An in vivo imaging system was developed to distinguish between two commercially available far-red dyes with identical spectra characteristics based upon fluorescent lifetime.

In order to demonstrate the applicability fluorescent lifetime methods to medical imaging, we developed a computer-based lifetime imaging sensor (CBLIS). The portable CBLIS features a supervising microcontroller, a pulse generation circuit, a 658nm laser diode, an avalanche photo diode, and a sampling circuit. Alexa Fluor 647(Invitrogen) and ATTO 647N (AttoTec) were first spectrally characterized and then tested in vitro using CBLIS. Then the ratio of fluorescence intensity at two time points was used to differentiate the dyes for in vivo measurements. Each dye was injected subcutaneously in the flank of nude mice. Point measurements was taken over each injection sight and classified as Alexa Fluor 657 or ATTO 647N.

Spectrally, the dyes were indistinguishable with absorption peaks at 648nm and 645nm respectively and emission peaks at 668nm and 664nm. Using CBLIS, the lowest noise measurements were obtained with a laser repetition rate of 20MHz and analog averaging of 200,000 samples in a 0.01s window. Using these parameters the emission light was measured for 64ns at 250ps yielding measurements of 1.1ns for AF 647 and 3.5 ns for ATTO 647N. The measured fluorescent lifetime responses were used to choose time points 3.5ns and 7ns after the end of the excitation pulse for in vivo sampling. These points can be sampled with a dynamic range of 30dB, sufficient to distinguish these dyes in vivo.

Dyes in optical imaging may be differentiated based upon fluorescent lifetime in vivo.

Optical imaging at multiple wavelengths and different fluorescent lifetimes creates a dozen independent channels, more than in any other imaging modality.