Quantifying Human Eosinophils Using Three-Dimensional Volumetric Images Collected With Multiphoton Fluorescence Microscopy

Eosinophilic esophagitis (EoE) is a relatively new disease with ~10-fold increase in prevalence over the past 20 years,1,2 and has been found in ~6.5% of the population undergoing upper endoscopy.3 This disease has become one of the leading causes of dysphagia and food impaction in adults. For diagnosis, an endoscopy is performed where multiple biopsies are collected at random throughout the length of the esophagus, including the proximal and distal regions. On histopathology, the primary feature of EoE is infiltration of eosinophils into the mucosa. These mediators of inflammation may contribute to the development of structural abnormalities of the esophagus, including edema, rings, furrows, and strictures.4 Clinical symptoms do not improve with high-dose proton pump inhibitor therapy, and the pH in distal esophagus is usually normal.5 However, the diagnostic criteria for this disease appear to lack clarity. EoE may be difficult to distinguish from GERD,6 which is also associated with increased eosinophilia but to a lesser extent, and the two diseases may be present at the same time. Eosinophils can also trigger allergic symptoms in other parts of the gastrointestinal tract.7 The degree of mucosal hypereosinophilia that defines EoE is controversial. While a diagnostic criteria of ≥15 eosinophils per high-power-field (hpf) on histology has been proposed,5 values as high as 30 eosinophils per hpf have been used, and no single number is widely accepted.8–10 Diagnostic uncertainty for this disease may be attributed in part to its patchy and focal nature. In addition, there is little known about the density or spatial distribution of eosinophils throughout the mucosa. Marked variability has been found within and between biopsy specimens of individual patients, resulting in a low sensitivity for detection. Currently, biopsy specimens are sectioned along a plane whose orientation to the mucosal surface is unknown. A non-uniform distribution of infiltrating eosinophils within the mucosa could result in a highly variable cell count that depends on the angle of sectioning, resulting in an inaccurate result. A novel method that can quickly and reliably quantify the number of cells over a 3-dimensional (3D) volume could be used to overcome this tissue processing limitation. Human eosinophils contain granules that produce an intense autofluorescence in comparison to surrounding squamous epithelium.11–13 There is evidence to support flavin adenine dinucleotide (FAD) as the source of this endogenous fluorescence.14 FAD is a coenzyme in the mitochondrial electron transport chain that has a maximum absorption at 445 nm, resulting in a peak fluorescence emission of 525 nm.15 Multi-photon microscopy (MPM) is a powerful method for collecting fluorescence images from cells and tissues,16 and has been used to perform in vivo imaging of FAD from squamous epithelium in animals.17,18 MPM imaging has inherent 3D resolution, uses near-infrared excitation for superior tissue penetration, has lower photobleaching effects, and is capable of providing quantitative information.19 We have previously demonstrated the use of MPM imaging as a highly accurate method for identifying and quantifying human eosinophils from mucosal smears of patients with allergic rhinitis.20 In this study, we aim to demonstrate the use of MPM to detect eosinophils within squamous epithelium, characterize the distribution of eosinophils with depth below the mucosal surface, and quantify the number of eosinophils within a 3D volume.