System characterization of time-resolved mesoscopic fluorescence molecular tomography

Computational macroscopic lifetime imaging and concentration unmixing of autofluorescence

“…Of note, for this experiment the lifetime of the Riboflavin probe appeared higher than what was reported and validated through both SP-MAFLI and MFMT systems for individually measured phores. To understand whether this change came from the probe itself or indicated a misestimation of the SP-MAFLI workflow, a well sample of the Riboflavin probe used for this experiment was measured by the MFTM system reported in [38]. The MFMT lifetime estimation of Riboflavin was in accordance to the one reported with the SP-MAFLI system as shown in Figure A8D.…”

“…First the current experiments have shown that in terms of lifetime, concentrations greater than 25 μm can be correctly quantified, however this might not be true for concentrations lower than this. In this regard, as the used illumination power density for a widefield illumination scheme covering a 3.4 Â 3.4 cm area is $0.24 mW/cm 2 , that is at least 500 times lower than that typically used in raster scanning methods [7,38], illumination power improvement is necessary. We hypothesize this will produce an increase in the amount of excited autofluorescence specially for low concentrations and hence the amount of detected signal.…”

“…Individual autofluorophores Riboflavin, FAD and PPIX were measured and results are displayed in the main text. Their lifetime was correlated to results from a Time-Domain Mesoscopic Fluorescence Molecular Tomography system reported in [38]. Of note, the MFMT works at the mesoscopic regime, therefore the signals from the sample are less prompt to noise and low-power effects as commonly expected for the macroscopic regime, however the covered FOV À0.15 Â 0.15 cm is at least 1/5 of the one covered by the proposed platform À3.4 Â 3.4 cm, hence only an example well is measured for lifetime quantification purposes, rather than the full FOV of the sample as measured with the SP-MAFLI platform.…”

“…The lifetimes of all wells were averaged, obtaining mean values of and for Ribloflavin, PPIX and FAD, respectively. As the literature reports a broad range of lifetimes for autofluorescence, as seen in [33], individual lifetime characterization of the autofluorophores was also performed through a Time‐Domain Mesoscopic Fluorescence Imager (TD‐MFMT) [38] and Alligator‐based lifetime quantification [36]. One well per autofluorophore was measured from the same stock solutions and after lifetime calculation, linear regression of averaged lifetime values obtained with both systems yielded p‐value of 2.3e‐3, = 1, intercept = −0.23 and slope = 1.13, indicating high correlation between the estimated values for both systems, as shown in Appendix B (B) ‐ Figure A3.…”

“…For FAD the estimated lifetimes behave constant across concentration, however fit residuals are still higher than PPIX for higher concentration wells. Note that the illumination power density used in these experiments is at least 500 times lower than that typically used in raster scanning methods [7, 38]. Hence, it is expected that the lower concentration threshold of the SP‐MAFLI platform can be greatly improved with the availability of higher illumination power, as the illumination is distributed in wide‐field configuration.…”