By offering rapid, perturbation-free, volumetric imaging capable of following both cellular- and nodular-level photodynamic therapy responses, OCT is a natural partner for imaging, understanding, and optimizing PDT. This is especially true for in vitro applications, where the resolution, depth range, scanning speed, and longitudinal capabilities of OCT facilitate new studies that can directly probe therapeutic responses deep inside complex 3D model systems. The studies presented and discussed in this review are only the first steps in utilizing these abilities for the selection and optimization of PDT regimens, with many more exciting opportunities waiting to be explored.
Efforts are underway to expand the capabilities of OCT by creating a robust image analysis toolkit that can mine high-resolution data for four-dimensional analysis. Specifically, routines must be developed that can identify key cellular and nodular events, and track the evolution of such events over long periods of time. Three-dimensional image segmentation algorithms must be developed to identify and quantify in vitro model factors, including volume, shape, growth rate, scattering intensity, and fragmentation, to name a few. These tools must include the ability to track cellular fate over the entire time-lapse experiment, including cellular migration following treatment. As seen above, the patterns of treatment response are highly dependent on the agent used, its concentration, uptake rate, and localization in tissue. Analysis routines must be developed to track treatment response patterns and make early predictions regarding treatment response. These routines can be applied for real-time analysis during data acquisition. High-throughput, high-content screening applications are likely a future use of OCT and TL-OCT systems, especially when used alongside increasingly relevant in vitro model systems. As mentioned earlier, TL-OCT is entirely compatible with multiwell culture systems where tens or hundreds of separate experiments can be plated and run simultaneously. While current studies investigating tumor response use similar methods, multiplex TL-OCT measurements would allow for the tracking of the natural history of 3D model treatment response for quantitative analysis.
This will be especially important for the optimization of PDT and PDT combination treatment approaches in the future. Combining the precise spatiotemporal knowledge gained through OCT with simultaneously acquired images or measurements of molecular responses would open the door to new intervention strategies. A multiplexed TL-OCT imaging platform combined with automated media collection and analysis, for example, would allow for the exploration and temporal mapping of numerous simultaneous factors to find time periods of peak sensitivity for combination therapeutic regimens.
While this review has focused on the use of OCT in the visualization of in vitro
culture systems, there are numerous means of extracting and culturing live tissues ex vivo
. Such approaches have several advantages over creating complex in vitro
models, most importantly that the tissue of interest can be harvested to preserve the original tissue architecture with its numerous differentiated cell types.
These live culture approaches provide an even more biologically relevant ex vivo
system than in vitro
models, especially for tissues not normally accessible for long-term high-resolution monitoring. OCT and TL-OCT are highly advantageous for imaging and following these large, complex models, especially for monitoring therapy. Tissue engineering experiments and bioreactor work would also benefit from the longitudinal, 3D, non-perturbative imaging capabilities of OCT.
Considerable work has been done in the field of OCT to improve equipment and detection schemes, aiming to boost image resolution and depth in order to obtain more detailed tissue structural information. The TL-OCT system used in the studies described here made use of older SD-OCT configurations optimized to provide long-term stability. Newer OCT technologies have since been developed and commercialized, enabling a new generation of long-term OCT acquisition tools. Swept-source OCT systems have advantages over fixed bandwidth systems, as they offer much improved spectral detection, enabling deeper imaging and oversampling for improved signal-to-noise contrast. Swept-source approaches used to be restricted to low bandwidths and thus had poor (>5 μm) axial imaging resolutions. Newer swept-source light sources can now achieve greater than 100 nm of bandwidth, opening the door for clearer, deeper TL-OCT imaging.
Deeper imaging can also be achieved by moving to longer wavelengths of light that are less attenuated by tissue. OCT systems operating at 1040 nm,
and now 1700 nm
can reach increasingly deeper into biological tissue, though the resolution of some of these systems is still too low for many cellular-level imaging applications. Of great interest for future TL-OCT designs has been the recent creation of highly stable μOCT systems using extremely broadband light for submicron imaging resolutions.
Furthermore, integration of advanced Doppler capabilities into TL-OCT will likely aid in the study of therapeutic effects, especially in advanced 3D cultures incorporating artificial vasculature.
A potential downside to OCT is its restriction to structural imaging. Unlike approaches that possess molecular sensitivity, such as multiphoton fluorescence microscopy and photoacoustic tomography, standard OCT systems are restricted to scattering-based contrast only. Several innovations, particularly by Boppart and colleagues,
might open the door to functional OCT with molecular sensitivity. Magnetic bead approaches,
and plasmonically-resonant targeted-nanoparticles,
are potential methods for enabling molecular sensitivity deep within 3D cultures for improved longitudinal therapeutic monitoring.