Results of TD Imaging

The unique benefits of the TD optical imaging strategy employed by the Optix system include accurate depth and concentration information of the embedded fluorescent material, lifetime analysis, and functional 3D reconstruction.

Depth and Concentration Recovery

An important issue in fluorescence imaging is signal localization. Without accurate depth information, quantification is not possible unless many assumptions are made. For example, a CW fluorescence system cannot distinguish between an intensely fluorescent, deep inclusion and a weakly fluorescent inclusion at a shallower depth without using tomography, as illustrated below.

Fluorescent inclusions A and B of different strengths embedded at different depths have the same appearance in CW measurements.

With bioluminescence systems, estimates of depth are possible using indirect strategies that provide approximate rather than direct measurements of depth.

TD systems, such as the Optix instrument, resolve the depth of a fluorophore by relying on the unique properties of the TPSF. Shifts in the time that correspond to the maximum in peak fluorescence intensity are used as a measure of inclusion depth, as shown below.

The delay in the peak of the photon arrival distribution indicates that the inclusion is deeper in the tissue, since the flight time from excitation pulse to emission detection is longer with increased depth. Concentration does not affect the peak position.

Once the fluorophore’s intensity and depth are known, they can both be taken into account in computing its concentration through a simple equation.

Fluorescence Lifetime

A unique characteristic of time domain technology — nanosecond temporal point resolution — enables researchers to acquire in vivo fluorescence lifetime measurements. This additional parameter measures the average time a fluorophore emits light. Lifetime is affected by changes in its biochemical environment and can be used to separate bound from label-free agents. Fluorescence lifetime is essentially independent of fluorophore concentration and is less affected by photo-bleaching processes.

Fluorescence lifetimes are calculated based on exponential fits on a given TPSF, using either a single or dual exponential model. A typical output from a dual exponential fit in OptiView is shown below. The original and fitted curves, fitting residual, as well as the fitting parameters and results are shown on the top left, bottom left, and right respectively  in the TPSF Fit viewer.

Typical applications of lifetime recovery include:

  • Monitoring of drug combination therapy. This is possible due to the capability of TD systems to evaluate two lifetimes simultaneously.
  • pH and oxygenation level sensing, since changes in fluorescence lifetime are also known to occur with varying tissue pH and oxygenation levels.
  • Separation of bound from label-free agents.
  • Differentiation of healthy and ischemic tissues.
  • Isolation of the expression of two biochemical reporters with similar spectra.

The results shown below and obtained using the Optix system, demonstrate that different fluorophores with similar emission spectra, Atto680 and AF680/BSA, can be separated based on their fluorescent lifetimes, even when they are excited with the same light source. The graph below the images shows the distinct lifetime populations of each fluorophore.

Functional 3D Reconstruction

One of the goals of optical imaging is the generation of functional 3D images. While typical CW systems use data from multiple angular projections to generate 3D volume images of a specimen, TD systems acquiring data in reflection mode such as the Optix instrument,  need only a single projection to generate 3D images. The reconstruction process itself is based on incorporating data from several TPSFs, and requires the solution to a mathematical problem that satisfies certain physical constraints and the modeling of photon migration in scattering media and fluorescence.

To date, the Optix system has demonstrated remarkable abilities in providing accurate tomographic images at low concentrations with a single-view planar scan. Reconstructed 3D volumetric representations of fluorophore distribution can recover the shape, location, and concentration of fluorescent inclusions within the animal’s interior. As shown below, 3D reconstruction can be displayed as 2D slices in all axes or in a Plane viewer that displays all axis representations simultaneously.

3D reconstructed volumes can be exported as DICOM files from OptiView and then co-registered with high-resolution microCT images.