Summer L. Gibbs

Fluorescence Imaging In Vivo: Raster Scanned Point-Source Imaging Provides More Accurate Quantification than Broad Beam Geometries

Two fluorescence imaging systems were compared for their ability to quantify mean fluorescence intensity from surface-weighted imaging of tissue. A broad beam CCD camera system was compared to a point sampling system that raster scans to create the image. The effects of absorption and scattering in the background tissue volume were shown to be similar in their effect upon the signal, but the effect of the three-dimensional shape of the tissue was shown to be a significant distortion upon the signal. Spherical phantoms with Intralipid and blood for absorber and scatterer were used with a fixed concentration of aluminum phthalocyanine fluorophore to illustrate that the mean intensity observed with the broad beam system increased with size, while the mean intensity observed with the raster scanned system was not as significantly affected. Similar results were observed in vivo with mice injected with the fluorophore and imaged multiple times to observe the pharmacokinetics of the drug. The fluorescence in the tumor observed with the broad beam system was higher than that observed with the raster scanned system. Based upon the phantom and animal observations in this study, it should be concluded that using broad beam fluorescence imaging systems to quantify fluorescence in vivo may be problematic when comparing tissues with different three dimensional characteristics. In particular, the ratio of fluorescence from tumor to normal tissue can yield inaccurate results when the tumor is large. However, similar measurements with a narrow beam system that is raster scanned to create the images are not as significantly affected by the three dimensional shape of the tissue. Raster scanned imaging appears to provide a more uniform and accurate way to quantify fluorescence signals from distributed tissues in vivo.

Protoporphyrin IX Level Correlates with Number of Mitochondria, But Increase in Production Correlates with Tumor Cell Size

Abstract Protoporphyrin IX (PpIX) is produced in cells via the heme synthesis pathway, from the substrate aminolevulinic acid (ALA), and can be used for tumor detection, monitoring or photodynamic therapy. PpIX production varies considerably between tumor cell types, and determining the cell types and methods to optimize production is a central issue in properly utilizing this drug. A panel of eight cancer cell types was examined for PpIX production capacity, including breast, prostate, and brain cancer tumors, and the production varied up to 10-fold among cell types. A positive correlation was seen between mitochondrial content and naturally occurring PpIX prior to ALA administration, but mitochondrial content did not correlate to the yield of PpIX resulting from the addition of ALA. Interestingly, total cell size was positively correlated to the yield of PpIX from ALA administration. Addition of an iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed, thereby further increasing the yield of PpIX. Those cell types that had the lowest ALA to PpIX production without L1 showed the largest percentage increase in production with L1. The study indicates that use of L1 in tumors with a lower innate production of PpIX with ALA alone may be the most productive approach to this combined delivery.

Topical dual-stain difference imaging for rapid intra-operative tumor identification in fresh specimens

Assessing tumor margin status during surgery is critical to ensure complete resection of cancer tissue; however, current approaches are ineffective and often result in repeat surgery. We present an optical imaging approach for margin assessment using topical application of two fluorescent stains, one targeted to a tumor biomarker and the other a nontargeted reference, to freshly excised specimens. Computing a normalized difference image from fluorescence images of the targeted and untargeted stains suppresses the confounding effects of nonspecific uptake. Applying this approach in excised breast tumor models produced promising tumor-to-normal tissue contrasts that were significantly higher than single-targeted-stain imaging.

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries. However, applications of SMLM in preserved tissues has been limited. Through adaptation of the immunofluorescence workflow on FFPE sections milled at histological thickness, cellular architecture can now be visualized on the nanoscale using SMLM including individual mitochondria, undulations in the nuclear lamina, and the HER2 receptor on membrane protrusions in human breast cancer specimens. Using astigmatism imaging, these structures can also be resolved in three dimensions to a depth of ~800 nm. These results demonstrate the utility of SMLM in efficiently uncovering ultrastructural information of archived clinical samples, which may offer molecular insights into the physiopathology of tissues to assist in disease diagnosis and treatment using conventional sample preparation methods.

Polymeric Micelles as Carriers for Nerve-Highlighting Fluorescent Probe Delivery

Nerve damage during surgery is a common morbidity experienced by patients that leaves them with chronic pain and/or loss of function. Currently, no clinically approved imaging technique exists to enhance nerve visualization in the operating room. Fluorescence image-guided surgery has gained in popularity and clinical acceptance over the past decade with a handful of imaging systems approved for clinical use. However, contrast agent development to complement these fluorescence-imaging systems has lagged behind with all currently approved fluorescent agents providing untargeted blood pool information. Nerve-specific fluorophores are known, however translations of these agents to the clinic has been complicated by their lipophilic nature, which necessitates specialized formulation strategies for successful systemic administration. To date the known nerve-specific fluorophores have only been demonstrated preclinically due to the necessity of a dimethyl sulfoxide containing formulation for solubilization. In the current study, a polymeric micellar (PM) formulation strategy was developed for a representative nerve-specific fluorophore from the distyrylbenzene family, BMB. The PM formulation strategy was able to solubilize BMB and demonstrated improved nerve-specific accumulation and fluorescence intensity when the same fluorophore dose was administered to mice utilizing the previous formulation strategy. The success of the PM formulation strategy will be important for moving toward clinical translation of these novel nerve-specific probes as it is nontoxic and biodegradable and has the potential to decrease the necessary dose for imaging while also improving the safety profile.

Sub-surface fluorescence imaging of Protoporphyrin IX with B-Scan mode tomography

A non-contact fluorescence diffuse optical tomography (DOT) system capable of producing B-scan-type images of localized fluorescence regions up to depths of 15mm is presented. The B-Scan mode is analogous to ultrasound where the excitation and remission signals are delivered from the same surface of the tissue. This optical fluorescence system utilizes a 635 nm diode laser and two orthogonal galvanometers to raster scan the position of the source along the tissue surface. Using Protoporphyrin IX (PpIX) as a fluorescent agent, the amplitude of the remitted signal is separated by a 650 nm long pass filter and the fluorescence is then detected by a cooled CCD camera. Images are acquired for all source positions along the surface of the tissue, providing remission intensity images for each. This volume data set is then used in image reconstruction of the sub-surface volume, via a finite element method of modeling the fluorescence diffusion. The optimal remission imaging geometry, in terms of depth sensitivity, computation time, and image contrast-to-noise, was determined by performing sensitivity and singular-value decomposition analysis of the Jacobian for various source/detector combinations. The simulated results indicate that the fluorophore concentration and the inclusion size can not be recovered accurately in this mode; however, it is shown that the inclusion depth can accurately be predicted. Finally, by performing simulations on a mesh created from an MR image, we show that our system may prove useful in predicting the regions of local tissue fluorescence in the application of resection of residual brain tumor tissue under fluorescence guidance. This non-contact diagnostic system is being calibrated and finalized for potential use in this application of sub-surface imaging in the brain.

Methodology for Quantitative Characterization of Fluorophore Photoswitching to Predict Superresolution Microscopy Image Quality

Single-molecule localization microscopy (SMLM) image quality and resolution strongly depend on the photoswitching properties of fluorophores used for sample labeling. Development of fluorophores with optimized photoswitching will considerably improve SMLM spatial and spectral resolution. Currently, evaluating fluorophore photoswitching requires protein-conjugation before assessment mandating specific fluorophore functionality, which is a major hurdle for systematic characterization. Herein, we validated polyvinyl alcohol (PVA) as a single-molecule environment to efficiently quantify the photoswitching properties of fluorophores and identified photoswitching properties predictive of quality SMLM images. We demonstrated that the same fluorophore photoswitching properties measured in PVA films and using antibody adsorption, a protein-conjugation environment analogous to labeled cells, were significantly correlated to microtubule width and continuity, surrogate measures of SMLM image quality. Defining PVA as a fluorophore photoswitching screening platform will facilitate SMLM fluorophore development and optimal image buffer assessment through facile and accurate photoswitching property characterization, which translates to SMLM fluorophore imaging performance.

Varied Length Stokes Shift BODIPY-Based Fluorophores for Multicolor Microscopy

Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)-based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches.

Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery

Rationale: Positive margin status due to incomplete removal of tumor tissue during breast conserving surgery (BCS) is a prevalent diagnosis usually requiring a second surgical procedure. These follow-up procedures increase the risk of morbidity and delay the use of adjuvant therapy; thus, significant efforts are underway to develop new intraoperative strategies for margin assessment to eliminate re-excision procedures. One strategy under development uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI). DDSI uses a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent companion imaging agent topically applied to fresh resected specimens, where the fluorescence from each probe is imaged and a normalized difference image is computed to identify tumor-target distribution in the specimen margins. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and further validation. Methods: In the present study, we used breast cancer xenografts and receiver operator characteristic (ROC) curve analysis to evaluate a wide range of staining and imaging parameters, and completed a prospective validation study on multiple tumor phenotypes with different target expression. Imaging fluorophore-probe pair, concentration, and incubation times were systematically optimized using n=6 tissue specimen replicates per staining condition. Resulting tumor vs. normal adipose tissue diagnostic performance were reported and staining patterns were validated via receptor specific immunohistochemistry colocalization. Optimal staining conditions were tested in receptor positive and receptor negative cohorts to confirm specificity. Results: The optimal staining conditions were found to be a one minute stain in a 200 nM probe solution (area under the curve (AUC) = 0.97), where the choice of fluorescent label combination did not significantly affect the diagnostic performance. Using an optimal threshold value determined from ROC curve analysis on a training data set, a prospective study on xenografts resulted in an AUC=0.95 for receptor positive tumors and an AUC = 0.50 for receptor negative (control) tumors, confirming the diagnostic performance of this novel imaging technique. Conclusions: DDSI provides a robust, molecularly specific imaging methodology for identifying tumor tissue over benign mammary adipose tissue. Using a dual probe imaging strategy, nonspecific accumulation of targeted probe was corrected for and tumor vs. normal tissue diagnostic potential was improved, circumventing difficulties with ex vivo tissue specimen staining and allowing for rapid clinical translation of this promising technology for tumor margin detection during BCS procedures.

Far-Red and Near-Infrared Seminaphthofluorophores for Targeted Pancreatic Cancer Imaging

Molecular probes that selectively highlight pancreatic cancer (PC) tissue have the potential to improve pancreatic ductal adenocarcinoma (PDAC) margin assessment through the selective highlighting of individual PC cells. Herein, we report a simple and unique family of systematically modified red and near-infrared fluorescent probes that exhibit a field-effect-derived redshift. Two of thirteen probes distributed to the normal mouse pancreas following systemic administration. One selectively accumulated in genetically modified mouse models of PDAC. The probe exhibited intracellular accumulation and enabled visualization of four levels of the structure, including the whole organ, resected tissue, individual cells, and subcellular organelles. In contrast to the small-molecule probes reported previously, it possesses an inherent affinity toward PDAC cells and thus does not require conjugation to any targeting agent. The fluorescent probe can thus promote new strategies not only for precision image-guided surgery, but also for PC detection, monitoring of therapeutic outcomes, and basic research.