Project Summary/AbstractAnal cancer incidence has been increasing by four times over the last three decades. The incidence rate isunacceptably high among HIV-positive homosexual men 131 per 100000. The main challenge in anal cancerdiagnosis and prevention is the lack of a standardized screening program. In vivo optical microscopytechnologies such as confocal microscopy and optical coherence tomography have the potential to visualizecellular morphologic changes associated with the anal malignancy but their clinical adaptation has beenchallenging. In this Trailblazer R21 project we propose to develop a low-cost scattering-based light sheetmicroscopy (sLSM) device that can aid accurate diagnosis of anal cancer with the goal of ultimately reducingthe anal cancer-associated morbidity and mortality. Our sLSM device will uniquely address the unmet needs inexisting in vivo microscopy technologies (e.g. CM OCT) simultaneously achieving i) high lateral resolution (1-2 m) for examining cellular nuclear features in the epithelium ii) large FOV (~2.5 mm) iii) visualization of theentire epithelial thickness in a single image and iv) low device cost (~$2000). We expect that the sLSM devicewill reveal the key diagnostic cellular features in vivo which will facilitate detection of early-stage analmalignancy and timely initiation of adequate treatment. The sLSM device will also guide the biopsy tomalignant lesions with high sensitivity and specificity to increase the biopsy yield and reduce the morbidity andcomplication caused by the unnecessary biopsy of benign lesions. As the first step towards this goal we willdevelop the prototype handheld sLSM device and evaluate the accuracy of diagnosing anal malignancy in thefollowing aims: In Aim 1 we will develop a handheld sLSM probe for imaging anal mucosa. The sLSM devicewill provide cellular resolution in the epithelium and reveal architectural details in deeper regions in a singleimage. We will optimally design the illumination and detection optics to achieve the 1.4 m lateral resolutionover a 2.5-mm FOV 6.5 0.9 m axial resolution for the epithelium and 18 8 m axial resolution for thesuperficial lamina propria. In Aim 2 we will conduct a clinical study of imaging human anal tissues with thehandheld sLSM device. One hundred anal biopsies concerning for anal cancer will be first imaged with thesLSM device. sLSM images will be compared with the corresponding histologic images to identify cellular andarchitectural features visualized in sLSM images and to develop the sLSM diagnostic criteria. The sensitivityand specificity of the sLSM for diagnosing anal malignancy will be evaluated. Automated image analysisalgorithms will be developed to quantitatively evaluate the cellular and architectural features. In addition toproviding a low-cost microscopy tool that aids accurate diagnosis of anal malignancy the sLSM technologycan be used for in vivo diagnosis of other cancers (e.g. cervical oral skin cancers) intra-operativeassessment of the surgical margin and quality assurance during the core needle biopsy.