We propose an alignment strategy for millimeter spectroscopy of cornea that uses imaging to screen for sufficient alignment conditions. The performance of different corneal imaging objectives, in the presence of misalignment, is evaluated. The cornea is illuminated with a TEM00 Gaussian beam at 650 GHz and the beam is swept across the cornea. Images are generated by calculating the coupling between illumination and scattered beams for each illumination beam position and angle. The cornea is displaced at intervals of 500 microns in the transverse and axial directions and with new coupling coefficient maps generated at each misaligned position. Contrast in the misaligned cases are compared to the aligned case via zero normalized spatial cross correlation. The results show a maximum normalized cross correlation of 0.92 for a two-mirror scanning system and 0.74 for a one-mirror scanning objective. The analysis suggests that imaging contrast at 650 GHz can be used to screen for misalignment that would be difficult to detect with MMW.
As we know, fluorescence lifetime imaging has demonstrated the ability to accurately detect materials and tissue constituents1–3. Current fluorescence lifetime systems rely on accurate temporal sampling to capture the tails of the decaying emission. These data are often fit to an exponential decay model3,4. Although these methodologies are powerful tools but they are often implemented as point measurement systems and require significant postprocessing to compute decay times or coefficients5–8. In some applications these factors can hinder clinical translation. Based on these observations, our group has developed algorithms and built simple, fast, and wide field imaging system9,10. This method uses a gated charge-coupled device (CCD) and a liquid light cable guided LED to compare the decay-time image intensity vs excited state image intensity, thus generating a spatially resolved maps of relative differences in autofluorescence decay of tissue constituents. This approach ensures very fast updating speed (< 2 sec per frame), big field of view (20 mm x 20 mm), excellent depth of field (up to 6 mm) for surface curvature of interested target at reasonable working distance (~50 mm). This innovative imaging system has a temporal resolution of 0.16 nanosecond, spatial resolution of 70 μm and has proved the capability to differentiate visibly similar tissue types, which has been validated with both fluorescent dyes and ex vivo human tissue samples in comparison to commercially available FLIM microscope. This work establishes a foundation to confirm the utility of our upgraded DOCI system for intraoperative tissue differentiating/imaging. Validation with a larger number of samples is currently ongoing.
Dynamic optical contrast imaging (DOCI) is a novel optical imaging technology that rapidly generates image contrast from measurements of aggregate endogenous fluorescence lifetime in a clinically meaningful field of view. Recently, our use of this system in both human ex-vivo and in-vivo specimens generated statistically significant contrast between tumor and adjacent normal tissue in biopsies taken from patients undergoing surgery for head and neck squamous cell carcinoma and primary hyperthyroidism. In this work we evaluated the components and resolution of our next-generation DOCI system. We also standardized the quantitative output of our system against the fluorescence lifetime values of three dye standards with monoexponential decay using a commercial Leica two-photon fluorescence lifetime imaging microscope. Significantly, our system continued to demonstrate clinically meaningful contrast between tissue samples with multiexponential decay in near real-time.