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Brett A Davis, Pryntha V Rajasingam, Michael J Collins; Measuring Ultraviolet Autofluorescence (UVAF) and Sodium Fluorescein (NaFl) Emission Spectra.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2141.
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© ARVO (1962-2015); The Authors (2016-present)
After building a UVAF imaging system we measured the spectral emission characteristics of ocular UVAF. As a proof of concept we also measured NaFl tear film fluorescence spectra.
A spectroscope was built using the head of a Rodenstock slitlamp, engineering rail, Sony DSC-F717 digital camera, 500 lines/mm diffraction grating, macro bellows unit and an adjustable optical slit. The optical slit was aligned with one slitlamp eyepiece while a Nikon D90 DSLR camera and Plossl lens were attached to the other slitlamp ocular to aid focusing. Two 375 nm UV LEDs were used as excitation sources and 400 nm high pass cutoff beamsplitters were used to remove unwanted visible light. An Essilor CRIZAL FORTE UV plano lens blank was placed over the slitlamp aperture to filter reflected UV light. The patient’s bright UVAF region was located using the slitlamp’s 24x magnification. Four spectral images (5s, 400 ISO, F2.4) and one en-face image (1/10s, 6400 ISO, F1.8) were recorded for both UVAF and NaFl. Images of a cold cathode fluorescent lamp (CCFL) and the UV reflection off a prosthetic eye were also taken. All spectral images were processed in ImageJ (NIH, USA) to create intensity profiles (‘plot profile’ option). The gas emission lines in the CCFL profile were used to calibrate for wavelength.
The 375nm UV light gave UVAF and NaFl emission peaks at approx 475 nm and 525 nm respectively. NaFl spectra also had a weaker peak at approximately 475 nm, possibly from the background UVAF emission. NaFl peaks were more variable than UVAF, probably due to changes in the tear film. All UVAF and NaFl spectra had a small peak from the UV LED reflections (see images D,E, F) at approx 390 nm, not 375 nm. The spectral response of the camera’s RGB colour channels was the cause of the UV peak ‘shift’ and the small ‘notches’ in the spectra.
It is possible to measure emission spectra for in-vivo UVAF and NaFl. Improvements could include 1) a monochrome sensor, 2) increased magnification, 3) improved optical filters, 4) intensity calibration and 5) multiple excitation wavelengths.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Figure 1: A) White light and B) UVAF images from a UVAF imaging system. C), D), E) and F) are en-face images for CCFL, UV LED reflections, UVAF and NaFl respectively. G), H), I) and J) are the corresponding spectral images and intensity profiles.
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