Fluorescent contrast agents targeted to cancer biomarkers are increasingly being explored for cancer detection, surgical guidance, and response monitoring. Efforts have been underway to topically apply such biomarker-targeted agents to freshly excised specimen for detecting cancer cell receptors on the surface as a method for intraoperative surgical margin assessment, including dual-probe staining methods introduce a second ‘non-specific’ optical agent as a control to help compensate for heterogeneous uptake and normalize the imaging field. Still, such specimen staining protocols introduce multifaceted complexity with unknown variables, such as tissue-specific diffusion, cell-specific binding and disassociation rates, and other factors, affecting the interpreted cancer-biomarker distribution across the specimen surface. The ability to recover three-dimensional dual-probe biodistributions throughout whole-specimens could offer a ground-truth validation method for examining topical staining uptake behaviors. Herein, we report on a novel method for characterizing dual-probe accumulation with 3D depth-profiles observed from a dual-probe fresh-specimen staining experiment.
Characterizing an administered drug’s pathway from initial systemic uptake, to targeted tissue accumulation, and the eventual excretion route is an important component of clinical translation. For mapping such pharmacokinetic behaviors in a biologically-relevant system, fluorescently-tagged drugs are commonly administered and examined in preclinical animal models. Broadband fluorescence cryo-imaging offers a high-resolution, whole-animal technique for recovering such fluorescently-tagged biodistributions, although agent-specificity remains a challenge due to unknown levels of heterogeneous tissue autofluorescence. Herein, we report on a new hyperspectral multichannel fluorescence cryo-imaging system and demonstrate higher agent-specificity and signal-sensitivity compared to conventional broadband fluorescence.
MRI images of gadolinium-based contrast agents (GBCA’s) acquired before surgery are often registered to patients and used to guide surgical resection of intracranial tumors. Yet, the accuracy of these MR images in describing the surgical field degrades as surgery progresses; a well-recognized problem which has prompted efforts to develop new techniques that provide updated guidance information on residual tumor location. These efforts span a wide array of technologies, including image updating with deformation models, intraoperative MRI, and fluorescence guided surgery, among others. However, introduction of a straightforward technique that provides surgeons with a current view of GBCA distribution in real time remains an important goal. In this context, development of a fluorescent agent that recapitulates the kinetic behavior of GBCA’s could provide familiar information directly in the surgical field in real time. To advance this strategy, we have begun identifying fluorescent contrast agents that show similar kinetic behavior to GBCA’s. Using a novel hyperspectral whole body cryo-imaging system, we acquired highresolution 3-D volumes of the distribution of multiple candidate fluorophores in whole heads bearing orthotopic brain tumors. Preliminary results reveal significant differences in the distribution of candidate optical agents, some of which show strong similarity to the GBCA uptake. Identification and eventual translation of a reliable GBCAoptical analog could improve and simplify surgical resection of brain tumors.
Short-wave infrared (SWIR/NIR-II) fluorescence imaging has received increased attention for use in fluorescence-guided surgery (FGS) due to the potential for higher resolution imaging of subsurface structures and reduced autofluorescence compared to conventional NIR-I imaging. As with any fluorescence imaging modality introduced in the operating room, an appropriate accounting of contaminating background signal from other light sources in the operating room is an important step. Herein, we report the background signals in the SWIR and NIR-I emitted from commonly-used equipment in the OR, such as ambient and operating lights, LCD screens and surgical guidance systems. These results can guide implementation of protocols to reduce background signal.
The ability to directly measure whole-body fluorescence can enable tracking of labeled cells, metastatic spread, and drug bio-distribution. We describe the development of a new hyperspectral imaging whole body cryomacrotome designed to acquire 3-D fluorescence volumes in large specimens (whole animals) at high resolution. The use of hyperspectral acquisition provides full spectra at every voxel, enabling spectral decoupling of multiple fluorohpores and autofluorescence. We present examples of tissue spectra and spectral fitting in a rodent glioma xenograft.
The potential to image subsurface fluorescent contrast agents at high spatial resolution has facilitated growing interest in short-wave infrared (SWIR) imaging for biomedical applications. The early but growing literature showing improvements in resolution in small animal models suggests this is indeed the case, yet to date, images from larger animal models that more closely recapitulate humans have not been reported. We report the first imaging of SWIR fluorescence in a large animal model. Specifically, we imaged the vascular kinetics of an indocyanine green (ICG) bolus injection during open craniotomy of a mini-pig using a custom SWIR imaging instrument and a clinical-grade surgical microscope that images ICG in the near-infrared-I (NIR-I) window. Fluorescence images in the SWIR were observed to have higher spatial and contrast resolutions throughout the dynamic sequence, particularly in the smallest vessels. Additionally, vessels beneath a surface pool of blood were readily visualized in the SWIR images yet were obscured in the NIR-I channel. These first-in-large-animal observations represent an important translational step and suggest that SWIR imaging may provide higher spatial and contrast resolution images that are robust to the influence of blood.
The observed behavior of short-wave infrared (SWIR) light in tissue, characterized by relatively low scatter and subdiffuse photon transport, has generated considerable interest for the potential of SWIR imaging to produce high-resolution, subsurface images of fluorescence activity in vivo. These properties have important implications for fluorescence-guided surgery and preclinical biomedical research. Until recently, translational efforts have been impeded by the conventional understanding that fluorescence molecular imaging in the SWIR regime requires custom molecular probes that do not yet have proven safety profiles in humans. However, recent studies have shown that two readily available near-infrared (NIR-I) fluorophores produce measurable SWIR fluorescence, implying that other conventional fluorophores produce detectable fluorescence in the SWIR window. Using SWIR spectroscopy and wide-field SWIR imaging with tissue-simulating phantoms, we characterize and compare the SWIR emission properties of eight commercially available red/NIR-I fluorophores commonly used in preclinical and clinical research, in addition to a SWIR-specific fluorophore. All fluorophores produce measurable fluorescence emission in the SWIR, including shorter wavelength dyes such as Alexa Fluor 633 and methylene blue. This study is the first to report SWIR fluorescence from six of the eight conventional fluorophores and establishes an important comparative reference for developing and evaluating SWIR imaging strategies for biomedical applications.
Short-wave infrared imaging in tissue in the 1000-2000 nm range is characterized by reduced photon scatter and comparable or higher absorption compared to the NIR-I regime. These characteristics have implications for the performance of fluorescence molecular tomography (FMT) techniques, potentially improving the resolution of subsurface structure, possibly at the expense of depth sensitivity. To examine these questions, we have developed a SWIR small animal fluorescence tomography system. This instrument acquires multi-angle SWIR projection images of a stationary platform through a rotating gantry technique. These images are then processed for tomographic reconstruction of the SWIR fluorescence activity. Herein, we describe the development of this system and show multi-angle images from a mouse carcass containing a SWIR-specific fluorophore inclusion.
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