Otitis Media (OM) is one of the most common conditions of early childhood, accounting for a very high proportion of all pediatric physician office visits annually with national health care cost estimated to be greater than $2 billion. Despite the overuse and emergence of resistant pathogens, antibiotics remain the primary medical form of OM management. In fact, antibiotics for acute otitis media (AOM) are the most common prescriptions for children. In turn, tympanostomy tube placement to treat chronic otitis media (COM) is the most common pediatric surgical procedure requiring anesthesia in the United States. The most reliable and commonly used method for the diagnosis of OM in clinical practice is pneumotoscopy, which provides visible light illumination and mobility assessment of the tympanic membrane (TM). However, pediatricians and otolaryngologists correctly identify middle ear effusions at a rate of 51% and 73%, respectively highlighting the need for new diagnostic modalities in the primary care setting. To address the inability to diagnose otitis media adequately, we have developed three innovative methods leveraging novel optical modalities and molecular probes to target bacteria and cysteine proteases. For our first aim, we developed a shortwave infrared (SWIR) fluorescence otoscopy capable of imaging bacterial infections using a maltotriose probe that targets gram-positive and gram-negative bacteria via an ATP-binding cassette (ABC) transporter unique to prokaryotic and not eukaryotic cells. Identifying a bacterial otitis media infection can help reduce unnecessary antibiotic prescriptions, which contribute to multi-resistant bacteria. In our second aim, we have designed a Spatial Offset Raman Spectroscopy (SORS) probe able to be used with current ear speculums to provide molecular information regarding changes in the eardrum and middle ear fluid.. Understanding the molecular changes in the tympanic membrane can provide prognostic information regarding the progression or resolution of otitis media. Clinically we rely on otoscopy and significant visible changes to assess the middle ear. Understanding the optical signatures Raman spectroscopy provides can help us improve how we monitor otitis media. We will also collect middle ear samples to test our previously described algorithm to differentiate serous from mucoid middle ear effusions. Lastly, to address our goal of clinical translation, for our third aim, we have developed a shortwave infrared otoscope to diagnose middle ear effusion based on the increased absorption of light by water in the shortwave infrared wavelengths at around 1450nm. We will perform a pilot clinical trial in two institutions on the ability of shortwave infrared otoscopy to diagnose middle ear effusions. The proposed application will build on novel imaging modalities to refine non-invasive diagnostic imaging strategies for preclinical and clinical translation applications for otitis media.