High-quality Fluorescence Imaging of the Human Acrosyringium Using a Transparency: Enhancing Technique and an Improved, Fluorescent Solvatochromic Pyrene Probe

Abstract

Two-photon, excitation fluorescent microscopy featuring autofluorescence or immunofluorescence, combined with optical clearance using a transparency-enhancing technique, allows deep imaging of three-dimensional (3D) skin structures. However, it remains difficult to obtain high-quality images of individual cells or 3D structures. We combined a new dye with a transparency-enhancing technology and performed high-quality structural analysis of human epidermal structures, especially the acrosyringium. Human fingertip skin samples were collected, formalin-fixed, embedded in both frozen and paraffin blocks, sliced, stained with propidium iodide, optically cleared using a transparency-enhancing technique, and stained with a new fluorescent, solvatochromic pyrene probe. Microscopy revealed fine skin features and detailed epidermal structures including the stratum corneum (horny layer), keratinocytes, eccrine sweat glands, and peripheral nerves. Threedimensional reconstruction of an entire acrosyringium was possible in one sample. This new fluorescence microscopy technique yields high-quality epidermal images and will aid in histopathological analyses of skin disorders.

An Advanced Detection System for <i>In Situ</i> Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe

“…Indeed, FISH has proven to be a powerful method for the analysis of specific DNA, but it is not suitable for analysis of RNA because of high background. Although great achievements were done in multiplex detection and signal amplification, the current procedures are still complex, time consuming, and require experienced personnel [14,15,17,18,28,31,34]. Therefore, simpler and more-rapid detection tools for ISH are needed.…”

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

“…4 The samples were evaluated by two-photon excitation fluorescent microscopy (TPM), as described previously. 1 All procedures were performed with prior approval from the Ethics Committees of Ehime University Graduate School of Medicine. This study was conducted in accordance with the principles of the Declaration of Helsinki.…”

“…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 . All procedures were performed with prior approval from the Ethics Committees of Ehime University Graduate School of Medicine.…”

“…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.…”

Synthesis and photophysical properties of a new push–pull pyrene dye with green-to-far-red emission and its application to human cellular and skin tissue imaging

“…18,19 This feature is useful for studying various cellular phenomena related to membrane processes. 14,15 PK is currently commercially available, and more recently, it was found to efficiently stain the cell membranes in paraffin-embedded or frozen human skin tissue blocks, enabling the visualization of the individual cell morphologies in a three-dimensional manner through two-photon fluorescence microscopy (2PM); 20 this is useful for the rapid and accurate diagnosis of human skin diseases. 21 Notably, most existing pyrene-based push–pull dyes exhibit fluorescence in the blue-to-yellow region (Fig.…”