Lalwani Balasingam Olazarra Saffari

Introduction

This quarter, our team worked on characterizing fluorophores purchased by the Valdez Lab in the Department of Otolaryngology, Head and Neck Surgery at the Stanford School of Medicine to determine optimal materials for cholesteatoma visualization. The fluorophores we used in our measurements include elastin, keratin, NAD, and collagen. Our inspiration to pursue a project on cholesteatoma imaging was derived from work being done in the Valdez lab as well as oral imaging work being done by Dr. Joyce E. Farrell and Zheng Lyu in the Department of Electrical Engineering. We hope our work can shed some light on which fluorophores may be best for producing a label-free method in congenital cholesteatoma detection via autofluorescence microscopy. To aid these efforts, our teams will work on characterizing and analyzing such fluorophores to determine the optimal selections for clinical use given their respective absorption and emission spectra. Following these measurements, we will produce a fluorophore matrix via optimal response curve measurements. This will give users access to a matrix populated with the fluorophores used in earlier testing; however, the design of the matrix will enable users to customize to their desired fluorophore selections moving forward. While our project allowed us to gain a great deal of information about the fluorophores and expand upon our own understanding of how light interacts with materials, we plan on refining our methods and taking further measurements in the new year to come.

Background

In this section, we will provide both the clinical and technical background that inspired our motivation to pursue this project. Additionally, we will discuss the reasons we deemed fluorescence to be an ideal diagnostic tool for our given clinical objective.

Clinical Background

We were driven to find a label-free method for autofluorescence imaging of cholesteatomas. Cholesteatomas are non-malignant, non-cancerous growths that typically form behind the tympanic membrane [1, 2]. It is similar to a cyst, and often occurs in people who are prone to otitis media (or middle ear infections), and is often congenital [2, 3]. Our objective is to create a label-free method to characterize biomolecular changes in the keratinized epithelial tissue by exploiting the molecular specificity of autofluorescence spectroscopy. Specifically, we will measure the spectral signatures of biological molecules like NADH and elastin, both of which are naturally found in the body and can fluoresce upon absorption of ultraviolet (UV) or visible light. Ultimately, being able to quickly and reliably visualize these cholesteatomas is desirable for early diagnosis and complete resection of the lesions, in order to better preserve the patient’s hearing ability at the time of surgery.

Methods

We acquired four fluorophores (NADH, collagen, elastin, and keratin), then measured the fluorescence signal produced by each of them using both a fluorometer and the OralEye camera. The purpose of our initial fluorescence measurements, which were performed using a fluorometer, was to determine whether we were able to measure emission spectra consistent with literature values for each of our fluorophores. Secondary measurements, which were performed using the OralEye camera, sought to determine whether we were able to measure similar emission spectra using the 385-nm excitation light source associated with the OralEye camera.

Results

Results from Fluorometer Measurements

We found the following spectra for the NADH, collagen, elastin, and keratin fluorophores using Haribo’s fluorolog:

NADH

Collagen

Elastin

Keratin

Troubleshooting

Future Work

In the future, we plan to evaluate the effects of different concentrations on our ability to distinguish a fluorescence signal from the background reflected light signal. Our experiments with the fluorometer indicate that concentration can have a noticeable effect on our ability to measure fluorescence. Assuming a similar dependency on concentration in our OralEye camera set-up, we would like to perform many experiments to find the “sweet-spot” concentration value that gives us measurable fluorescence. By the same token, we would like to perform more measurements on the fluorometer by exciting our fluorophores at the 385 nm wavelength, to better compare the measurements between the OralEye Camera and the emission spectra. This will also help us find emission spectra that are consistent with those recorded in previous literature.

In order to acquire more meaningful data using the OralEye camera set-up, we must address the issue of measuring weak fluorescence in the presence of a relatively strong reflected light signal. One obvious way to do this is to implement a more effective filter in order to prevent excessive reflected light from “leaking” into our fluorescence measurements. One possibility would be to apply a filter with an even higher threshold, such as a Y48 or Y50 filter (with cut-off wavelengths of ~480 nm and ~500 nm, respectively), to further reduce the impact of the light reflected from the 325 nm light source. As mentioned earlier, another possibility would be to increase or decrease the concentration of the fluorophore samples we measure; doing so might increase the signal produced by the fluorophores, and thus reduce the issues associated with the weak signal we saw in our earlier measurements. By exploring these sorts of solutions, we feel confident that a robust set of measurements can be acquired for our selected fluorophores.

Appendix II

Anand and Persiana worked on characterizing the fluorophores by measuring the absorption and emission spectra of each fluorophore. They used a fluorescence spectrometer (fluorimeter) in the chemistry department that can measure wavelengths from the UV range to 1,000 nanometers as well as has a sub-nanometer image resolution. Following fluorimeter training, preparation of samples, and recording of measurements, Anand and Persiana analyzed the data and interpreted it for the writing of this report and the presentation.

Based on the results of the fluorophore absorption and emission spectra, Sofie and Ramya constructed a matrix containing the optimal water-soluble fluorophores. The design was modeled after that of Connect Four, and was 3D-printed on campus, in the Huang basement. Following construction of the matrix, Sofie and Ramya proceeded to take measurements with the OralEye Camera in Dr. Farrell’s lab. They then subsequently plotted their results using MATLAB scripts found in Appendix I.

We all contributed to the final in-class presentation, and Sofie, Persiana, and Ramya wrote up our work for the Wiki page submission.

Giving Thanks

We would like to express our appreciation for the countless hours that Professor Brian A. Wandell, Dr. Joyce E. Farrell, and Zheng Lyu spent on making this course truly exceptional. Across our team, the class has inspired several new interests, including presenting magneto-sensor research at the next imaging conference, exploring a career in ophthalmology, and continuing to refine our fluorophore results in the year to come. We are tremendously grateful for your time, energy, and investment in our team. It brought us great joy and honor to be your students.