Despite the high prevalence of middle ear diseases, such as otitis media and cholesteatoma (abnormal skin cell growth), accurate diagnosis of these pathological conditions in the clinical setting remains a challenge. Current diagnostic methods suffer from significant observer variability, providing only minimal understanding of the diseases' underlying biochemistries. Standard ear evaluations are based primarily on visual inspection using a white light otoscope, a technology that has remained virtually unchanged for more than a century. Moreover, the whole process of examining the normal structure, diagnosing disease, and providing critical input for therapy relies mostly on human recognition of morphologic patterns in vivo. This approach is the current basis for decision-making, and a lack of objective diagnosis may result in many patients being over-treated on the worst-case assumption. On the other hand, misdiagnosis may result in severe complications, revisits to the healthcare practitioner, and surgical procedures, leading to a significant decrease in the patient's quality of life. Consequently, it is imperative to develop new reliable tools that can provide non-invasive and real-time middle ear diagnosis without necessitating human interpretation.
To address this need, our research has focused on developing chemical imaging techniques, especially autofluorescence1and reflectance imaging.2 These optical tools demonstrate molecular specificity and an ability to perform real-time measurements in a non-perturbative manner. They potentially improve our understanding of tissue structure and chemistry, collectively yielding objective recognition of pathology, which is currently unattainable in clinical practice. As an example, biochemical modifications in surgical margins (normal tissue around the site of a growth) is often anecdotally observed to be a precursor of lesion development, and, therefore, a definition of this pathology in chemical terms would be highly valuable. In addition to segmentation of the pathologies, an important thrust in our research is the identification of molecular markers from specific spatial regions. These may help to establish new opportunities for early diagnosis (and detection of relapse) of middle-ear conditions.
As a case in point, cholesteatoma is a non-neoplastic, expansive, and destructive lesion, consisting of hyperproliferative keratinizing squamous epithelium in the middle ear and mastoid cavity. Congenital cholesteatoma is defined as epithelial inclusions behind an intact tympanic membrane in a patient without history of otitis media. At present, surgery is only the treatment course recommended. However, surgical removal of cholesteatoma is both challenging and fraught with the possibility of recurrence.
To provide surgeons with a better imaging modality that can ascertain the ‘ clean’ margins, we used fluorescence imaging to differentiate cholesteatoma from uninvolved middle ear tissue, based on the characteristic autofluorescence signals (those naturally emitted by the biological structures). We developed a multi-wavelength ‘ fluoro-otoscope’ with a video-rate imaging capability that exploits the endogenous contrast of the middle-ear constituents to identify cholesteatoma. Given the promise of our preliminary findings in a patient cohort exhibiting congenital cholesteatoma, we are now seeking to investigate its feasibility in a multicenter setting with a larger patient population. Successful translation of this incredibly simple system, the fluoro-otoscope, could enable low-cost, objective, and automated assessment of lesion margins intraoperatively, with significant further opportunities for application in other prevalent middle ear conditions, notably acute otitis media.
Figure 1 shows images acquired from a representative congenital cholesteatoma in vivo. Figure 1(A) shows a white light image, while Figure 1(B) is a fluorescence image obtained using the fluoro-otoscope with 405nm excitation. The white light image displays the clear presence of a cholesteatoma in the posterosuperior aspect, with increased vascularity of the tympanic membrane. The cholesteatoma shows a broad fluorescence pattern, but there is little or no interference from the autofluorescence emitted by the bony promontory, and there is a complete absence of any autofluorescence signal from the tympanic membrane. This is particularly promising from a clinical translation standpoint, as the fluorescence images provide clear differentiation between the cholesteatoma and the surrounding uninvolved tissues. Further, the presence of the blood vessels and—critically—the boundaries of the lesion, are better defined in the autofluorescence image.
Figure 1. Images of a congenital cholesteatoma on the superior anterior quadrant of the tympanic membrane (in the middle ear). (A) White light image. (B) Fluorescence image. The fluorescence images provide clear differentiation between the cholesteatoma and the surrounding uninvolved tissues.
Recently we used Raman spectroscopy, which has high molecular specificity, to differentiate between cholesteatoma and another middle ear lesion, myringosclerosis, by analyzing the spectral patterns of differentially expressed molecules.3 Differentiation of these two conditions is particularly challenging using white light otoscopy because of the similarity in their visual appearance. Using Raman spectroscopy, we revealed signatures consistent with the known pathobiology of these middle ear lesions, and observed the first evidence of the presence of carbonate and silicate substitutions in the calcium phosphate plaques found in myringosclerosis. This study shows the potential of Raman spectroscopy not only to provide a new understanding of the etiology of these conditions by defining objective molecular markers, but also to aid in margin assessment to improve surgical outcomes.
In summary, we developed techniques that exploit autofluorescence and reflectance to improve imaging and characterization of features of the middle ear. Potentially, this helps physicians to differentiate diseased tissue from the surroundings in vivo, and therefore may assist with the development of therapeutic strategies.
In future work, we envision combining the exquisite chemical specificity of Raman spectroscopy with wide-field fluorescence imaging to obtain a multimodal algorithm capable of differentiating a broader range of middle ear pathologies. Collectively, the identification of molecular targets from spatially localized regions offers much-desired quantifiable data to enable early detection and longitudinal monitoring of middle ear pathology.
Tulio A. Valdez, Rishikesh Pandey, Nicolas Spegazzini, Ramachandra R. Dasari
Massachusetts Institute of Technology
Tulio A. Valdez is a pediatric otolaryngologist at Connecticut Children's Medical Center, where he is associate chair of academic affairs in the Department of Surgery. He is also a visiting scientist at the Laser Biomedical Research Center.
Rishikesh Pandey is a postdoctoral associate at the Laser Biomedical Research Center. His current research interest is development and deployment of novel biophotonic technologies that interface and bridge Raman spectroscopy, clinical diagnostics, and physical chemistry.
Johns Hopkins University
1. T. A. Valdez, R. Pandey, N. Spegazzini, K. Longo, C. Roehm, R. R. Dasari, I. Barman, Multi-wavelength fluorescence otoscope for video-rate chemical imaging of middle ear pathology, Anal. Chem. 86, p. 10454-10460, 2014.
2. T. A. Valdez, N. Spegazzini, R. Pandey, K. Longo, C. Roehm, C. Grindle, D. Peterson, I. Barman, Exploring multi-color reflectance imaging for in vivo monitoring of middle ear pathophysiology, Anal. Bioanal. Chem. 407, p. 3277-3283, 2015.
3. R. Pandey, S. K. Paidi, J. W. Kang, N. Spegazzini, R. R. Dasari, T. A. Valdez, I. Barman, Discerning the differential molecular pathology of proliferative middle ear lesions using Raman spectroscopy, Nat. Sci. Rep. 5, p. 13305, 2015.