A combined fluorescence lifetime and depth imaging system for medical imaging and surgical guidance

Abstract

Fluorescence lifetime imaging (FLIM) is a valuable technique which can be used to provide label free contrast between different tissue types and provide information about their molecular makeup and local environment. FLIM systems based on single photon avalanche diode (SPAD) arrays are increasingly being used in applications such as medical imaging due to their high sensitivity and excellent temporal resolution [1]. Additionally, SPAD arrays are also commonly employed for time of flight (ToF) imaging techniques such as light detection and ranging (LiDAR) [2]. Here we demonstrate a system which employs both of these modalities into a single instrument, allowing us to acquire both depth and widefield FLIM images simultaneously using a single 32 x 32 pixel SPAD array operating in time correlated single-photon counting (TCSPC) mode with 50 ps temporal resolution. Initial results show that we can correctly measure depths and distances of sample objects with < 1 cm resolution while maintaining excellent and consistent fluorescence contrast. Lifetime is consistent over a distance of 10 cm with a standard deviation of < 0.5 ns, showing that it is possible to decouple depth and lifetime data. We believe this work is the first demonstration of a widefield FLIM system capable of 3D imaging. The next step will be the addition of a miniaturized system [1] and future applications for this technology include fields such as surgical guidance, endoscopy and diagnostic imaging.

Fluorescence lifetime imaging with distance and ranging using a miniaturised SPAD system

“…As mentioned previously, this group has recently demonstrated that time resolved SPAD sensors may be used to perform combined WFLIm and time of flight measurements to allow for simultaneous lifetime contrast and 3D scene reconstruction 5 , 15 . The means for doing so is by analysing the temporal position of the rising edge of the fluorescence signal for each pixel 5 .…”

“…1 c. The green/yellow sample (colorFabb fluorescent green) consisted of fluorophores distributed within a PLA/PHA matrix with fluorophores embedded, and the pink (Real Filament fluorescent pink) consisted of fluorophores distributed throughout a matrix of just PLA. Although the precise fluorophores used in these filaments has not been shared by the manufacturer, they offer excellent fluorescent intensity and lifetime contrast and have been used widely for WFLIm characterisation previously 4 , 5 , 15 .…”

“…Another common application of time gated SPAD arrays is in time-of-flight techniques such as LIDAR, where the time resolution is used to measure the round trip of photons scattered from objects, thus allowing for distances to be calculated. Recently, this group has demonstrated that WFLIm and ToF data may be extracted simultaneously from the same time resolved SPAD data to allow for 3D scene reconstruction and surface mapping of fluorescent objects 5 , 15 . This differs from most previous attempts to combine ToF and WFLIm, as these have tended to focus on locating exogenous fluorescent inclusions at depth in the sample 16 – 18 , and although fluorescence ToF/LIDAR is a well established technique, it has tended to look only at fluorescence intensity rather than lifetime 19 – 21 .…”

Combined fluorescence lifetime and surface topographical imaging of biological tissue

“…The FLIMera camera and associated software are both commercially available and have previously been demonstrated to give highly accurate fluorescent lifetimes [ 29 , 32 ]. These filaments have previously been used in WFLIm using the same excitation and emission settings as are used in this work [ 9 , 28 ].…”

“…Most recently, the authors of the current paper used a SPAD array in TCSPC mode to perform combined fluorescence lifetime and ToF imaging [ 28 ]. This work differed from the previous techniques detailed above, as it aimed to determine the distance of the sample surface to the camera as opposed to subsurface depth, allowing surface mapping of the sample.…”