Two-color diffuse in vivo flow cytometer.

Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal. Aim: The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs. Approach: A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo. Results: In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice. Conclusions: We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.

Evaluating Receptor-Specific Fresh Specimen Staining for Tumor Margin Detection in Clinical Breast Specimens.

PURPOSE: Reliable and rapid identification of tumor in the margins of breast specimens during breast-conserving surgery to reduce repeat surgery rates is an active area of investigation. Dual-stain difference imaging (DDSI) is one of many approaches under evaluation for this application. This technique aims to topically apply fluorescent stain pairs (one targeted to a receptor-of-interest and the other a spectrally distinct isotype), image both stains, and compute a normalized difference image between the two channels. Prior evaluation and optimization in a variety of preclinical models produced encouraging diagnostic performance. Herein, we report on a pilot clinical study which evaluated HER2-targeted DDSI on 11 human breast specimens. PROCEDURES: Gross sections from 11 freshly excised mastectomy specimens were processed using a HER2-receptor-targeted DDSI protocol shortly after resection. After staining with the dual-probe protocol, specimens were imaged on a fluorescence scanner, followed by tissue fixation for hematoxylin and eosin and anti-HER2 immunohistochemical staining. Receiver operator characteristic curves and area under the curve (AUC) analysis were used to assess diagnostic performance of the resulting images. Performance values were also compared to expression level determined from IHC staining. RESULTS: Eight of the 11 specimens presented with distinguishable invasive ductal carcinoma and/or were not affected by an imaging artifact. In these specimens, the DDSI technique provided an AUC = 0.90 ± 0.07 for tumor-to-adipose tissue and 0.81 ± 0.15 for tumor-to-glandular tissue, which was significantly higher than AUC values recovered from images of the targeted probe alone. DDSI values and diagnostic performance did not correlate with HER2 expression level, and tumors with low HER2 expression often produced high AUC, suggesting that even the low expression levels were enough to help distinguish tumor. CONCLUSIONS: The results from this preliminary study of rapid receptor-specific staining in human specimens were consistent with prior preclinical results and demonstrated promising diagnostic potential.

Dual probe difference specimen imaging for prostate cancer margin assessment.

Significance: Positive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative. Aim: Current intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients. Approach: Dual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images (i.e., DDSI) of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer. Results: Targeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues (prostate, adipose, muscle). Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues. Conclusions: Previous work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker.

A birefringent spectral demultiplexer enables fast hyper-spectral imaging of protoporphyrin IX during neurosurgery.

Hyperspectral imaging and spectral analysis quantifies fluorophore concentration during fluorescence-guided surgery1-6. However, acquisition of the multiple wavelengths required to implement these methods can be time-consuming and hinder surgical workflow. To this end, a snapshot hyperspectral imaging system capable of acquiring 64 channels of spectral data simultaneously was developed for rapid hyperspectral imaging during neurosurgery. The system uses a birefringent spectral demultiplexer to split incoming light and redirect wavelengths to different sections of a large format microscope sensor. Its configuration achieves high optical throughput, accepts unpolarized input light and exceeds channel count of prior image-replicating imaging spectrometers by 4-fold. Tissue-simulating phantoms consisting of serial dilutions of the fluorescent agent characterize system linearity and sensitivity, and comparisons to performance of a liquid crystal tunable filter based hyperspectral imaging device are favorable. The new instrument showed comparable, if not improved, sensitivity at low fluorophore concentrations; yet, acquired wide-field images at more than 70-fold increase in frame rate. Image data acquired in the operating room during human brain tumor resection confirm these findings. The new device is an important advance in achieving real-time quantitative imaging of fluorophore concentration for guiding surgery.

A new candidate agent for fluorescence guided neurosurgery produces high, persistent tumor contrast shortly after administration.

Neurosurgical fluorescence guidance relies on contrast agents to identify tumor regions to aid in increasing the extent of resection. Existing contrast agents for this indication each have their own limitation: unpredictable contrast from tumor heterogeneity, significant extravasation into the background brain and long incubation times. An ideal contrast agent should have high and rapid contrast that persists well into the surgical procedure. By using a whole animal hyperspectral cryo-imaging system several CAs were screened for these favorable properties and compared to the gold standard of gadolinium enhanced MR. Herein, we briefly report on the leading candidate Rd-PEG1k, which shows high contrast within minutes of administration that persists for at least 90 minutes.

Maternally transferred mAbs protect neonatal mice from HSV-induced mortality and morbidity.

Neonatal herpes simplex virus (nHSV) infections often result in significant mortality and neurological morbidity despite antiviral drug therapy. Maternally transferred herpes simplex virus (HSV)-specific antibodies reduce the risk of clinically overt nHSV, but this observation has not been translationally applied. Using a neonatal mouse model, we tested the hypothesis that passive transfer of HSV-specific human mAbs can prevent mortality and morbidity associated with nHSV. The mAbs were expressed in vivo via vectored immunoprophylaxis or recombinantly. Through these maternally derived routes or through direct administration to pups, diverse mAbs to HSV glycoprotein D protected against neonatal HSV-1 and HSV-2 infection. Using in vivo bioluminescent imaging, both pre- and post-exposure mAb treatment significantly reduced viral load in mouse pups. Together these studies support the notion that HSV-specific mAb-based therapies could prevent or improve HSV infection outcomes in neonates.

Examining the Feasibility of Quantifying Receptor Availability Using Cross-Modality Paired-Agent Imaging.

PURPOSE: The ability to noninvasively quantify receptor availability (RA) in solid tumors is an aspirational goal of molecular imaging, often challenged by the influence of non-specific accumulation of the contrast agent. Paired-agent imaging (PAI) techniques aim to compensate for this effect by imaging the kinetics of a targeted agent and an untargeted isotype, often simultaneously, and comparing the kinetics of the two agents to estimate RA. This is usually accomplished using two spectrally distinct fluorescent agents, limiting the technique to superficial tissues and/or preclinical applications. Applying the approach in humans using conventional imaging modalities is generally infeasible since most modalities are unable to routinely image multiple agents simultaneously. We examine the ability of PAI to be implemented in a cross-modality paradigm, in which the targeted and untargeted agent kinetics are imaged with different modalities and used to recover receptor availability. PROCEDURES: Eighteen mice bearing orthotopic brain tumors were administered a solution containing three contrast agents: (1) a fluorescent agent targeted to epidermal growth factor receptor (EGFR), (2) an untargeted fluorescent isotype, and (3) a gadolinium-based contrast agent (GBCA) for MRI imaging. The kinetics of all three agents were imaged for 1 h after administration using an MRI-coupled fluorescence tomography system. Paired-agent receptor availability was computed using (1) the conventional all-optical approach using the targeted and untargeted optical agent images and (2) the cross-modality approach using the targeted optical and untargeted MRI-GBCA images. Receptor availability estimates between the two methods were compared. RESULTS: Receptor availability values using the cross-modality approach were highly correlated to the conventional, single-modality approach (r = 0.94; p < 0.00001). CONCLUSION: These results suggest that cross-modality paired-agent imaging for quantifying receptor availability is feasible. Ultimately, cross-modality paired-agent imaging could facilitate rapid, noninvasive receptor availability quantification in humans using hybrid clinical imaging modalities.

A method for validating depth-resolved biodistributions in topically-stained specimen with multi-channel fluorescence cryo-imaging.

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.

Monitoring cancer cell surface receptor expression during anti-angiogenesis therapy in vivo.

Concurrent administration of cancer therapeutics with tumor vasculature targeting treatment has been shown to improve overall survival in multiple human cancer types, as such combinations aim to destroy different compartments of tumors. Anti-angiogenesis therapeutics designed to inhibit tumor induced vessel sprouting have also been shown to re-model the tumor vasculature through a transient vessel normalization effect, which leads to improved perfusion of oxygen and drug in tumor. However, the effects that this normalized vasculature has on the availability of cancer receptor, such as EGFR, is unknown. Herein, we examined the use of MRI-PAFT to estimate cancer surface receptor availability in response to anti-angiogenesis therapy, using MRI-coupled paired agent fluorescence tomography. Bevacizumab treated tumors showed increase in RA compared to control tumors, but this was not statistically significant.

Clinically relevant dual probe difference specimen imaging (DDSI) protocol for freshly resected breast cancer specimen staining.

BACKGROUND: Re-excision rates following breast conserving surgery (BCS) remain as high as ~ 35%, with positive margins detected during follow-up histopathology. Additional breast cancer resection surgery is not only taxing on the patient and health care system, but also delays adjuvant therapies, increasing morbidity and reducing the likelihood of a positive outcome. The ability to precisely resect and visualize tumor margins in real time within the surgical theater would greatly benefit patients, surgeons and the health care system. Current tumor margin assessment technologies utilized during BCS involve relatively lengthy and labor-intensive protocols, which impede the surgical work flow. METHODS: In previous work, we have developed and validated a fluorescence imaging method termed dual probe difference specimen imaging (DDSI) to accurately detect benign and malignant tissue with direct correlation to the targeted biomarker expression levels intraoperatively. The DDSI method is currently on par with touch prep cytology in execution time (~ 15-min). In this study, the main goal was to shorten the DDSI protocol by decreasing tissue blocking and washing times to optimize the DDSI protocol to < 10-min whilst maintaining robust benign and malignant tissue differentiation. RESULTS: We evaluated the utility of the shortened DDSI staining methodology using xenografts grown from cell lines with varied epidermal growth factor receptor (EGFR) expression levels, comparing accuracy through receiver operator characteristic (ROC) curve analyses across varied tissue blocking and washing times. An optimized 8-min DDSI methodology was developed for future clinical translation. CONCLUSIONS: Successful completion of this work resulted in substantial shortening of the DDSI methodology for use in the operating room, that provided robust, highly receptor specific, sensitive diagnostic capabilities between benign and malignant tissues.

Hyperspectral imaging and spectral unmixing for improving whole-body fluorescence cryo-imaging.

Whole-animal fluorescence cryo-imaging is an established technique that enables visualization of the biodistribution of labeled drugs, contrast agents, functional reporters and cells in detail. However, many tissues produce endogenous autofluorescence, which can confound interpretation of the cryo-imaging volumes. We describe a multi-channel, hyperspectral cryo-imaging system that acquires densely-sampled spectra at each pixel in the 3-dimensional stack. This information enables the use of spectral unmixing to isolate the fluorophore-of-interest from autofluorescence and/or other fluorescent reporters. In phantoms and a glioma xenograft model, we show that the approach improves detection limits, increases tumor contrast, and can dramatically alter image interpretation.

Improving the Usability of 5-Aminolevulinic Acid Fluorescence-Guided Surgery by Adding an Optimized Secondary Light Source.

BACKGROUND: Tumors that take up and metabolize 5-aminolevulinic acid emit bright pink fluorescence when illuminated with blue light, aiding surgeons in identifying the margin of resection. The adoption of this method is hindered by the blue light illumination, which is too dim to safely operate under and therefore necessitates switching back and forth from white-light mode. The aim of this study was to examine the addition of an optimized secondary illuminant adapter to improve usability of blue-light mode without degrading tumor contrast. METHODS: Color science methods were used to evaluate the color of the secondary illuminant and its impact on color rendering index as well as the tumor-to-background color contrast in data collected from 7 patients with high-grade gliomas (World Health Organization grade III and IV). A secondary illuminant adapter was built to provide 475-600 nm light the intensity of which can be controlled by the surgeon and was evaluated in 2 additional patients. RESULTS: Secondary illuminant color had opposing effects on color rendering index and tumor-to-background color contrast; providing surgeon control of intensity allows this trade-off to be balanced in real time. Demonstration in 2 high-grade glioma cases confirms this, showing that additional visibility adds value when intensity can be controlled by the surgeon. CONCLUSIONS: Addition of a secondary illuminant may mitigate surgeon complaints that the operative field is too dark under the blue light illumination required for 5-aminolevulinic acid fluorescence guidance by providing improved color rendering index without completely sacrificing tumor-to-background color contrast.

Heterogeneity of circulating tumor cell dissemination and lung metastases in a subcutaneous Lewis lung carcinoma model.

Subcutaneous (s.c.) tumor models are widely used in pre-clinical cancer metastasis research. Despite this, the dynamics and natural progression of circulating tumor cells (CTCs) and CTC clusters (CTCCs) in peripheral blood are poorly understood in these models. In this work, we used a new technique called 'diffuse in vivo flow cytometry' (DiFC) to study CTC and CTCC dissemination in an s.c. Lewis lung carcinoma (LLC) model in mice. Tumors were grown in the rear flank and we performed DiFC up to 31 days after inoculation. At the study endpoint, lungs were excised and bioluminescence imaging (BLI) was performed to determine the extent of lung metastases. We also used fluorescence macro-cryotome imaging to visualize infiltration and growth of the primary tumor. DiFC revealed significant heterogeneity in CTC and CTCC numbers amongst all mice studied, despite using clonally identical LLC cells and tumor placement. Maximum DiFC count rates corresponded to 0.1 to 14 CTCs per mL of peripheral blood. In general, CTC numbers did not necessarily increase monotonically over time and were poorly correlated with tumor volume. However, there was a good correlation between CTC and CTCC numbers in peripheral blood and lung metastases. We attribute the differences in CTC numbers primarily due to growth patterns of the primary tumor. This study is one of the few reports of CTC shedding dynamics in sub-cutaneous metastasis models and underscores the value of in vivo methods for continuous, non-invasive CTC monitoring.

Noninvasive quantification of target availability during therapy using paired-agent fluorescence tomography.

Immuno-oncological treatment strategies that target abnormal receptor profiles of tumors are an increasingly important feature of cancer therapy. Yet, assessing receptor availability (RA) and drug-target engagement, important determinants of therapeutic efficacy, is challenging with current imaging strategies, largely due to the complex nonspecific uptake behavior of imaging agents in tumors. Herein, we evaluate whether a quantitative noninvasive imaging approach designed to compensate for nonspecific uptake, MRI-coupled paired-agent fluorescence tomography (MRI-PAFT), is capable of rapidly assessing the availability of epidermal growth factor receptor (EGFR) in response to one dose of anti-EGFR antibody therapy in orthotopic brain tumor models. Methods: Mice bearing orthotopic brain tumor xenografts with relatively high EGFR expression (U251) (N=10) or undetectable human EGFR (9L) (N=9) were considered in this study. For each tumor type, mice were either treated with one dose of cetuximab, or remained untreated. All animals were scanned using MRI-PAFT, which commenced immediately after paired-agent administration, and values of RA were recovered using a model-based approach, which uses the entire dynamic sequence of agent uptake, as well as a simplified "snapshot" approach which requires uptake measurements at only two time points. Recovered values of RA were evaluated between groups and techniques. Hematoxylin & eosin (H&E) and immunohistochemical (IHC) staining was performed on tumor specimens from every animal to confirm tumor presence and EGFR status. Results: In animals bearing EGFR(+) tumors, a significant difference in RA values between treated and untreated animals was observed (RA = 0.24 ± 0.15 and 0.61 ± 0.18, respectively, p=0.027), with an area under the curve - receiver operating characteristic (AUC-ROC) value of 0.92. We did not observe a statistically significant difference in RA values between treated and untreated animals bearing EGFR(-) tumors (RA = 0.18 ± 0.19 and 0.27 ± 0.21, respectively; p = 0.89; AUC-ROC = 0.55), nor did we observe a difference between treated EGFR(+) tumors compared to treated and untreated EGFR(-) tumors. Notably, the snapshot paired-agent strategy quantified drug-receptor engagement within just 30 minutes of agent administration. Examination of the targeted agent alone showed no capacity to distinguish tumors either by treatment or receptor status, even 24h after agent administration. Conclusions: This study demonstrated that a noninvasive imaging strategy enables rapid quantification of receptor availability in response to therapy, a capability that could be leveraged in preclinical drug development, patient stratification, and treatment monitoring.

Tracking tumor radiotherapy response in vivo with Cherenkov-excited luminescence ink imaging.

This study demonstrates remote imaging for in vivo detection of radiation-induced tumor microstructural changes by tracking the diffusive spread of injected intratumor UV excited tattoo ink using Cherenkov-excited luminescence imaging (CELI). Micro-liter quantities of luminescent tattoo ink with UV absorption and visible emission were injected at a depth of 2 mm into mouse tumors prior to receiving a high dose treatment of radiation. X-rays from a clinical linear accelerator were used to excite phosphorescent compounds within the tattoo ink through Cherenkov emission. The in vivo phosphorescence was detected using a time-gated intensified CMOS camera immediately after injection, and then again at varying time points after the ink had broken down with the apoptotic tumor cells. Ex vivo tumors were imaged post-mortem using hyperspectral cryo-fluorescence imaging to quantify necrosis and compared to Cherenkov-excited light imaging of diffusive ink spread measured in vivo. Imaging of untreated control mice showed that ink distributions remained constant after four days with less than 3% diffusive spread measured using full width at 20% max. For all mice, in vivo CELI measurements matched within 12% of the values estimated by the high-resolution ex vivo sliced luminescence imaging of the tumors. The tattoo ink spread in treated mice was found to correlate well with the nonperfusion necrotic core volume (R2 = 0.92) but not well with total tumor volume changes (R2 = 0.34). In vivo and ex vivo findings indicate that the diffusive spread of the injected tattoo ink can be related to radiation-induced necrosis, independent of total tumor volume change. Tracking the diffusive spread of the ink allows for distinguishing between an increase in tumor size due to new cellular growth and an increase in tumor size due to edema. Furthermore, the imaging resolution of CELI allows for in vivo tracking of subtle microenvironmental changes which occur earlier than tumor shrinkage and this offers the potential for novel, minimally invasive radiotherapy response assay without interrupting a singular clinical workflow.

Topical dual-probe staining using quantum dot-labeled antibodies for identifying tumor biomarkers in fresh specimens.

PURPOSE: Rapid, intra-operative identification of tumor tissue in the margins of excised specimens has become an important focus in the pursuit of reducing re-excision rates, especially for breast conserving surgery. Dual-probe difference specimen imaging (DDSI) is an emerging approach that uses the difference in uptake/clearance kinetics between a pair of fluorescently-labeled stains, one targeted to a biomarker-of-interest and the other an untargeted isotype, to reveal receptor-specific images of the specimen. Previous studies using antibodies labeled with either enhanced Raman particles or organic fluorophores have shown promising tumor vs. normal diagnostic performance. Yet, the unique properties of quantum dot-labeled antibody complexes (QDACs), which provide spectrally-distinct fluorescence emission from a common excitation source, make them ideal candidates for this application. Herein, we evaluate the diagnostic performance of QDAC-based DDSI in excised xenografts. PROCEDURES: Excised fresh specimens of normal tissue and human tumor xenografts with elevated expression of HER2 were stained with a HER2-targeted QDAC and an untargeted QDAC isotype. Stained specimens were imaged on a custom hyperspectral imaging system capable of spectrally separating the quantum dot signatures, and images processed using the DDSI approach. The diagnostic performance of this technique under different incubation temperatures and probe concentrations was evaluated using receiver-operator characteristic analysis. RESULTS: HER2-targeted QDAC-DDSI was able to distinguish HER2(+) tumors from normal tissue with reasonably high diagnostic performance; however, this performance was sensitive to temperature during the staining procedure. Area under the curve values were 0.61 when staining at room temperature but increased to over 0.81 when staining at 37 °C. Diagnostic performance was not affected by increasing stain concentration. CONCLUSIONS: This study is the first to report dual-probe difference imaging of specimens using QDACs and hyperspectral imaging. Our results show promising diagnostic performance under certain conditions, and compel further optimization and evaluation of this intra-operative margin assessment technique.

A paired-agent fluorescent molecular imaging strategy for quantifying antibody drug target engagement in in vivo window chamber xenograft models.

A paired-agent fluorescent molecular imaging strategy is presented as a method to measure drug target engagement in whole tumor imaging. The protocol involves dynamic imaging of a pair of targeted and control imaging agents prior to and following antibody therapy. Simulations demonstrated that antibody "drug target engagement" can be estimated within a 15%-error over a wide range of tumor physiology (blood flow, vascular permeability, target density) and antibody characteristics (affinity, binding rates). Experimental results demonstrated the first in vivo detection of binding site barrier, highlighting the potential for this methodology to provide novel insights in drug distribution/binding imaging.

Estimating paired-agent uptake in altered tumor vasculature using MRI-coupled fluorescence tomography.

Angiogenesis inhibiting cancer therapy has become a standard treatment for many cancer types. The ability to examine the effects of these drugs in tumors noninvasively could help assess efficacy early in the treatment course or identify optimal times to introduce other combinatorial treatments. Herein, we examine whether a paired agent MRI-coupled fluorescence tomography approach can be used to monitor the effects of anti-angiogenesis therapy. Using small animal models bearing orthotopic glioma xenografts, we demonstrate noninvasive quantification of paired-agent uptake in response to anti-angiogenesis therapy in vivo. The result provides insights on receptor targeted drug delivery in altered vasculature, a potential important development for treatment monitoring and combinatorial strategies.

Developing a novel hyperspectral imaging cryomacrotome for whole body fluorescence imaging.

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 cryo-macrotome 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.