Fluorescence Optical Imaging Research Articles

Fundamental Limits on Measuring the Rotational Constraint of Single Molecules Using Fluorescence Microscopy.

Optical fluorescence imaging is capable of measuring both the translational and rotational dynamics of single molecules. However, unavoidable measurement noise will result in inaccurate estimates of rotational dynamics, causing a molecule to appear to be more rotationally constrained than it actually is. We report a mathematical framework to compute the fundamental limit of accuracy in measuring the rotational mobility of dipolelike emitters. By applying our framework to both in-plane and three-dimensional methods, we provide a means to choose the optimal orientation-measurement technique based on experimental conditions.

Uncovering the biology of myelin with optical imaging of the live brain.

Myelin has traditionally been considered a static structure that is produced and assembled during early developmental stages. While this characterization is accurate in some contexts, recent studies have revealed that oligodendrocyte generation and patterns of myelination are dynamic and potentially modifiable throughout life. Unique structural and biochemical properties of the myelin sheath provide opportunities for the development and implementation of multimodal label-free and fluorescence optical imaging approaches. When combined with genetically encoded fluorescent tags targeted to distinct cells and subcellular structures, these techniques offer a powerful methodological toolbox for uncovering mechanisms of myelin generation and plasticity in the live brain. Here, we discuss recent advances in these approaches that have allowed the discovery of several forms of myelin plasticity in developing and adult nervous systems. Using these techniques, long-standing questions related to myelin generation, remodeling, and degeneration can now be addressed.

Gold nanocluster-loaded hybrid albumin nanoparticles with fluorescence-based optical visualization and photothermal conversion for tumor detection/ablation

Gold nanoclusters (AuNCs) are viewed as effective hyperthermal agents for the treatment of tumors. Whereas AuNCs formed by the agglomeration of several to tens of gold atoms (<1–2 nm) possess significant fluorescence, they have a negligible hyperthermal effect, while AuNCs comprised of spherical gold nanoparticles (AuNPs > a few nanometers) have a marked hyperthermic effect but lose their inherent fluorescence and obstruct the intensity of neighboring fluorescent dyes due to Forster resonance energy transfer (FRET). To achieve both hyperthermia and fluorescence-based optical visualization, we generated hybrid albumin nanoparticles containing AuNCs (~88 nm) comprising AuNPs (~4.5 nm). We generated a series of formulated AuNCs and optimized the size, morphology, NIR absorbance (600–900 nm), hyperthermal activity, and fluorescence spectral characters of the resulting hybrid albumin nanoparticles (AuNCs/BSA-NPs) by considering the interparticle distance between the AuNPs and Cy5.5. Among these, AuNCs/BSA-NPs (formula D) had a strong hyperthermic effect and had well-preserved fluorescence intensity (from the attached Cy5.5) due to localized surface plasmon resonance (LSPR) and a reduction in FRET. These AuNCs/BSA-NPs were able to elevate the surface tumor temperature of HCT116-bearing mice to >50 °C following 808 nm laser irradiation (1.5 W/cm2, 10 min), which remarkably suppressed tumor growth (17.8 ± 16.9 mm3vs. PBS and AuNCs/BSA-NPs (formula E): ~1850 and ~1250 mm3, respectively). Also, Cy5.5-modified AuNCs/BSA-NPs (formula D) showed good performance in optical fluorescence imaging of target tumors in HCT116 tumor-bearing mice. Together, our results indicate that the interparticle distance between albumin or Cy5.5 and AuNPs/AuNCs can be optimized to achieve both hyperthermia and fluorescence emission by striking a balance between LSPR and FRET effects. We believe that the AuNC/BSA-NPs formulation presented here can serve as a potential platform for both optically visualizing and treating colon cancers.

Association between baseline clinical and imaging findings and the development of digital ulcers in patients with systemic sclerosis

ObjectiveSystemic sclerosis (SSc) can lead to ischemic complications such as digital ulcers (DUs). The aim of the study was to find predictors of DUs by clinical and new imaging methods.Patients and methodsAll 79 SSc patients included in the study received a clinical, colour Doppler ultrasound (CDUS), fluorescence optical imaging (FOI) and capillaroscopy examination at baseline, and their capacity to predict new DU development was analysed in 76 patients at 12 months follow-up.ResultsTwenty-two of 76 patients (28.9%) developed new ulcers during follow-up (diffuse SSc 48.1%; limited SSc 18.4%). Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.7576 for DU development, with a specificity of 87% and a sensitivity of 54.6% (p = 0.0003, OR = 8.1 [95%CI 2.5–25.6]) at a cut-off of ≥ 21 points (ACR/EULAR classification criteria for SSc). Capillaroscopy and CDUS had high sensitivity (100% and 95.5%) but low specificity (28.9% and 22.2%) for ulcer occurrence when used alone, but better specificity (46.3%) when combined (OR = 18.1 [95%CI 2.3–144.4]; p = 0.0004). Using FOI, fingers with pathologic staining had a higher risk for new ulcer development in the same finger (p = 0.0153). General future DU (i.e. DU also in other fingers) was associated with a missing FOI signal in the right digit III at baseline (p = 0.048).ConclusionNew imaging modalities can predict digital ulcer development in SSc patients with high sensitivity for capillaroscopy and CDUS and enhanced specificity when combined. A missing signal of FOI in the right digit III at baseline was associated with general future DU.

Comparative study of one-step and two-step quantitative fluorescence photoacoustic tomography.

Fluorescence optical tomography (FOT) is a well-known imaging technique, where fluorescent biological markers are injected to tag targeted tissues (tumors, proteins), and the absorption coefficient of fluorophore is reconstructed to provide contrast-enhanced images. Conventional FOT is known to have lack of stability to noise and shallow imaging depth due to strong optical scattering in biological tissue. Photoacoustic tomography (PAT) has been previously proposed to combine with FOT to resolve this issue. We propose a fully nonlinear one-step reconstruction in a diffuse-approximation modeled fluorescence photoacoustic tomographic (FPAT) setting, where the absorption coefficient of exogenous fluorophore is recovered directly from the photoacoustic data. Computational validations in two dimensions in single- and dual-grid reconstruction settings using full as well as partial data have been provided in support of the proposed algorithm. One-step schemes are particularly useful with respect to dual representations of field (optical and pressure) variables and optical parameters, especially in limited-data settings, which effectively help in constraining the optimization search space. We have compared the results of one- and two-step FPAT schemes and concluded that the one-step reconstructions are superior as compared with the corresponding two-step reconstructions. To the best of our knowledge, these are the first comparisons of one-step and two-step reconstructions in FPAT.

Functional Organotypic Cultures of Prostate Tissues: A Relevant Preclinical Model that Preserves Hypoxia Sensitivity and Calcium Signaling

In prostate cancer research, there is a lack of valuable preclinical models. Tumor cell heterogeneity and sensitivity to microenvironment signals, such as hypoxia or extracellular calcium concentration, are difficult to reproduce. Here, we developed and characterized an exvivo tissue culture model preserving these properties. Prostate tissue slices from 26 patients were maintained exvivo under optimized culture conditions. The expression of markers associated with proliferation, androgen-receptor signaling, and hypoxia was assessed by immunostaining. A macroscope was used to achieve real-time calcium fluorescence optical imaging. Tissue morphology was maintained successfully without necrosis for 5 days. Compared with native tumors and tissue cultured with androgens, androgen deprivation in the medium led to decreased expression of both androgen receptor and its target gene products, prostate specific antigen (PSA) and ETS-related gene (ERG). Exvivo cultured slices also were sensitive to hypoxia because carbonic anhydrase IX and zinc finger E-box binding homeobox 1 (Zeb1) protein levels increased in 1% oxygen. Exposure of slices to supraphysiological extracellular Ca2+ concentration induced a robust and rapid Ca2+ entry, with a greater response in tumor compared with nontumor tissue. This exvivo model reproduces the morphologic and functional characteristics of human prostate cancer, including sensitivity to androgen deprivation and induced response to hypoxia and extracellular Ca2+. It therefore could become an attractive tool for drug response prediction studies.

Feasibility, inter-reader reliability, and comparison of fluorescence optical imaging enhancement in patients with erosive hand osteoarthritis and rheumatoid arthritis

Feasibility, inter-reader reliability, and comparison of fluorescence optical imaging enhancement in patients with erosive hand osteoarthritis and rheumatoid arthritis

Optical fluorescence imaging in oral cancer and potentially malignant disorders: A systematic review.

This study aimed to systematically review the efficacy of direct optical fluorescence imaging as an adjunct to comprehensive oral examination in the clinical evaluation, risk assessment and surgical management of oral cancer and potentially malignant disorders. Studies adopting autofluorescence devices, evaluating the efficacy of comprehensive oral examination and optical fluorescence imaging in detection, visualisation or management of oral squamous cell carcinoma or oral potentially malignant disorders, as well as discriminating oral epithelial dysplasia from other mucosal lesions, were included in the literature search across bibliographic databases until October 2018. Twenty-seven studies were found to be eligible for inclusion in qualitative analysis. Of these, only six studies demonstrated a low risk of bias across all domains of the methodological assessment tool (QUADAS-2). Optical fluorescence imaging demonstrated positive results, with higher sensitivity scores, increased lesion detection and visualisation than comprehensive oral examination alone in the clinical evaluation of oral squamous cell carcinoma and oral potentially malignant disorders. This review provides promising evidence for the utilisation of optical fluorescence imaging as an adjunct to comprehensive oral examination in varying clinical settings. It is important that devices utilising optical fluorescence imaging are viewed strictly as clinical adjuncts and not specifically as diagnostic devices.

Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain.

Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy.

Development of silicon microneedle arrays with spontaneously generated micro-cavity ring for transdermal drug delivery

Transdermal drug delivery techniques based on microneedle arrays can painlessly administer controlled amount of drugs through penetration of the epidermis. Solid microneedles arrays can be employed in this process after coating the tip with the liquid agent before insertion. In this work, we report a low cost technique for the formation of pyramidal microneedle arrays based on crystalline silicon. A combination of crystallographic etching, thermal oxidation and subsequent HNA treatment was employed. Optimization of the process parameters led to the generation of pyramidal microneedle arrays where the tip diameter can be reduced without loss of overall structural robustness. Micro-cavities with size 4–10 μm in diameter can be reproducibly generated in a ring surrounding the tip, while maintaining an otherwise smooth microneedle facet wall. Optical fluorescence imaging indicates that fluids can be exclusively trapped in these microcavities, with implications in transdermal drug delivery.

Multifunctional far-field luminescence nanoscope for studying single molecules and quantum dots: (50th anniversary of the Institute of Spectroscopy, Russian Academy of Sciences)

Far-field fluorescence spectromicroscopy of single quantum emitters (SQEs) (single molecules, quantum dots, color centers in crystals) is an actively developing field of modern photonics, which is in widespread demand in various applications in physics, chemistry, material sciences, life sciences, and quantum technologies. In this paper, we present a description of a multifunctional experimental setup which was developed in recent years at the Institute for Spectroscopy of the Russian Academy of Sciences. It allows measuring optical spectra and fluorescence images of SQEs, as well as their temporal behavior and luminescence kinetics, in a broad range of temperatures (from cryogenic to ambient). It is shown that the spatial coordinates of SQEs can be reconstructed with subdiffractional accuracy (up to a few angstroms). Some examples of the developed methods for multiparameter superresolution microscopy (nanoscopy) of materials and nanostructures are presented.

04:12 PM Abstract No. 226 Glypican-3 optical molecular imaging enables in vivo detection and monitoring of response to adoptive immunotherapy

We have previously described synthesis of a bispecific fusion protein for catheter-directed targeting of glypican-3 (GPC3) expressed on hepatocellular carcinoma (HCC) tumors and CD3 T-cell receptors (GPC3-CD3-bsAb) (1). We hypothesized that in vivo optical fluorescent imaging of GPC3 in mouse models of human HCC would enable effective monitoring of adoptive immunotherapy mediated by GPC3-CD3-bsAb. A modified GPC3 specific small peptide was labeled with Hylite488 or Cylyte5 fluorescent dyes (GPC3-FL) and used for in-vitro and in vivo imaging, respectively. In-vitro competitive binding assay was performed to assess specific probe binding to GPC3 on human HCC tumor cells (HepG2 (GPC3+) and SNU449 (GPC3-)). For in vivo imaging, HCC mouse xenograft models were created through implantation of HepG2 and SNU449 cells in the flanks of nude mice (n=5 each group). Optical imaging was performed after tail-vein injection of 6.25μg of GPC3-FL on IVIS Spectrum in vivo imaging system. Mice were imaged at baseline, followed by GPC3 blockage using higher concentrations of non-labeled GPC3 specific peptide and after adoptive immunotherapy with concurrent injection of GPC3-CD3-bsAb and activated human T-cells. In-vitro assays confirmed dose dependent blockage of GPC3-FL binding to GPC3+ HepG2 cells by GPC3 specific antibodies. Optical imaging of HepG2 and SNU449 tumor bearing mice demonstrated a significantly higher target to background ratio (TBR) for GPC3+ HepG2 tumors compared to GPC3- SNU449 tumors (2.99±0.36 vs 1.16±1.15, p=0.01). Concurrent injection of non-labeled GPC3 specific peptide and GPC-FL showed a marked decrease in tumor TBRs in GPC3+ HepG2 tumors (1.45±0.18 vs. 2.99±0.36, p=0.004). Following an adoptive immunotherapy treatment course, fluorescent signal in treated tumors markedly decreased in the treatment group compared to controls (4.36±0.9 before treatment vs 1.8±0.4, p=0.049). Optical imaging using GPC3-FL is feasible for in vivo detection of GPC3 positive HCC cells and allows non-invasive monitoring of tumor response to adoptive immunobtherapy, which cannot be achieved using available morphological methods of imaging.

The rheumatoid hand in the light of fluorescence: a diagnostic technique of the future?

Fluorescence spectroscopy is usually applied in physics, chemistry and related sciences. However, in recent years we can observe a growing interest in fluorescence spectroscopy for medical diagnostics. Currently, it is beginning to be used in the monitoring of rheumatoid arthritis (RA) activity. As the knowledge on RA increases, growing importance is being placed on the evaluation of synovitis. Today, it is difficult to imagine contemporary rheumatology without ultrasound (US) and magnetic resonance imaging (MRI). However, it turns out that these are not the only methods allowing one to visualise subclinical lesions, particularly synovitis. Fluorescence optical imaging (FOI) is also useful for the evaluation of inflammatory lesions in the joints. In the future, FOI may become competitive with “traditional” imaging studies. It is characterised by low cost, short duration and similar sensitivity to US.

Self-crosslinked fibrous collagen/chitosan blends: Processing, properties evaluation and monitoring of degradation by bi-fluorescence imaging.

Porous collagen/chitosan scaffolds with different Collagen:Chitosan (Coll:Ch) ratios were prepared by freeze-drying followed by self-crosslinking via dehydrothermal treatment (DHT) and characterized as biomaterials for tissue engineering. Cy7 and Cy5.5 fluorochromes were covalently grafted to collagen and chitosan, respectively. Thus, it was possible, using optical fluorescence imaging of the two fluorochromes, to simultaneously track their in vivo biodegradation, in a blend scaffold form. The fluorescence signal evolution, due to the bioresorption, corroborated with histological analysis. In vitro cytocompatibility of Coll:Ch blend scaffolds were evaluated with standardized tests. In addition, the scaffolds showed a highly interconnected porous structure. Extent of crosslinking was analyzed by convergent analysis using thermogravimetry, Fourier Transform Infrared Spectroscopy and PBS uptake. The variations observed with these techniques indicate strong interactions between collagen and chitosan (covalent and hydrogen bonds) promoted by the DHT. The mechanical properties were characterized to elucidate the impact of the different processing steps in the sample preparation (DHT, neutralization and sterilization by β-irradiation) and showed a robust processing scheme with low impact of Coll:Ch composition ratio.

UMUC1-Targeting Magnetic Resonance Imaging of Therapeutic Response in an Orthotropic Mouse Model of Colon Cancer.

Noninvasive assessment of chemotherapeutic response in colon cancer would tremendously aid in therapeutic intervention of cancer patients and improve outcomes. The aim of the study was to evaluate the feasibility of a noninvasive assessment of chemotherapeutic response by magnetic resonance imaging utilizing underglycosylated mucin 1 (uMUC1) tumor antigen as a biomarker of therapeutic response in a colon cancer mouse model. The study was performed by applying molecular imaging approach based on targeting uMUC1 with specific dual-modality imaging probe (MN-EPPT). The probe consisted of dextran-coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1-specific peptide (EPPT) and was used for magnetic resonance imaging (MRI) and fluorescence optical imaging. An orthotopic murine model of colon cancer expressing human uMUC1 peptide (MC38 MUC1) was created along with the control model devoid of the antigen (MC38 neo). Animals received chemotherapy with 5-fluorouracil (5-FU) followed by MN-EPPT-enhanced MR and optical imaging. In vivo imaging of animals with uMUC1 expressing tumors after 5-FU therapy showed that the average deltaT2 was reduced by 7.27ms (p = 0.045) compared with animals in control groups indicating lower accumulation of MN-EPPT caused by uMUC1 downregulation. In vivo optical imaging, biodistribution, and fluorescence microscopy confirmed the MRI findings. Interestingly, we found that the group of animals that did not respond to chemotherapy ("progressive disease" per RECIST) showed higher accumulation of MN-EPPT compared to the group of responders ("stable disease") consistent with proliferating tumor cells and increased antigen availability. We believe that in application to over 50% of human cancers expressing uMUC1, our results could provide insight into overall assessment of therapeutic response based on its expression as defined by non-invasive MN-EPPT-enhanced MRI.

Lysosome-Targeted Bioprobes for Sequential Cell Tracking from Macroscopic to Microscopic Scales.

Longitudinal tracking of living cells is crucial to understanding the mechanism of action and toxicity of cell-based therapeutics. To quantify the presence of administered cells in the host tissue without sacrifice of animals, labeling of the target cells with a nontoxic and stable contrast agent is a prerequisite. However, such long-term live cell tracking is currently limited by the lack of fluorophores with steady optical and physicochemical properties in the near-infrared (NIR) window. Herein, for the first time, the design of fixable cell-tracking NIR fluorophores (CTNFs) with high optical properties, excellent cell permeation and retention, and high stability against chemical treatments is reported. Efficient cellular labeling and tracking of CTNFs using intraoperative optical fluorescence imaging by following the fate of NIR-labeled cells from the time of injection into animals to ex vivo cellular analysis after resection of the target tissue is demonstrated. Due to the lipophilic cationicity and primary amine docking group, CTNF126 outperforms the other tested fluorophores with rapid diffusion into the cytoplasmic membrane and sequestration inside the lysosomes, which prevents cellular efflux and improves cellular retention. Thus, CTNF126 will be useful to track cells in living organisms for the mechanism of action at the single cell level.

A mathematical model and iterative inversion for fluorescent optical projection tomography

Solving the fluorophore distribution in a tomographic setting has been difficult because of the lack of physically meaningful and computationally applicable propagation models. This study concentrates on the direct modelling of fluorescence signals in optical projection tomography (OPT), and on the corresponding inverse problem. The reconstruction problem is solved using emission projections corresponding to a series of rotational imaging positions of the sample. Similarly to the bright field OPT bearing resemblance with the transmission x-ray computed tomography, the fluorescent mode OPT is analogous to x-ray fluorescence tomography (XFCT). As an improved direct model for the fluorescent OPT, we derive a weighted Radon transform based on the XFCT literature. Moreover, we propose a simple and fast iteration scheme for the slice-wise reconstruction of the sample. The developed methods are applied in both numerical experiments and inversion of fluorescent OPT data from a zebrafish embryo. The results demonstrate the importance of propagation modelling and our analysis provides a flexible modelling framework for fluorescent OPT that can easily be modified to adapt to different imaging setups.

Inverse Optical Tomography through PDE Constrained Optimization $L^\infty$

Fluorescent optical tomography (FOT) is a new biomedical imaging method with wider industrial applications. It is currently intensely researched since it is very precise and with no side effects for humans, as it uses nonionizing red and infrared light. Mathematically, FOT can be modeled as an inverse parameter identification problem, associated with a coupled elliptic system with Robin boundary conditions. Herein we utilize novel methods of calculus of variations in $L^{\infty}$ to lay the mathematical foundations of FOT which we pose as a PDE-constrained minimization problem in $L^{p}$ and $L^{\infty}$.

Multifunctional drug carrier on the basis of 3d-4f Fe/La-MOFs for drug delivery and dual-mode imaging.

Multifunctional drug carriers for simultaneous imaging and drug delivery have emerged as an important new direction for the treatment of cancer. In this study, 3d-4f heterometallic Fe/La-MOFs, with excellent fluorescence and superior positive magnetic resonance imaging with high resolution, were synthesized successfully. The feasibility of Fe/La-MOFs for use in multifunctional drug delivery was demonstrated using doxorubicin hydrochloride (DOX) as the drug model. After modification with tunable amino modified silica layers, the drug loading increased to 150.2 mg g-1 from 23.9 mg g-1, and the pH responsive drug release climbed 80% from 10% upon regulating the pH from 5.8 to 7.4. T2-Weighted magnetic resonance imaging (MRI) and fluorescence optical imaging (FOI) both showed a clear concentration-dependent contrast enhancement, indicating potential application as a MRI/FOI dual mode imaging agent. In addition, the FOI of 4T1 cells loaded with Fe/La-MOFs demonstrated their capacity for imaging living cells. The particles were tracked by MRI, and the transverse relativity (r2) ranks among the highest reported for Fe3+complexes (100.49 mM-1 s-1). In summary, this is the first report on 3d-4f MOF particles displaying pH-responsive delivery and MRI/FOI dual mode imaging.

Multimodal Molecular Imaging Strategies using Functionalized Nano Probes

Cancer prognosis was mainly done by imaging and biopsy with histological analysis of solid tumours. Identifying tumour margins and staging of the tumour are critical for choosing appropriate treatments so that the risk of reoccurrence could be minimized. Imaging modalities have their strengths and limitations. Hence multimodality imaging that takes advantage of strengths from two or more imaging techniques may serve improved diagnostic and therapeutic monitoring abilities. Radiolabelled small molecules have been used as contrast agents to detect tumour for prognosticating therapeutic interventions. However, these probes lack tissue specificity and stability for optimal usage. Quantum dots (QDs) are fluorescent probes for optical imaging, which are capable of tunable optical properties, ability to target tumours when their surface-functionalized with high stability. Recently, a wide application of biocompatible surface-functionalized QDs is evidenced as multimodal imaging probes. However, their pathway to clinical translation is not yet fully explored. In this review, different biocompatible functionalized nanoprobes, their in-vivo application in animal model followed by their future possible clinical applications, recent developments of optical fluorescence imaging probes and its integration with other imaging modalities such as X-ray computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and ultrasonography (US) are discussed.