NIR Fluorescence lifetime macroscopic imaging with a time-gated SPAD camera.

The performance of SwissSPAD2 (SS2), a large scale, widefield time-gated CMOS SPAD imager developed for fluorescence lifetime imaging, has recently been described in the context of visible range and fluorescence lifetime imaging microscopy (FLIM) of dyes with lifetimes in the 2.5 - 4 ns range. Here, we explore its capabilities in the NIR regime relevant for small animal imaging, where its sensitivity is lower and typical NIR fluorescent dye lifetimes are much shorter (1 ns or less). We carry out this study in a simple macroscopic imaging setup based on a compact NIR picosecond pulsed laser, an engineered diffuser-based illumination optics, and NIR optimized imaging lens suitable for well-plate or small animal imaging. Because laser repetition rates can vary between models, but the synchronization signal frequency accepted by SS2 is fixed to 20 MHz, we first checked that a simple frequency-division scheme enables data recording for different laser repetition rates. Next, we acquired data using different time gate widths, including gates with duration longer than the laser period, and analyzed the resulting data using both standard nonlinear least-square fit (NLSF) and phasor analysis. We show that the fixed synchronization rate and large gate widths characterizing SS2 (10 ns and over) are not an obstacle to accurately extracting lifetime in the 1 ns range and to distinguishing between close lifetimes. In summary, SS2 and similar very large gated SPAD imagers appear as a versatile alternative to other widefield time-resolved detectors for NIR fluorescence lifetime imaging, including preclinical molecular applications.

High compression deep learning based single-pixel hyperspectral macroscopic fluorescence lifetime imaging in vivo.

Single pixel imaging frameworks facilitate the acquisition of high-dimensional optical data in biological applications with photon starved conditions. However, they are still limited to slow acquisition times and low pixel resolution. Herein, we propose a convolutional neural network for fluorescence lifetime imaging with compressed sensing at high compression (NetFLICS-CR), which enables in vivo applications at enhanced resolution, acquisition and processing speeds, without the need for experimental training datasets. NetFLICS-CR produces intensity and lifetime reconstructions at 128 × 128 pixel resolution over 16 spectral channels while using only up to 1% of the required measurements, therefore reducing acquisition times from ∼2.5 hours at 50% compression to ∼3 minutes at 99% compression. Its potential is demonstrated in silico, in vitro and for mice in vivo through the monitoring of receptor-ligand interactions in liver and bladder and further imaging of intracellular delivery of the clinical drug Trastuzumab to HER2-positive breast tumor xenografts. The data acquisition time and resolution improvement through NetFLICS-CR, facilitate the translation of single pixel macroscopic flurorescence lifetime imaging (SP-MFLI) for in vivo monitoring of lifetime properties and drug uptake.

Assessing patterns for compressive fluorescence lifetime imaging.

A novel hyperspectral single pixel system was used to compare different compressive basis patterns for intensity imaging, lifetime imaging, and FRET quantification. Six popular basis patterns were compared experimentally in a phantom containing two fluorescent dyes. The basis patterns that performed best for lifetime quantification were used to measure FRET occurrence in well-plate samples with varying acceptor-donor ratios. The ABS-WP approach using Haar patterns and the compressive sensing approach with Hadamard Ranked patterns displayed the best overall performances at a 50% compression ratio.

Time Domain Fluorescent Diffuse Optical Tomography: analytical expressions.

Light propagation in tissue is known to be favored in the Near Infrared spectral range. Capitalizing on this fact, new classes of molecular contrast agents are engineered to fluoresce in the Near Infrared. The potential of these new agents is vast as it allows tracking non-invasively and quantitatively specific molecular events in-vivo. However, to monitor the bio-distribution of such compounds in thick tissue proper physical models of light propagation are necessary. To recover 3D concentrations of the compound distribution, it is necessary to perform a model based inverse problem: Diffuse Optical Tomography. In this work, we focus on Fluorescent Diffuse Optical Tomography expressed within the normalized Born approach. More precisely, we investigate the performance of Fluorescent Diffuse Optical Tomography in the case of time resolved measurements. The different moments of the time point spread function were analytically derived to construct the forward model. The derivation was performed from the zero order moment to the second order moment. This new forward model approach was validated with simulations based on relevant configurations. Enhanced performance of Fluorescent Diffuse Optical Tomography was achieved using these new analytical solutions when compared to the current formulations.

Metabolism-enhanced tumor localization by fluorescence imaging: in vivo animal studies.

We present a high-sensitivity near-infrared optical imaging system for noninvasive cancer detection and localization based on molecularly labeled fluorescent contrast agents. This frequency-domain system utilizes the interferencelike pattern of diffuse photon density waves to achieve high detection sensitivity and localization accuracy for the fluorescent heterogeneity embedded inside the scattering media. A two-dimensional localization map is obtained through reflectance probe geometry and goniometric reconstruction. In vivo measurements with a tumor-bearing mouse model by use of the novel Cypate-mono-2-deoxy-glucose fluorescent contrast agent, which targets the enhanced tumor glycolysis, demonstrate the feasibility of detection of a 2-cm-deep subsurface tumor in the tissuelike medium, with a localization accuracy within 2-3 mm.

Interfering diffusive photon-density waves with an absorbing-fluorescent inhomogeneity.

This work reports an investigation of the fluorescent field re-emitted by an object embedded in a highly scattering media illuminated by two-interfering sources. Simulations in the frequency domain with a finite difference method solving the diffusion equation were performed. The media considered had features typical of a soft-compressed breast. An absorbing-fluorescent inhomogeneity was embedded in the center of the slab. A qualitative study of the re-emitted field was achieved. The re-emitted field was found to possess unique features characteristic of the two-interfering sources excitation, i.e. null intensity when the object was between the two sources and a 180 degrees transition crossing this position. Those features, when performing a scan of the two sources, permitted accurate localization of the inhomogeneity. Moreover, even when the detector was not placed on the mid-plane of the two sources, the re-emitted field still exhibited the interfering characteristic pattern, which was not seen at the excitation wavelength. Thus, for such configurations, the re-emitted field still possessed the specific sensitivity of phased array emission conversely to the excitation wavelength.

Signal-to-noise analysis for detection sensitivity of small absorbing heterogeneity in turbid media with single-source and dual-interfering-source.

Previous studies have suggested that the phased-array detection can achieve high sensitivity in detecting and localizing inhomogeneities embedded in turbid media by illuminating with dual interfering sources. In this paper, we analyze the sensitivity of single-source and dual-interfering-source (phased array) systems with signal-to-noise ratio criteria. Analytical solutions are presented to investigate the sensitivity of detection using different degrees of absorption perturbation by varying the size and contrast of the object under similar configurations for single- and dual-source systems. The results suggest that dual-source configuration can provide higher detection sensitivity. The relation between the amplitude and phase signals for both systems is also analyzed using a vector model. The results can be helpful for optimizing the experimental design by combining the advantages of both single- and dual-source systems in object detection and localization.