Quantitative Quantum Yield Measurements Using Flourescents Modulated Imaging
Background: Modulated imaging (MI) is a non-contact imaging modality that employs broadband, spatially modulated illumination capable of wide-field imaging, depth sectioning of turbid media, and the simultaneous extraction of the optical absorption (µa) and reduced scattering (µs') properties. The technique relies on extracting the depth and optical properties encoded in the spatial modulation transfer function of turbid media. Sinusoidal patterns of various spatial frequencies are used to illuminate the sample. Intensity data at each frequency (3 phase images per frequency) are demodulated, calibrated, and fit using a diffusion approximation of the radiative transfer equation. The differential contrast observed as illumination frequency increases is the basis for the quantitative separation of absorption and scattering. From these maps, chromophore concentrations can be derived. Technology: Researchers at the University of CA Irvine are advancing fluorescent Modulated Imaging (fMI). Fluorescence Modulated Imaging (fMI) provides quantitative and qualitative fluorescence wide-field images in turbid media including features such as (1) optical property, chromophore concentration, and quantum yield maps and (2) improved spatial resolution and depth sectioning.
Qualitative imaging is enhanced by Modulated Imaging due to the introduction of depth sectioning and improved spatial resolution. Three 1 mm beads tagged with 1 µM Cy5.5 equivalence were placed on the surface, as well as at a depths of 1 mm and 2mm, in a medium with a fluorescence background (0.1 µM Cy5.5 equivalence). All three beads at all three depth locations are visible although the beads appear to be of different sizes due to resolution limitations. The introduction of a higher spatial frequency illumination suppresses background fluorescence as well as the deeper 2 mm bead while decreasing the apparent bead size due to improvement in resolution. Finally, the introduction of an even higher spatial frequency isolates the surface structures dramatically and further suppresses background fluorescence. These images demonstrate the low pass spatial frequency characteristic of tissue as well as the depth sectioning provided by Modulated Imaging. As a result, surface structures can be isolated at higher frequencies while deeper perturbations can also be isolated with simple image processing. In addition, resolution improves with an increase in illumination spatial frequency.
Future directions of this work will include the determination of the fundamental resolution limits, further model development for quantitative calculations, and tomographic reconstruction algorithms.
Application: Here, we present fluorescent Modulated Imaging (fMI), an imaging modality that provides simultaneous (1) quantitative mapping of quantum yield and fluorophore concentrations and (2) qualitative depth sectioning and spatial localization. The combination of these quantitative and qualitative properties could be crucial in clinical situations. For example, this information could become a tremendous intra-operative tool as a means to identify tumor margins during surgery as well as determining tissue/fluorophore states.
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