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Updated: Jul 27, 2025
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Published on: December 9, 2013
Dual-ratio approach for detection of point fluorophores in biological tissue
Giles Blaney1, Fernando Ivich2, Angelo Sassarolia1
1Tufts University, Department of Biomedical Engineering, Medford, MA USA, 02155.
Significance:
Diffuse in-vivo Flow Cytometry (DiFC) is an emerging fluorescence sensing method to non-invasively detect labeled circulating cells in-vivo. However, due to Signal-to-Noise Ratio (SNR) constraints largely attributed to background tissue autofluorescence, DiFC's measurement depth is limited. multiplies Aim: The Dual-Ratio (DR) / dual-slope is a new optical measurement method that aims to suppress noise and enhance SNR to deep tissue regions. We aim to investigate the combination of DR and Near-InfraRed (NIR) DiFC to improve circulating cells' maximum detectable depth and SNR.
Approach:
Phantom experiments were used to estimate the key parameters in a diffuse fluorescence excitation and emission model. This model and parameters were implemented in Monte-Carlo to simulate DR DiFC while varying noise and autofluorescence parameters to identify the advantages and limitations of the proposed technique.
Results:
Two key factors must be true to give DR DiFC an advantage over traditional DiFC; first, the fraction of noise that DR methods cannot cancel cannot be above the order of 10% for acceptable SNR. Second, DR DiFC has an advantage, in terms of SNR, if the distribution of tissue autofluorescence contributors is surface-weighted.
Conclusions:
DR cancelable noise may be designed for (e.g. through the use of source multiplexing), and indications point to the autofluorescence contributors' distribution being truly surface-weighted in-vivo. Successful and worthwhile implementation of DR DiFC depends on these considerations, but results point to DR DiFC having possible advantages over traditional DiFC.
