Abstract
The detailed mechanisms associated with the influence of scattering and absorption properties on the fluorescence intensity sampled by a single optical fiber have recently been elucidated based on Monte Carlo simulated data. Here we develop an experimental single fiber fluorescence (SFF) spectroscopy setup and validate the Monte Carlo data and semi-empirical model equation that describes the SFF signal as a function of scattering. We present a calibration procedure that corrects the SFF signal for all system-related, wavelength dependent transmission efficiencies to yield an absolute value of intrinsic fluorescence. The validity of the Monte Carlo data and semi-empirical model is demonstrated using a set of fluorescent phantoms with varying concentrations of Intralipid to vary the scattering properties, yielding a wide range of reduced scattering coefficients (μ's = 0-7 mm (-1)). We also introduce a small modification to the model to account for the case of μ's = 0 mm (-1) and show its relation to the experimental, simulated and theoretically calculated value of SFF intensity in the absence of scattering. Finally, we show that our method is also accurate in the presence of absorbers by performing measurements on phantoms containing red blood cells and correcting for their absorption properties.
Highlights
Fluorescence detection and quantification is important for many biomedical applications
We have developed a single fiber based spectroscopic method, that allows a full quantification of the intrinsic fluorescence in a turbid medium
We have presented a calibration procedure that corrects for system transmission properties, enabling measurement of the intrinsic fluorescence in a turbid medium
Summary
Fluorescence detection and quantification is important for many biomedical applications. Developed methods to extract intrinsic fluorescence spectra involve the acquisition of a paired measurement of fluorescence and white-light reflectance, where the latter is used to inform a correction of the influence of optical properties on fluorescence. This general approach has been extensively investigated for multi-fiber fluorescence probes, with separate source(s) and detectors [7,8,9,10,11,12,13,14,15]. An alternative approach for fluorescence measurements is to use small fiber optic probes that utilize a single optical fiber to both deliver excitation light and collect emitted fluorescence [16,17,18,19,20]; such a measurement results in a localized sampling volume, with the majority of the collected signal originating very close to the probe face [18]
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