Preclinical whole body time domain fluorescence lifetime multiplexing of fluorescent proteins

Abstract

Abstract. The application of time domain (TD) fluorescence lifetime multiplexing for the detection of fluorescent proteins (FPs) in whole animals, in the presence of a strong background tissue autofluorescence and excitation light leakage is discussed. Tissue autofluorescence (AF) exhibits a nonexponential temporal response, distinct from the mono-exponential decay of FPs. This allows a direct separation of FP fluorescence from AF using a dual basis function approach. We establish the detection limits of this approach using in vitro and in vivo measurements. We also demonstrate, using an experimental model of lymph node metastasis, that FP-AF lifetime multiplexing provides a greater than 30-fold improvement in contrast-to-background ratio compared with continuous wave data. In addition, we show that TD detection can simultaneously discriminate between up to three red shifted FPs placed under the skin of a nude mouse based on their distinct fluorescence lifetimes.

In Vivo Tomographic Imaging of Deep-Seated Cancer Using Fluorescence Lifetime Contrast

“…2j), allows the use of a dual basis function approach (18,21) to eliminate AF from the transmission TD fluorescence images. In this approach, the decay portion of the TD fluorescence was fit with a dual-basis function (Eq.…”

“…2f,g,j. The dual basis function approach allows a dramatic increase in the contrast to background ratio (defined as the ratio of either the CW intensity or decay amplitude inside the lungs to that of the surrounding tissue (21)) of more than 20-fold by separating the iRFP720 fluorescence and tissue AF. Unlike the CW images (Figs.…”

“…We have recently demonstrated the tomographic lifetime multiplexing of anatomically targeted fluorophores in living mice (20). We have also shown that fluorescence lifetime contrast allows for a greater than 25-fold increase in sensitivity over traditional continuous wave (CW) techniques for detecting subcutaneous tumors expressing GFP (21). Based on the NIR spectral properties of iRFPs and the dramatic increase in sensitivity afforded by fluorescence lifetime contrast, we investigated whether the pairing of these two technologies provides a viable option for deep tissue imaging in intact mice.…”

Experimental Comparison of Continuous-Wave and Frequency-Domain Fluorescence Tomography in a Commercial Multi-Modal Scanner

“…As a result, time-dependent methods may provide more information for mathematical reconstruction to identify 3-D fluorescent target location and strength [18], [19]. In addition, time-dependent measurements offer the only opportunity for 3-D reconstruction of fluorescent lifetime which can be developed for sensing tissue environments, receptor-probe interactions, and evaluating molecular kinetics in vivo [20]- [23]. However, these time-dependent methods, whether are conducted with pulsed excitation light in time-domain (TD) measurements or with modulated excitation light in frequency-domain (FD) measurements, require more complicated instrumentation, are more greatly influenced by noise, and have longer data acquisition times than CW technique.…”

Multiplexed fluorescence mediated tomography with temporal and spectral data

“…17 To address this limitation, we and others have explored the idea of jointly using both fluorophore emission lifetime and spectral data in FMT. [18][19][20][21][22] In particular, organic NIR fluorophores typically have lifetimes on the order of 0.5 to 2 ns (with emission lifetimes usually being shorter at longer wavelengths). Our previous studies 23,24 suggested that joint use of spectral and temporal data provides more accurate demixing performance than either alone.…”