the boundaries between EMPD lesions and surrounding normal skin. This study demonstrated that use of fluorescent dyes and TPM allows effective identification of Paget cells in frozen and paraffin-embedded tissues, as well as in tissues closer to living conditions. Our method facilitates 3D evaluation of Paget cells across the full thickness of the epidermis, addressing challenges associated with conventional histological techniques. This approach holds promise for applications in EMPD and other cutaneous and non-cutaneous malignancies. However, future challenges include improving dye permeability in live tissue and developing new fluorescent dyes.
This technique has the potential to be applied to Mohs micrographic surgery by improving the permeability of fluorescent dyes into tissues, leading to immediate microscopic evaluation of all excised margins. Addressing these challenges will guide the direction of further research.
V. Abbreviations
BDV, brightness difference value; H&E, hematoxylin-and-eosin; EMPD, extramammary Paget’s disease; TPM, two-photon excitation microscopy; 3D, three dimensions; OCTET, optical clearance using transparency-enhancing technology; MMS, master mix solution; D-MEM, Dulbecco’s Modified Eagle Medium.
VI. Conflicts of Interest
All authors declare that they have no competing interests.
VII. Acknowledgments
The authors thank Dr. Tasuku Nishihara, Department of Anesthesia and Perioperative Medicine, Ehime University Graduate School of Medicine, Ehime, Japan, for his technical support. This work was supported by JSPS KAKENHI Grant Number 22H04926, 22K06960, 23K07767, and a Grant-in-Aid for Scientific Research on Innovative Areal Platforms for Advanced Technologies and Research Resources from the “Advanced Bioimaging Support” program. This work was also supported by JST A-STEP Grant Number JPMJTR234G, Japan.
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