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Background The development and optimization of therapies for rheumatoid arthritis (RA) is currently hindered by a lack of methods for early non-invasive monitoring of treatment response. Annexin A2, an inflammation-associated protein whose presence and phosphorylation levels are upregulated in RA, represents a potential molecular target for tracking RA treatment response. Methods LS301, a near-infrared dye-peptide conjugate that selectively targets tyrosine 23-phosphorylated annexin A2 (pANXA2), was evaluated for its utility in monitoring disease progression, remission, and early response to drug treatment in mouse models of RA by fluorescence imaging. The intraarticular distribution and localization of LS301 relative to pANXA2 was determined by histological and immunohistochemical methods. Results In mouse models of spontaneous and serum transfer-induced inflammatory arthritis, intravenously administered LS301 showed selective accumulation in regions of joint pathology including paws, ankles, and knees with positive correlation between fluorescent signal and disease severity by clinical scoring. Whole-body near-infrared imaging with LS301 allowed tracking of spontaneous disease remission and the therapeutic response after dexamethasone treatment. Histological analysis showed preferential accumulation of LS301 within the chondrocytes and articular cartilage in arthritic mice, and colocalization was observed between LS301 and pANXA2 in the joint tissue. Conclusions We demonstrate that fluorescence imaging with LS301 can be used to monitor the progression, remission, and early response to drug treatment in mouse models of RA. Given the ease of detecting LS301 with portable optical imaging devices, the agent may become a useful early treatment response reporter for arthritis diagnosis and drug evaluation.

Background: HER2 activating mutations occur in 2-5% of metastatic breast cancer (MBC) patients, and three phase II or basket clinical trials have shown that the irreversible pan-HER tyrosine kinase inhibitor, neratinib, has good single agent efficacy for HER2 mutated MBC patients. Current trials are combining neratinib with other targeted therapies to increase response rate and progression free survival for these patients. Methods: We established patient derived xenografts (PDX) and organoids from two patients with HER2 mutated, non-amplified MBC and used them to test neratinib with the antibody drug conjugates (ADC’s), trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), both in 3D culture and in vivo. Real time, in vivo uptake of these ADC’s was visualized with a near infrared fluorophore. Results: PDX lines WHIM51 and WHIM64 were established from ER+, HER2 non-amplified MBC patients that had HER2 activating mutations. WHIM51 has HER2 exon 20 insertion mutation at amino acid 776 (ERBB2 A775_G776insYVMA) and WHIM64 has a HER2 L869R missense mutation, both of which are located in the HER2 tyrosine kinase domain. Both of these HER2 mutations have been previously characterized and are known activating mutations. Organoids were established from both PDX’s and were grown in 3D culture. Drug combination testing of neratinib with T-DM1 in 3D culture showed strong synergy and the mechanism was explored. We demonstrate that neratinib and other irreversible HER2 inhibitors increase the endocytic uptake of T-DM1, but this effect does not occur with the reversible HER2 inhibitors, tucatinib and lapatinib. Real time, in vivo uptake of T-DM1 was measured by labeling the ADC with a near infrared fluorophore and we observed statistically significant increase in T-DM1 uptake with neratinib pre-treatment. Combining neratinib with T-DM1 increased apoptosis at day 3 post-treatment and enhanced tumor shrinkage. With the FDA approval of T-DXd at the end of 2019, we hypothesized that this same mechanism may apply to neratinib combined with T-DXd. We have tested both the combinations of neratinib + T-DXd and neratinib + T-DM1 in vivo in both HER2 mutant PDX’s and observed statistically significant tumor regression with the neratinib + ADC combinations as compared to either T-DXd or T-DM1 on its own. Conclusions: Neratinib increases the endocytosis of trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), thereby increasing tumor cell kill and causing greater tumor regression in HER2 mutated MBC. These data provide preclinical justification for trials of neratinib plus HER2 ADCs including T-DXd or T-DM1 in HER2 mutant or HER2+ MBC. Further, this mechanism of neratinib stimulated HER2 endocytosis may also apply to HER2 low MBC. Citation Format: Ron Bose, Shunqiang Li, Tina M. Primeau, Maureen K. Highkin, Ashley R. Tipton, Nagalaxmi Vemalapally, Xuefeng Gao, Gail Sudlow, Irmina Diala, Yu Tao, Jingqin Luo, Ian Hagemann, Chieh-Yu Lin, Richard P. Bryce, Alshad S. Lalani, Samuel Achilefu, Cynthia X. Ma. Irreversible inhibition of HER2 activating mutations with neratinib enhances the pre-clinical efficacy of trastuzumab emtansine and trastuzumab deruxtecan [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-13.

The heterogeneity and continuous genetic adaptation of tumours complicate their detection and treatment via the targeting of genetic mutations. However, hallmarks of cancer such as aberrant protein phosphorylation and calcium-mediated cell signalling provide broadly conserved molecular targets. Here, we show that, for a range of solid tumours, a cyclic octapeptide labelled with a near-infrared dye selectively binds to phosphorylated Annexin A2 (pANXA2), with high affinity at high levels of calcium. Because of cancer-cell-induced pANXA2 expression in tumour-associated stromal cells, the octapeptide preferentially binds to the invasive edges of tumours and then traffics within macrophages to the tumour's necrotic core. As proof-of-concept applications, we used the octapeptide to detect tumour xenografts and metastatic lesions, and to perform fluorescence-guided surgical tumour resection, in mice. Our findings suggest that high levels of pANXA2 in association with elevated calcium are present in the microenvironment of most solid cancers. The octapeptide might be broadly useful for selective tumour imaging and for delivering drugs to the edges and to the core of solid tumours.

Multidrug-resistant organisms (MDROs) represent a continuing healthcare crisis with no definitive solution to date. An alternative to antibiotics is the development of therapies and vaccines using biocompatible physical methods such as ultrashort pulsed (USP) lasers, which have previously been shown to inactivate pathogens while minimizing collateral damage to human cells, blood proteins, and vaccine antigens. Here we demonstrate that visible USP laser treatment results in bactericidal effect (≥ 3-log load reduction) against clinically significant MDROs, including methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL)-producing Escherichia coli. Bacillus cereus endospores, which are highly resistant to conventional chemical and physical treatments, were also shown to be effectively inactivated by USP laser treatment, resulting in sporicidal (≥ 3-log load reduction) activity. Furthermore, we demonstrate that administration of USP laser-inactivated E. coli whole-cell vaccines at dosages as low as 105 cfu equivalents without adjuvant was able to protect 100% of mice against subsequent lethal challenge. Our findings open the possibility for application of USP lasers in disinfection of hospital environments, therapy of drug-resistant bacterial infections in skin or bloodstream via pheresis modalities, and in the production of potent bacterial vaccines. This article is protected by copyright. All rights reserved.

Quantifying solid tumor margins with fluorescence-guided surgery approaches is a challenge, particularly when using near infrared (NIR) wavelengths due to increased penetration depths. An NIR dual wavelength excitation fluorescence (DWEF) approach was developed that capitalizes on the wavelength-dependent attenuation of light in tissue to determine fluorophore depth. A portable dual wavelength excitation fluorescence imaging system was built and tested in parallel with an NIR tumor-targeting fluorophore in tissue mimicking phantoms, chicken tissue, and in vivo mouse models of breast cancer. The system showed high accuracy in all experiments. The low cost and simplicity of this approach make it ideal for clinical use.

LS301 is a promising NIR fluorescence probe for targeting brain glioma cancer. The co-localization of LS31 and 5-ALA induced PpIX in vitro and in vivo enables LS301 to guide PpIX fluorescence image surgery. Moreover, LS301 showed no negative impact on 5-ALA-PpIX PDT outcome in brain glioma cells and mouse tumor model. Therefore, the implementation of LS301 and PpIX has potential to improve tumor resection surgery and PDT treatment for better tumor outcomes.

Dynamic thermal imaging has improved bulk tissue characterization, but fails to capture subtle thermal property differences in heterogeneous systems. We present focal dynamic thermal imaging (FDTI), a simple, label-free, and high-resolution technology for delineating tissue heterogeneity. Stimulation of focal regions of absorptive materials with a narrow beam, low power, and low cost 405 nm laser locally perturbs the thermal equilibrium. Measurement of phantoms, ex vivo tissue, and in vivo animal models of cancer reveals finite structures of materials and delineates diseased from healthy tissue. Portable FDTI holds promise to capture the heterogeneous nature of malignant tissue.

Drug resistance and dose-limiting toxicities are significant barriers for treatment of multiple myeloma (MM). Bone marrow microenvironment (BMME) plays a major role in drug resistance in MM. Drug delivery with targeted nanoparticles have been shown to improve specificity and efficacy and reduce toxicity. We aim to improve treatments for MM by (1) using nanoparticle delivery to enhance efficacy and reduce toxicity; (2) targeting the tumor-associated endothelium for specific delivery of the cargo to the tumor area, and (3) synchronizing the delivery of chemotherapy (bortezomib; BTZ) and BMME-disrupting agents (ROCK inhibitor) to overcome BMME-induced drug resistance. We find that targeting the BMME with P-selectin glycoprotein ligand-1 (PSGL-1)-targeted BTZ and ROCK inhibitor-loaded liposomes is more effective than free drugs, non-targeted liposomes, and single-agent controls and reduces severe BTZ-associated side effects. These results support the use of PSGL-1-targeted multi-drug and even non-targeted liposomal BTZ formulations for the enhancement of patient outcome in MM., The tumour microenvironment (TME) has a major role in chemoresistance in multiple myeloma. The authors show that a nanoparticle targeted to TME and loaded with bortezomib (BTZ) and Y27632 is more effective than free drugs, non-targeted and single-agent controls and reduces BTZ-related side effects.

Microcapsules are emerging as promising microsize drug carriers due to their remarkable deformability. Shape plays a dominant role in determining their vascular transportation. Herein, we explored the effect of the shape of the microcapsules on the in vivo biodistribution for rational design of microcapsules to achieve optimized targeting efficiency. Silk fibroin, a biocompatible, biodegradable, and abundant material, was utilized as a building block to construct biconcave discoidal and spherical microcapsules with diameter of 1.8 μm and wall thickness of 20 nm. We have compared the cytocompatibility, cellular uptake, and biodistribution of both microcapsules. Both biconcave and spherical microcapsules exhibited excellent cytocompatibility and internalization into cancer cells. During blood circulation in mice, both microcapsules showed retention in liver and kidney and most underwent renal clearance. However, we observed significantly higher accumulation of biconcave silk microcapsules in lung compared with spherical microcapsules, and the accumulation was found to be stable in lung even after 3 days. The higher concentration of biconcave discoidal microcapsules found in lung arises from pulmonary environment, margination dynamics, and enhanced deformation in bloodstream. Red blood cell (RBC)-mimicking silk microcapsules demonstrated here can potentially serve as a promising platform for delivering drugs for lung diseases.

Evolution from static to dynamic label-free thermal imaging has improved bulk tissue characterization, but fails to capture subtle thermal properties in heterogeneous systems. Here, we report a label-free, high speed, and high-resolution platform technology, focal dynamic thermal imaging (FDTI), for delineating material patterns and tissue heterogeneity. Stimulation of focal regions of thermally responsive systems with a narrow beam, low power, and low cost 405 nm laser perturbs the thermal equilibrium. Capturing the dynamic response of 3D printed phantoms, ex vivo biological tissue, and in vivo mouse and rat models of cancer with a thermal camera reveals material heterogeneity and delineates diseased from healthy tissue. The intuitive and non-contact FDTI method allows for rapid interrogation of suspicious lesions and longitudinal changes in tissue heterogeneity with high-resolution and large field of view. Portable FDTI holds promise as a clinical tool for capturing subtle differences in heterogeneity between malignant, benign, and inflamed tissue.

Significance: The blood–brain barrier (BBB) is a major obstacle to detecting and treating brain tumors. Overcoming this challenge will facilitate the early and accurate detection of brain lesions and guide surgical resection of tumors. Aim: We generated an orthotopic brain tumor model that simulates the pathophysiology of gliomas at early stages; determine the BBB integrity and breakdown over the time course of tumor progression using generic and cancer-targeted near-infrared (NIR) fluorescent molecular probes. Approach: We developed an intracranial tumor xenograft model that rapidly reestablished BBB integrity and monitored tumor progression by bioluminescence imaging. Sham control mice were injected with phosphate-buffered saline only. Fluorescence molecular tomography (FMT) was used to quantify the uptake of tumor-targeted and passive NIR fluorescent imaging agents in orthotopic glioma (U87-GL-GFP PDE7B H217Q cells) tumor model. Cancer-induced and transient (with focused ultrasound, FUS) disruption of BBB integrity was monitored with NIR fluorescent dyes. Results: Stereotactic injection of 50,000 cells into mouse brain allowed rapid reestablishment of BBB integrity within a week, as determined by the inability of both tumor-targeted and generic NIR imaging agents to extravasate into the brain. Tumor-induced BBB disruption was observed 7 weeks after tumor implantation. FUS achieved a similar effect at any time point after reestablishing BBB integrity. While tumor uptake and retention of the passive NIR dye, indocyanine green, was negligible, both actively tumor-targeting agents exhibited selective accumulation in the tumor region. The tumor-targeting molecular probe that clears rapidly from nontumor brain tissue exhibits higher contrast than the analogous vascular-targeting agent and helps delineate tumors from sham control. Conclusions: We highlight the utility of FMT imaging for longitudinal assessment of brain tumors and the interplay between the stages of BBB disruption and molecular probe retention in tumors, with potential application to other neurological diseases.

Near-infrared (NIR) dye-peptide conjugates are widely used for tissue-targeted molecular fluorescence imaging of pathophysiologic conditions. However, the significant contribution of both dye and peptide to the net mass of these bioconjugates implies that small changes in either component could alter their photophysical and biological properties. Here, we synthesized and conjugated a type I collagen targeted peptide, RRANAALKAGELYKCILY, to either a hydrophobic (LS1000) or hydrophilic (LS1006) NIR fluorescent dye. Spectroscopic analysis revealed rapid self-assembly of both LS1000 and LS1006 in aqueous media to form stable dimeric/H aggregates, regardless of the free dye's solubility in water. We discovered that replacing the cysteine residue in LS1000 and LS1006 with acetamidomethyl cysteine to afford LS1001 and LS1107, respectively, disrupted the peptide's self-assembly and activated the previously quenched dye's fluorescence in aqueous conditions. These results highlight the dominant role of the octadecapeptide, but not the dye molecules, in controlling the photophysical properties of these conjugates by likely sequestering or extruding the hydrophobic or hydrophilic dyes, respectively. Application of the compounds for imaging collagen-rich tissue in an animal model of inflammatory arthritis showed enhanced uptake of all four conjugates, which retained high collagen-binding affinity, in inflamed joints. Moreover, LS1001 and LS1107 improved the arthritic joint-to-background contrast, suggesting that reduced aggregation enhanced the clearance of these compounds from non-target tissues. Our results highlight a peptide-driven strategy to alter the aggregation states of molecular probes in aqueous solutions, irrespective of the water-solubilizing properties of the dye molecules. The interplay between the monomeric and aggregated forms of the conjugates using simple thiol-modifiers lends this peptide-driven approach to diverse applications, including the effective imaging of inflammatory arthritis joints.

Most cancer patients succumb to disseminated disease because conventional systemic therapies lack spatiotemporal control of their toxic effects in vivo, particularly in a complicated milieu such as bone marrow where progenitor stem cells reside. Here, we demonstrate the treatment of disseminated cancer by photoactivatable drugs using radiopharmaceuticals. An orthogonal-targeting strategy and a contact-facilitated nanomicelle technology enabled highly selective delivery and co-localization of titanocene and radiolabelled fluorodeoxyglucose in disseminated multiple myeloma cells. Selective ablation of the cancer cells was achieved without significant off-target toxicity to the resident stem cells. Genomic, proteomic and multimodal imaging analyses revealed that the downregulation of CD49d, one of the dimeric protein targets of the nanomicelles, caused therapy resistance in small clusters of cancer cells. Similar treatment of a highly metastatic breast cancer model using human serum albumin-titanocene formulation significantly inhibited cancer growth. This strategy expands the use of phototherapy for treating previously inaccessible metastatic disease., Most of the systemic cancer therapies lack spatiotemporal control. Here, the authors show targeted activation of a light-sensitive drug by radiopharmaceuticals in disseminated cancer cells as potential in vivo treatment of metastatic diseases with reduced off-target toxicity.

Aim: CaCO3 nanoparticles (nano-CaCO3) can neutralize the acidic pHe of solid tumors, but the lack of intrinsic imaging signal precludes noninvasive monitoring of pH-perturbation in tumor microenvironment. We aim to develop a theranostic version of nano-CaCO3 to noninvasively monitor pH modulation and subsequent tumor response. Materials & methods: We synthesized ferromagnetic core coated with CaCO3 (magnetite CaCO3). Magnetic resonance imaging (MRI) was used to determine the biodistribution and pH modulation using murine fibrosarcoma and breast cancer models. Results: Magnetite CaCO3-MRI imaging showed that nano-CaCO3 rapidly raised tumor pHe, followed by excessive tumor-associated acid production after its clearance. Continuous nano-CaCO3 infusion could inhibit metastasis. Conclusion: Nano-CaCO3 exposure induces tumor metabolic reprogramming that could account for the failure of previous intermittent pH-modulation strategies to achieve sustainable therapeutic effect.

Cyclic arginine-glycine-aspartic acid (cRGD) peptides are well known to target ανβ3 integrin expressed on cancer cells and neovasculature. Conjugation of these peptides with dyes, drugs, antibodies and other biomolecules through covalent linkers provides a facile way to deliver these products to tumor cells for targeted cancer therapy and diagnosis. Click chemistry and acid-amine couplings are widely used conjugation strategies. However, the effects of different linkers and the distance between the cRGD and the conjugates on the binding of cRGD ligand with ανβ3 has been underexplored. In this present study, we prepared cRGD-conjugates using different linkers and determined how they altered the tumor targeting efficiency in vitro and in vivo. The results demonstrate that different linkers significantly altered the pharmacokinetics of the cRGD conjugates and the tumor uptake kinetics. Unlike large antibodies, this preliminary finding shows that linkers used to attach drugs and fluorescent molecular probes to small peptides play a major role in the accuracy of tumor targeting and treatment outcomes. As a result, considerable attention should be paid to the nature of linkers used in the design of molecular probes and targeted therapeutics.

Enhanced glycolysis and poor perfusion in most solid malignant tumors create an acidic extracellular environment, which enhances tumor growth, invasion, and metastasis. Complex molecular systems have been explored for imaging and treating these tumors. Here, we report the development of a small molecule, LS662, that emits near-infrared (NIR) fluorescence upon protonation by the extracellular acidic pH environment of diverse solid tumors. Protonation of LS662 induces selective internalization into tumor cells and retention in the tumor microenvironment. Noninvasive NIR imaging demonstrates selective retention of the pH sensor in diverse tumors, and two-photon microscopy of ex vivo tumors reveals significant retention of LS662 in tumor cells and the acid tumor microenvironment. Passive and active internalization processes combine to enhance NIR fluorescence in tumors over time. The low background fluorescence allows tumors to be detected with high sensitivity, as well as dead or dying cells to be delineated from healthy cells. In addition to demonstrating the feasibility of using small molecule pH sensors to image multiple aggressive solid tumor types via a protonation-induced internalization and retention pathway, the study reveals the potential of using LS662 to monitor treatment response and tumor-targeted drug delivery.

The detection of the pectoral muscle boundary in the medio-lateral oblique view of mammograms is essential to improving the computer-aided diagnosis of breast cancer. In this study, a shape-based detection method is proposed for accurately extracting the boundary of the pectoral muscle in mammograms. A shape-based enhancement mask is applied to the mammogram and the initial boundary is then defined using morphological operators. The seed point is then detected on the initial boundary and the pectoral boundary is evolved from candidate points produced using a shape-based growth strategy. A cubic polynomial fitting function is implemented to obtain the final pectoral muscle boundary. The proposed method was applied to 322 mammograms from the mini Mammographic Image Analysis Society database. A 97.2 % acceptable rate from expert radiologists and assessment results based on the false positive rate, false negative rate, and Hausdorff distance demonstrate the robustness and effectiveness of the proposed shape-based detection method.

Single photon emission computed tomography (SPECT) radionuclide pairs having distinct decay rates and different energy maxima enable simultaneous detection of dual gamma signals and real-time assessment of dynamic functional and molecular processes in vivo. Here, we report image acquisition and quantification protocols for a single molecule labeled with two different radionuclides for functional SPECT imaging.LS370 and LS734 were prepared using modular solid phase peptide synthesis. Each agent has a caspase-3 cleavable reporting motif, flanked by a tyrosine residue and a chelator at the opposite end of molecule. Cell uptake and efflux were assessed in human MDA-MB-231 breast cancer cells. Biodistribution studies were conducted in tumor naive and orthotopic 4T1 metastatic breast cancer tumor mice. NanoSPECT dual-imaging validation and attenuation correction parameters were developed using phantom vials containing varying radionuclide concentrations. Proof-of-principle SPECT imaging was performed in MMTV-PyMT transgenic mice.LS370 and LS734 were singly or dually radiolabeled with (125)I and (111)In or (99m)Tc. Cell assays demonstrated 11-fold higher percent uptake (P 0.001) of [(125)I]LS734 (3.6 ± 0.5) compared to [(125)I]LS370 (0.3 ± 0.3) at 2 h. Following chemotherapy, cellular retention of [(125)I]LS734 was 3-fold higher (P 0.05) than untreated cells. Pharmacokinetics at 1 h postinjection demonstrated longer blood retention (%ID/g) for [(125)I]LS734 (3.2 ± 0.9) compared to [(125)I]LS370 (1.6 ± 0.1). In mice bearing bilateral orthotopic 4T1 tumors, the uptake (%ID/g) was 2.4 ± 0.3 for [(125)I]LS734 and 1.2 ± 0.03 for [(125)I]LS370. The iodinated tyrosine peptide residue label was stable under in vitro conditions for up to 24 h; rapid systemic deiodination (high thyroid uptake) was observed in vivo. Phantom studies using standards demonstrated deconvolution of radionuclide signals based on different gamma ray energies. In MMTV-PyMT mice imaged with dual-labeled [(111)In]-[(125)I]LS734, the gamma signals were separable and quantifiable.Image processing protocols were developed for quantitative signal separation resulting from a caspase-3 responsive dual-radiolabeled SPECT probe. Crosstalk unmixing was obtained for multiradionuclide NanoSPECT imaging. In vitro and in vivo data demonstrated structure-activity relationships for developing functional agents for ratiometric SPECT imaging.

The combination of light and photosensitizers for phototherapeutic interventions, such as photodynamic therapy, has transformed medicine and biology. However, the shallow penetration of light into tissues and the reliance on tissue oxygenation to generate cytotoxic radicals have limited the method to superficial or endoscope-accessible lesions. Here we report a way to overcome these limitations by using Cerenkov radiation from radionuclides to activate an oxygen-independent nanophotosensitizer, titanium dioxide (TiO2). We show that the administration of transferrin-coated TiO2 nanoparticles and clinically used radionuclides in mice and colocalization in tumours results in either complete tumour remission or an increase in their median survival. Histological analysis of tumour sections showed the selective destruction of cancerous cells and high numbers of tumour-infiltrating lymphocytes, which suggests that both free radicals and the activation of the immune system mediated the destruction. Our results offer a way to harness low-radiance-sensitive nanophotosensitizers to achieve depth-independent Cerenkov-radiation-mediated therapy.

A gold nanoparticle was radiolabeled with (125)I and (111)In and functionalized with an MMP9-cleavable peptide to form a multispectral SPECT imaging contrast agent. Peptide cleavage from the nanoprobe by MMP9 was observed in vitro, and distinct pharmacokinetic properties of the contrast agent were observed between tumors with high or low MMP9 expression.

Near-infrared (NIR) water-dispersible fluorescent tags are of big importance for biomedical imaging. Bright, stable, biocompatible NIR fluorescent nanoparticles have great translation potential to improve diagnosis of early stages of different diseases. Here we report on the synthesis of exceptionally bright (“ultrabright”) fluorescent meso(nano)porous silica nanoparticles of 28 ± 3 nm in diameter. The NIR fluorescent dye LS277 is encapsulated inside these silica nanoparticles. The wavelengths of the maximum excitation/ fluorescence of the particles are 804/815 nm. The absorptivity coefficient of the particles is 2.1 × 10(8) M(−1) cm(−1) at 805 nm and the quantum yield of the dye increased by a factor of 5 after encapsulating to 1.5%. The fluorescent brightness of these particles is more than 2000× higher than the fluorescence of one molecule of LS277 in water. When exited in NIR spectral region (>700 nm), these particles are up to 4× brighter than QD800 commercial quantum dots emitting at 800 nm. We demonstrate that the synthesized NIR mesoporous silica nanoparticles easily internalize 4T1luc breast tumor cells, and remain bright for more than 9 weeks whereas the dye is completely bleached by that time.

With the development of three-dimensional television (3DTV), the fatigue associated with watching 3DTV has received considerable attention. To study how watching 3DTV influences mental fatigue and to investigate the related objective indicators, we use physiological detectors to record EEGs of 10 individual subjects both before and after watching 2D or 3D television. The 2D/3D changes of both energy values of EEG in the four wavebands, namely alpha (α), beta (β), theta (θ), delta (δ), and four fatigue-related algorithms (α + θ)/β, α/β, (α + θ)/(α + β) and θ/β, were compared and analyzed. The results show that the parameters for the 3DTV group, except theta rhythm, changed more significantly than those for the 2DTV groups in some brain regions. For most subjects watching 3DTV, the energy in alpha and beta frequency bands significantly decrease while delta activity shows a significant increase, and theta activity remains largely unchanged. All four algorithms show a significant increase with algorithm (α + θ)/β showing a greater increase (p

Inspired by the visual system of the morpho butterfly, we have designed, fabricated, tested and clinically translated an ultra-sensitive, light weight and compact imaging sensor capable of simultaneously capturing near infrared (NIR) and visible spectrum information. The visual system of the morpho butterfly combines photosensitive cells with spectral filters at the receptor level. The spectral filters are realized by alternating layers of high and low dielectric constant, such as air and cytoplasm. We have successfully mimicked this concept by integrating pixelated spectral filters, realized by alternating silicon dioxide and silicon nitrate layers, with an array of CCD detectors. There are four different types of pixelated spectral filters in the imaging plane: red, green, blue and NIR. The high optical density (OD) of all spectral filters (OD>4) allow for efficient rejections of photons from unwanted bands. The single imaging chip weighs 20 grams with form factor of 5mm by 5mm. The imaging camera is integrated with a goggle display system. A tumor targeted agent, LS301, is used to identify all spontaneous tumors in a transgenic PyMT murine model of breast cancer. The imaging system achieved sensitivity of 98% and selectivity of 95%. We also used our imaging sensor to locate sentinel lymph nodes (SLNs) in patients with breast cancer using indocyanine green tracer. The surgeon was able to identify 100% of SLNs when using our bio-inspired imaging system, compared to 93% when using information from the lymphotropic dye and 96% when using information from the radioactive tracer.

Background Current cancer management faces several challenges, including the occurrence of a residual tumor after resection, the use of radioactive materials or high concentrations of blue dyes for sentinel lymph node biopsy, and the use of bulky systems in surgical suites for image guidance. To overcome these limitations, we developed a real-time, intraoperative imaging device that, when combined with near infrared fluorescent molecular probes, can aid in the identification of tumor margins, guide surgical resections, map sentinel lymph nodes, and transfer acquired data wirelessly for remote analysis. Methods We developed a new compact, wireless, wearable, and battery-operated device that allows for hands-free operation by surgeons. A charge-coupled device–based, consumer-grade night vision viewer was used to develop the detector portion of the device, and the light source portion was developed from a compact headlamp. This piece was retrofitted to provide both near infrared excitation and white light illumination simultaneously. Wireless communication was enabled by integrating a battery-operated, miniature, radio-frequency video transmitter into the system. We applied the device in several types of oncologic surgical procedures in murine models, including sentinel lymph node mapping, fluorescence-guided tumor resection, and surgery under remote expert guidance. Results Unlike conventional imaging instruments, the device displays fluorescence information directly on its eyepiece. When employed in sentinel lymph node mapping, the locations of sentinel lymph nodes were visualized clearly, even with tracer level dosing of a near infrared fluorescent dye (indocyanine green). When used in tumor resection, tumor margins and small nodules invisible to the naked eye were visualized readily. In a simulated, point-of-care setting, tumors were located successfully and removed under remote guidance using the wireless feature of the device. Importantly, the total cost of this prototype system ($1200) is substantially less than existing imaging instruments. Conclusion Our results demonstrate the feasibility of using the new device to aid surgical resection of tumors, map sentinel lymph nodes, and facilitate telemedicine.

Image-guided surgery can enhance cancer treatment by decreasing, and ideally eliminating, positive tumor margins and iatrogenic damage to healthy tissue. Current state-of-the-art near-infrared fluorescence imaging systems are bulky and costly, lack sensitivity under surgical illumination, and lack co-registration accuracy between multimodal images. As a result, an overwhelming majority of physicians still rely on their unaided eyes and palpation as the primary sensing modalities for distinguishing cancerous from healthy tissue. Here we introduce an innovative design, comprising an artificial multispectral sensor inspired by the Morpho butterfly’s compound eye, which can significantly improve image-guided surgery. By monolithically integrating spectral tapetal filters with photodetectors, we have realized a single-chip multispectral imager with 1000 × higher sensitivity and 7 × better spatial co-registration accuracy compared to clinical imaging systems in current use. Preclinical and clinical data demonstrate that this technology seamlessly integrates into the surgical workflow while providing surgeons with real-time information on the location of cancerous tissue and sentinel lymph nodes. Due to its low manufacturing cost, our bio-inspired sensor will provide resource-limited hospitals with much-needed technology to enable more accurate value-based health care.

The inability to identify microscopic tumors and assess surgical margins in real-time during oncologic surgery leads to incomplete tumor removal, increases the chances of tumor recurrence and necessitates costly repeat surgery. To overcome these challenges, we have developed a wearable goggle augmented imaging and navigation system (GAINS) that can provide accurate intraoperative visualization of tumors and sentinel lymph nodes in real-time without disrupting normal surgical workflow. GAINS projects both near-infrared fluorescence from tumors and the natural color images of tissue onto a head-mounted display without latency. Aided by tumor-targeted contrast agents, the system detected tumors in subcutaneous and metastatic mouse models with high accuracy (sensitivity = 100%, specificity = 98% ± 5% standard deviation). Human pilot studies in breast cancer and melanoma patients using a near-infrared dye show that the GAINS detected sentinel lymph nodes with 100% sensitivity. Clinical use of the GAINS to guide tumor resection and sentinel lymph node mapping promises to improve surgical outcomes, reduce rates of repeat surgery and improve the accuracy of cancer staging.

The large size of many near-infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. In this study, we developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from 500 to 1200 nm and a QD core diameter between 1.5 and 9 nm. Conjugation of a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys) resulted in monodisperse, water-soluble QDs (hydrodynamic diameter < 10 nm) without loss of the peptide's high binding affinity to tumor-associated integrins (KI = 1.8 nM/peptide). Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and noncytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

We have developed a wearable, fluorescence goggle based system for intraoperative imaging of tumors and image guidance in oncologic surgery. Our system can detect fluorescence from cancer selective near infra-red (NIR) contrast agent, facilitating intraoperative visualization of surgical margins and tumors otherwise not apparent to the surgeon. The fluorescence information is displayed directly to the head mounted display (HMD) of the surgeon in real time, allowing unhindered surgical procedure under image guidance. This system has the potential of improving surgical outcomes in oncologic surgery and reduce the chances of cancer recurrence.

We report two methods for quantitatively determining maximal imaging depth from thick tissue images captured using all–near-infrared (NIR) multiphoton microscopy (MPM). All-NIR MPM is performed using 1550 nm laser excitation with NIR detection. This method enables imaging more than five-fold deep in thick tissues in comparison with other NIR excitation microscopy methods. In this study, we show a correlation between the multiphoton signal along the depth of tissue samples and the shape of the corresponding empirical probability density function (pdf) of the photon counts. Histograms from this analysis become increasingly symmetric with the imaging depth. This distribution transitions toward the background distribution at higher imaging depths. Inspired by these observations, we propose two independent methods based on which one can automatically determine maximal imaging depth in the all-NIR MPM images of thick tissues. At this point, the signal strength is expected to be weak and similar to the background. The first method suggests the maximal imaging depth corresponds to the deepest image plane where the ratio between the mean and median of the empirical photon-count pdf is outside the vicinity of 1. The second method suggests the maximal imaging depth corresponds to the deepest image plane where the squared distance between the empirical photon-count mean obtained from the object and the mean obtained from the background is greater than a threshold. We demonstrate the application of these methods in all-NIR MPM images of mouse kidney tissues to study maximal depth penetration in such tissues.

Analysis of the relationship between transcriptional activators and chromatin organization has focused largely on lower levels of chromatin structure. Here we describe striking remodeling of large-scale chromatin structure induced by a strong transcriptional activator. A VP16-lac repressor fusion protein targeted the VP16 acidic activation domain to chromosome regions containing lac operator repeats. Targeting was accompanied by increased transcription, localized histone hyperacetylation, and recruitment of at least three different histone acetyltransferases. Observed effects on large-scale chromatin structure included unfolding of a 90-Mbp heterochromatic chromosome arm into an extended 25–40-μm chromonema fiber, remodeling of this fiber into a novel subnuclear domain, and propagation of large-scale chromatin unfolding over hundreds of kilobase pairs. These changes in large-scale chromatin structure occurred even with inhibition of ongoing transcription by α-amanitin. Our results suggest a functional link between recruitment of the transcriptional machinery and changes in large-scale chromatin structure. Based on the observed long-range propagation of changes in large-scale chromatin structure, we suggest a possible rationale for the observed clustering of housekeeping genes within Mbp-sized chromosome bands.

Recently we described a new method for in situ localization of specific DNA sequences, based on lac operator/repressor recognition (Robinett, C.C., A. Straight, G. Li, C. Willhelm, G. Sudlow, A. Murray, and A.S. Belmont. 1996. J. Cell Biol. 135:1685-1700). We have applied this methodology to visualize the cell cycle dynamics of an approximately 90 Mbp, late-replicating, heterochromatic homogeneously staining region (HSR) in CHO cells, combining immunostaining with direct in vivo observations. Between anaphase and early G1, the HSR extends approximately twofold to a linear, approximately 0.3-mum-diam chromatid, and then recondenses to a compact mass adjacent to the nuclear envelope. No further changes in HSR conformation or position are seen through mid-S phase. However, HSR DNA replication is preceded by a decondensation and movement of the HSR into the nuclear interior 4-6 h into S phase. During DNA replication the HSR resolves into linear chromatids and then recondenses into a compact mass; this is followed by a third extension of the HSR during G2/ prophase. Surprisingly, compaction of the HSR is extremely high at all stages of interphase. Preliminary ultrastructural analysis of the HSR suggests at least three levels of large-scale chromatin organization above the 30-nm fiber.

Optical imaging enables real-time visualization of intrinsic and exogenous contrast within biological tissues. Applications in human medicine have demonstrated the power of fluorescence imaging to enhance visualization in dermatology, endoscopic procedures, and open surgery. Although few optical contrast agents are available for human medicine at this time, fluorescence imaging is proving to be a powerful tool in guiding medical procedures. Recently, intraoperative detection of fluorescent molecular probes that target cell-surface receptors has been reported for improvement in oncologic surgery in humans. We have developed a novel system, optical projection of acquired luminescence (OPAL), to further enhance real-time guidance of open oncologic surgery. In this method, collected fluorescence intensity maps are projected onto the imaged surface rather than via wall-mounted display monitor. To demonstrate proof-of-principle for OPAL applications in oncologic surgery, lymphatic transport of indocyanine green was visualized in live mice for intraoperative identification of sentinel lymph nodes. Subsequently, peritoneal tumors in a murine model of breast cancer metastasis were identified using OPAL after systemic administration of a tumor-selective fluorescent molecular probe. These initial results clearly show that OPAL can enhance adoption and ease-of-use of fluorescence imaging in oncologic procedures relative to existing state-of-the-art intraoperative imaging systems.

We have developed a near-infrared (NIR) fluorescence goggle system based on the complementary metal–oxide–semiconductor active pixel sensor imaging and see-through display technologies. The fluorescence goggle system is a compact wearable intraoperative fluorescence imaging and display system that can guide surgery in real time. The goggle is capable of detecting fluorescence of indocyanine green solution in the picomolar range. Aided by NIR quantum dots, we successfully used the fluorescence goggle to guide sentinel lymph node mapping in a rat model. We further demonstrated the feasibility of using the fluorescence goggle in guiding surgical resection of breast cancer metastases in the liver in conjunction with NIR fluorescent probes. These results illustrate the diverse potential use of the goggle system in surgical procedures.

Real-time image guidance in the operating room is needed to improve instantaneous surgical decisions. Toward this goal, we utilized a new fluorescence goggle system and a near-infrared fluorescent dye approved for human use, indocyanine green, to demonstrate the feasibility of detecting liver tumors intraoperatively. The fluorescence goggle provided successful imaging of multifocal breast cancer metastases in mouse liver. Diffused tumor deposits as small as 0.8 mm in diameter were detected, which were not obvious without the fluorescence goggle. A combination of surface-weighted fluorescence imaging and deep tissue-sensitive ultrasound imaging allowed comprehensive image guidance with the fluorescence goggle system for tumor resection in a rabbit VX2 liver metastasis model. This multimodal detection and guided surgical intervention strategy using ultrasonic imaging and real-time intraoperative fluorescence guidance is a promising and innovative technology platform for improving surgical outcome of human patients with primary or metastatic liver cancer.

We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.

Fluorescence microscopy offers high sensitivity for disease diagnosis. However, little structural information is revealed by this method, requiring another technique to localize the source of fluorescence. We developed a complementary fluorescence-polarization microscope. We used a division-of-focal-plane charge coupled device polarization sensor to enable real-time video rate polarization imaging without any moving parts. The polarization information provided by the microscope enabled detection of structural element and complements the fluorescence information. Application of this multimodal system for cancer imaging using a tumor selective molecular probe revealed the association of diminished structural integrity of tumor tissue with high fluorescence of the imaging agent compared to surrounding normal tissue. This study demonstrates a new paradigm to improve cancer detection and diagnosis.

We have developed a fluorescence goggle device for intraoperative oncologic imaging. With our system design, the surgeon can directly visualize the fluorescence information from the eyepieces in real time without any additional monitor, which can improve one's coordination and surgical accuracy. In conjunction with targeting fluorescent dyes, the goggle device can successfully detect tumor margins and small nodules that are not obvious to naked eye. This can potentially decrease the incidence of incomplete resection.

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.

Intracellular trafficking of the epidermal growth factor receptor (EGF-R) is regulated by receptor occupancy. To investigate this, we developed an assay to study endosomal sorting under steady-state conditions. Using a cell line transfected with EGF-R variants, we found that the fraction of internalized EGF.EGF-R complexes sorted to lysosomes was a function of the number of intracellular complexes and required sequences in the cytoplasmic domain of the receptor. As the number of intracellular occupied wild-type receptors increased from 3 x 10(2) to 2 x 10(5)/cell, the fraction of internalized EGF that was degraded dropped from 70 to 20%. Transforming growth factor-alpha, which dissociates from the EGF-R at endosomal pH, was degraded to a uniform extent of approximately 50% at all intracellular ligand concentrations. EGF internalized by receptors lacking a cytoplasmic domain (c'647) was degraded to an extent of only 5-10% independent of the number of intracellular complexes. Mutant receptors truncated either at residues 1022 or 973 displayed sorting patterns intermediate between wild-type and c'647 receptors. Despite large differences in their internalization rates, the fractional sorting patterns of c'1022 and c'973 receptors were indistinguishable. Receptor tyrosine kinase activity appeared to have a small effect on sorting pattern, but only in the context of full-length receptors. Our results indicate that the default pathway of internalized receptors is rapidly recycling and that lysosomal targeting of occupied EGF-R is due to endosomal retention that is both specific and saturable. In addition, internalization and endosomal retention of EGF-R appear to be mediated by distinct structural elements.

In the past decade, researchers at Seoul National University of South Korea have validated the existence of the primo-vascular system using animal models, which was originally reported by Bonghan Kim in the 1960s. After exploring a variety of dye stains, Kwang-Sup Soh, et al. found that trypan blue is an effective dye for staining structures of the primo vessels and nodes. Most of the studies reported so far have used relatively large animal models such as rabbits. In this study, we explored the potential of visualizing primo-vascular system in murine models. As compared to conventional models used for primo-vascular studies, murine models are more versatile and affordable. In mice, we found primo ­vessels extended to and from abdominal viscera, often disappearing in layers of adipose tissue before resurfacing in a more distal region. In addition, we found potential primo-vascular structures on the tumor surface. These structures were loosely attached to the surface of the tumor and some segments appear to be within the clear serosal tissue or invading into the tumor. The experience of identifying ­primo-vascular system in rodents empowers us to investigate the detailed relationship of primo-vessels and cancer in future studies. A breakthrough in ­elucidating the complete anatomical and physiological description of the primo-vascular system holds great promise of unraveling the molecular basis of various human diseases.

Cancer stem cells (CSCs) have been successfully isolated from solid tumors and are believed to be initiating cells of primary, metastatic and recurrent tumors. Imaging and therapeutic reagents targeted to CSCs have potential to detect subclinical tumors and completely eradicate the disease. Previously, we have demonstrated that Mab CC188 binds to colon cancer CD133- and CD133+ (CSCs) cells. In this study, we examined the reactivity of Mab CC188 to ovarian cancer cells including CD133+ cells and primary tumor tissues using immunofluorescence staining methods and tissue microarray technique. We also explored the feasibility of using NIR dye-labeled Mab CC188 probe to image ovarian tumors in vivo. Mab CC188 stains both CD133- and CD133+ cells of ovarian cancer. Tissue microarray analysis reveals that 75% (92/123) of ovarian cancer cases are positively stained with Mab CC188. Weak positive (±), positive (+), strong positive (++) and very strong positive (+++) stains are 14.8%, 3.7%, 11% and 24.4% respectively. In contrast, Mab CC188 staining is low in normal cells and tissues. In vivo study show that significant amounts of the probe accumulates in the excretion organs in the early period post injection. At 24 hours, the imaging probes have largely accumulates in the tumor, while the intensity of the imaging probe decreases in the liver. The tumor uptake was still evident at 120 hours post injection. Our work suggests that Mab CC188 based imaging and therapeutic reagents are capable of detecting early stage ovarian tumors and effectively treating the tumor.

Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic arginine-glycine-aspartic acid (cRGD) peptide targeting α(v)β(3) integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibits high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pK(a) of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.

We have developed a novel real-time intraoperative fluorescence imaging device that can detect near-infrared (NIR) fluorescence and map sentinel lymph nodes (SLNs). In contrast to conventional imaging systems, this device is compact, portable, and battery-operated. It is also wearable and thus allows hands-free operation of clinicians. The system directly displays the fluorescence in its goggle eyepiece, eliminating the need for a remote monitor. Using this device in murine lymphatic mapping, the SLNs stained with indocyanine green (ICG) can be readily detected. Fluorescence-guided SLN resection under the new device was performed with ease. Ex vivo examination of resected tissues also revealed high fluorescence level in the SLNs. Histology further confirmed the lymphatic nature of the resected SLNs.

Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6 ± 1.65) and flat lesions (12.1 ± 1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.42) Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy.

Publisher Summary In situ hybridization methods have been pivotal in allowing the exploration of chromosome and gene arrangements within interphase nuclei. However, the requirement of DNA denaturation for in situ hybridization is incompatible with direct in vivo observations. Moreover, the harsh conditions required for DNA denaturation are antagonistic to ultrastructural preservation. This has led to a large gap between primarily light microscopy studies, using in situ hybridization to examine the locations of specific chromosomal regions or loci, and ultrastructural analysis of higher order, “generic” chromatin structure, primarily by electron microscopy. In an attempt to bridge these two structural approaches, this chapter explores the use of protein–DNA recognition as a tag for localizing specific chromosome regions to provide an alternative to in situ hybridization. The chapter uses the lac operator/repressor system for an initial test case for several reasons. The lac repressor had been demonstrated as having the capability of binding in vitro to an operator sequence packaged within nucleosomes with only a small decrease in binding affinity. Later in vivo experiments confirmed the specific binding of lac repressor to lac operator within eukaryotic cells.

The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100 μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500 μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms.

We have developed a fiber-based, video-rate fluorescence diffuse optical tomography (DOT) system for noninvasive in vivo sentinel lymph node (SLN) mapping. Concurrent acquisition of fluorescence and reference signals allowed the efficient generation of ratio-metric data for 3D image reconstruction. Accurate depth localization and high sensitivity to fluorescent targets were established in to depths of >10 mm. In vivo accumulation of indocyanine green (ICG) dye was imaged in the region of the SLN following intradermal injection into the forepaw of rats. These results suggest that video-rate fluorescence DOT has significant potential as a clinical tool for noninvasive mapping of SLN.

Cancer-related enzyme activity can be detected noninvasively using activatable fluorescent molecular probes. In contrast to "always-on" fluorescent molecular probes, activatable probes are relatively nonfluorescent at the time of administration due to intramolecular fluorescence resonance energy transfer (FRET). Enzyme-mediated hydrolysis of peptide linkers results in reduced FRET and increase of fluorescence yield. Separation of signal from active and inactive probe can be difficult with conventional intensity-based fluorescence imaging. Fluorescence lifetime (FLT) measurement is an alternative method to detect changes in FRET. Thus, we investigate FLT imaging for in vivo detection of FRET-based molecular probe activation in an orthotopic breast cancer model. Indeed, the measured FLT of the enzyme-activatable molecular probe increases from 0.62 ns just after injection to 0.78 ns in tumor tissue after 4 h. A significant increase in FLT is not observed for an always-on targeted molecular probe with the same fluorescent reporter. These results show that FLT contrast is a powerful addition to preclinical imaging because it can report molecular activity in vivo due to changes in FRET. Fluorescence lifetime imaging exploits unique characteristics of fluorescent molecular probes that can be further translated into clinical applications, including noninvasive detection of cancer-related enzyme activity.

We demonstrate the development and application of a broad spectrum near-infrared molecular probe for imaging primary and metastatic tumors in small animals, with a goal to clinical translation.

We developed a fluorescence goggle system for intraoperative imaging and surgical navigation. This system enables visualization of fluorescence information directly from the eyepieces in real time, which improves surgical accuracy.