Characterizing Fluorophores for Clinical Applications in Otolaryngology

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.

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

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.