Oncologic surgery can greatly benefit from imaging techniques for the accurate identification of tumor-positive margins both intraoperatively and in resection specimens immediately following surgery. We have demonstrated clinically that fluorescence lifetime can significantly improve the accuracy for tumor vs. normal classification compared to fluorescence intensity in multiple cancer types using tumor targeted agents. Ongoing efforts by our group towards the translation of fluorescence lifetime imaging for intraoperative image guidance using exogenous agents will also be discussed.
We performed synthesis and testing of Fab’-based NIR fluorescent probes with the goal of achieving sufficiently high tumor/background (muscle) ratios at the earlier time points after intravenous injection in comparison to the full-size IgG or larger fragments. Conjugation of NIRDye 800CW to Fab’ resulted in partially quenched imaging probes. Fluorescence intensity imaging of orthotopic xenografts in both EGFR-attenuated and wild-type triple negative breast cancer tumors using Fab’-800CW allowed earlier detection of EGFR expression in vivo.
Fluorescence imaging can result in poor tumor contrast due to non-specific probe accumulation of receptor targeted probes. Here we show using preclinical and clinical studies that fluorescence lifetime (FLT) imaging can significantly improve the sensitivity and specificity for tumor detection using epidermal growth factor receptor (EGFR) targeted near infrared probes. We also show that FLTs in tissue are highly correlated with receptor expression levels, thereby enabling quantification of receptor quantification in vivo. Ongoing efforts in our group towards the translation of FLT imaging for intraoperative image guidance during head and neck surgeries will also be discussed.
Programed death ligand-1 (PD-L1) expression is currently the only predictive biomarker for cancer immunotherapy. Since PD-L1 expression in tumors is largely heterogeneous, in-vivo detection and quantification of PD-L1 in intact tumors is of major interest. Here we employ fluorescence lifetime (FLT) imaging for in-vivo detection and quantification of PD-L1 expression using an anti-PD-L1 antibody conjugated to IRDye800CW (αPDL1-800). We show that FLT imaging accurately identifies heterogeneous PD-L1 expression in tumors. Tumor areas of high PD-L1 levels were spatially correlated to significantly longer FLTs of αPDL1-800 and the distribution of PD-L1 in deep-seated (>1cm depth) tumors was achieved using FLT tomography.
To accurately reconstruct fluorophore concentrations in-vivo in fluorescence lifetime-based tomography, it is necessary to accurately model and optimize, both the propagation of light in tissue in complex geometries and the system parameters involved in the entire optical chain from the source to the detector. This paper proposes a comprehensive methodology for accurately modelling a time domain tomography system along entire the optical chain, including the time-gated intensifier. Based on this modelling, we optimize the system parameters to obtain signals with high SNR at a faster acquisition rate. We validate the model using simulations and in-vivo experimental studies.
A study evaluating the use of multimodal widefield fluorescence imaging with nonlinear optical microscopy for detection of oral neoplasia was carried out using human clinical samples alongside a preclinical model with the goal of optimizing the most relevant endpoint measures to facilitate development and translation. Samples (in vivo buccal mucosa and surgical samples of oral cancer) were imaged by WF imaging using filter selection of red and green spectral windows, multispectral WF imaging to obtain WF spectral characteristics, and multiphoton autofluorescence microscopy. Features between preclinical and human samples were compared. Similarities and relevant endpoints for pursuing further development of a multimodal workflow will be discussed.
The well-known red shift of fluorescence spectra in thick tissue causes significant cross-talk and inaccurate quantification of fluorophores using multispectral imaging (MSI). However, fluorescence lifetimes (FLT) are largely unaffected by tissue light propagation and results in superior quantitation accuracy. Here we present a systematic experimental comparison of quantitative MSI and FLT of multiple fluorescent molecules in tissue. In experiments using one or two fluorescence species in mixture with tissue autofluorescence, MSI showed significantly higher error rates (>20%) than FLT (<5%). These results show promise for in vivo FLT multiplexing based imaging of receptor expression in cancer and other diseases.
The recognized need to develop better clinical approaches for detection of epithelial cancers and potentially malignant lesions than currently used has motivated work in development of noninvasive fluorescence imaging devices. While individual large area imaging and microscopic techniques are promising, recent trends have explored combinations that could merge strengths. The study will discuss a workflow to combine strengths of label-free nonlinear optical microscopy (NLOM) which has shown promise for optical biopsy but is limited in scannable area with widefield autofluorescence microscopy providing large surface area assessment, in studies conducted in both a hamster model for oral neoplasia and inflammation and in surgical oral cancer specimens.
Fluorescence imaging of cancers using continuous wave (CW) detection of receptor targeted probes offer poor sensitivity and specificity due to background autofluorescence and non-specific probe accumulation. Here we show that fluorescence lifetime (FLT) imaging can significantly improve tumor contrast using epidermal growth factor receptor (EGFR) and programed death ligand 1 (PD-L1) targeted probes in a preclinical model of human breast cancer. Our results suggest that these probes have significantly longer FLTs in tumors than in normal tissue and the FLT enhancement is receptor dependent. We also show potential for simultaneous quantification of EGFR and PD-L1 using in vivo FLT multiplexing.
Breast cancer is a highly heterogeneous disease comprising a variety of genotypes and phenotypes of varying levels of aggressiveness. This presents significant challenges to clinical management of early-stage cancers. In this paper, we describe the use of multimodal optical technologies including near-infrared (NIR) spectroscopy, diffuse correlation spectroscopy (DCS) and indocyanine green (ICG) fluorescence imaging to evaluate the aggressiveness and progression of two patient-derived xenograft models of human breast cancer. Optical markers reveal distinctive features between low- and high-aggressiveness tumors that could potentially be translated for clinical use.
Significance: Early detection of epithelial cancers and precancers/neoplasia in the presence of benign lesions is challenging due to the lack of robust in vivo imaging and biopsy guidance techniques. Label-free nonlinear optical microscopy (NLOM) has shown promise for optical biopsy through the detection of cellular and extracellular signatures of neoplasia. Although in vivo microscopy techniques continue to be developed, the surface area imaged in microscopy is limited by the field of view. FDA-approved widefield fluorescence (WF) imaging systems that capture autofluorescence signatures of neoplasia provide molecular information at large fields of view, which may complement the cytologic and architectural information provided by NLOM.
Aim: A multimodal imaging approach with high-sensitivity WF and high-resolution NLOM was investigated to identify and distinguish image-based features of neoplasia from normal and benign lesions.
Approach:In vivo label-free WF imaging and NLOM was performed in preclinical hamster models of oral neoplasia and inflammation. Analyses of WF imaging, NLOM imaging, and dual modality (WF combined with NLOM) were performed.
Results: WF imaging showed increased red-to-green autofluorescence ratio in neoplasia compared to inflammation and normal oral mucosa (p < 0.01). In vivo assessment of the mucosal tissue with NLOM revealed subsurface cytologic (nuclear pleomorphism) and architectural (remodeling of extracellular matrix) atypia in histologically confirmed neoplastic tissue, which were not observed in inflammation or normal mucosa. Univariate and multivariate statistical analysis of macroscopic and microscopic image-based features indicated improved performance (94% sensitivity and 97% specificity) of a multiscale approach over WF alone, even in the presence of benign lesions (inflammation), a common confounding factor in diagnostics.
Conclusions: A multimodal imaging approach integrating strengths from WF and NLOM may be beneficial in identifying oral neoplasia. Our study could guide future studies on human oral neoplasia to further evaluate merits and limitations of multimodal workflows and inform the development of multiscale clinical imaging systems.
Fluorescence diffuse optical tomography has traditionally employed near infrared (NIR) light (700 nm - 850 nm), owing to the lower absorption, and consequently, deeper penetration through thick biological tissue in these wavelengths. However, tissue scattering is a major impediment that has limited spatial resolution. We demonstrate that tomography using light in the short-wave infrared (SWIR) spectrum (>1000 nm), characterized by lower tissue scattering, can provide a several-fold improvement in spatial resolution compared to that using NIR light. We also show that the use of SWIR light for both excitation and detection provides the improved spatial resolution enhancement compared to using SWIR detection alone. Using Monte Carlo simulations and phantom experiments, we characterize the tomographic spatial resolution performance across both the NIR and SWIR spectral regions. We also validate the application of SWIR tomography in complex shaped, heterogeneous biological tissue using mouse cadavers with embedded fluorescent inclusions in the brain. These results suggest that SWIR tomography will offer a powerful new approach for non-invasive, depth resolved, 3D tomographic imaging in whole animals.
Volumetric Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia representing the complex microstructural organization of mucosa, potentially providing high specificity for detection of neoplasia, but is limited by small imaging area. Large area fluorescence methods on the other hand show high sensitivity appropriate for screening but are hampered by low specificity. In this study, we apply MPAM-SHGM following guidance from large area fluorescence, by either autofluorescence or a targeted metabolic fluorophore, as a potentially clinically viable approach for detection of oral neoplasia.
Sites of high neoplastic potentially were identified by large area red/green autofluorescence or by a fluorescently labelled deoxy-glucose analog, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to highlight areas of high glucose uptake across the buccal pouch of a hamster model for OSCC. Follow-up MPAM-SHGM was conducted on regions of interests (ROIs) to assess whether microscopy would reveal microscopic features associated with neoplasia to confirm or exclude large area fluorescence findings. Parameters for analysis included cytologic metrics, 3D epithelial connective tissue interface metrics (MPAM-SHGM) and intensity of fluorescence (widefield). Imaged sites were biopsied and processed for histology and graded by a pathologist. A small sample of human ex vivo tissues were also imaged.
A generalized linear model combining image metrics from large area fluorescence and volumetric MPAM-SHGM indicated the ability to delineate normal and inflammation from neoplasia.
The majority of oral cancers are comprised of oral squamous cell carcinoma in which neoplastic epithelial cells invade across the epithelial connective tissue interface (ECTI). Invasion is preceded by a multi-component process including epithelial hyperproliferation, loss of cell polarity, and remodeling of the extracellular matrix. Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia. In particular, volumetric imaging by these methods can reveal aspects of the 3D microstructure that are not possible by other methods and which could both further our understanding of neoplastic transformation and be explored for development of diagnostic approaches in this disease having only 55% 5-year survival rate. MPAM-SHG were applied to reveal the 3D structure of the critical ECTI interface that plays an integral part toward invasion.
Epithelial dysplasia was induced in an established hamster model. MPAM-SHGM was applied to lesion sites, using 780 nm excitation (450-600nm emission) for autofluroescence of cellular and extracellular components; 840 nm using 420 nm bandpass filter for SHG. The ECTI surface was identified as the interface at which SHG signal began following the epithelium and was modeled as a 3D surface using Matlab. ECTI surface area and cell features at sites of epithelial expansion where ECTI was altered were measured; Imaged sites were biopsied and processed for histology.
ROC analysis using ECTI image metrics indicated the ability to delineate normal from neoplasia with high sensitivity and specificity and it is noteworthy that inflammation did not significantly alter diagnostic potential of MPAM-SHGM .
Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) have shown the potential for noninvasive assessment of oral precancers and cancers. We have explored a combination of these nonlinear optical microscopic imaging techniques with widefield fluorescence imaging to assess morphometry similar to that of pathologic evaluation as well as information from endogenous fluorophores, which are altered with neoplastic transformation. Widefield fluorescence revealed areas of interest corresponding to sites with precancers or early tumors, generally resulting in a decrease in green emission or increase in red emission. Subsequent microscopy revealed significant differences in morphology between normal, dysplastic/neoplastic mucosa for all layers. Combination of a widefield and a microscopic technique provides a novel approach for tissue morphometric analysis along with large area assessment of tissue autofluorescence properties.
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