Enhanced Fluorescence Imaging Research Articles

Multi-Color Enhanced Fluorescence Imaging of a Breast Cancer Cell with A Hole-Arrayed Plasmonic Chip.

Breast cancer cells of MDA-MB-231 express various types of membrane proteins in the cell membrane. In this study, two types of membrane proteins in MDA-MB-231 cells were observed using a plasmonic chip with an epifluorescence microscope. The targeted membrane proteins were epithelial cell adhesion molecules (EpCAMs) and epidermal growth factor receptor (EGFR), and Alexa®488-EGFR antibody and allophycocyanin (APC)-labeled EpCAM antibody were applied to the fluorescent detection. The plasmonic chip used in this study is composed of a two-dimensional hole-array structure, which is expected to enhance the fluorescence at different resonance wavelengths due to two kinds of grating pitches in a square side and a diagonal direction. As a result of multi-color imaging, the enhancement factor of Alexa®488-EGFR and APC-EpCAM was 13 ± 2 and 12 ± 2 times greater on the plasmonic chip, respectively. The excited wavelength or emission wavelength of each fluorescent agent is due to consistency with plasmon resonance wavelength in the hole-arrayed chip. The multi-color fluorescence images of breast cancer cells were improved by the hole-arrayed plasmonic chip.

Cubic POSS engineering of photosensitizer-doped semiconducting polymer nanoparticles for enhanced fluorescence imaging and amplified photodynamic therapy

PorPOSSC12 -doped semiconducting polymer nanoparticles with simultaneously enhanced fluorescence brightness and amplified singlet oxygen generation ability were designed for imaging-guided photodynamic therapy.

Abstract P3-03-40: A prospective two arm comparative study of indo-cyanine green (ICG) enhanced fluorescence imaging vs conventional methods (blue dye and radiocolloid/hand held gamma probe) for sentinel lymph node detection in breast cancer - Going beyond the horizon

Abstract Background: The dual technique with radio colloid and blue dye is the gold standard in sentinel lymph node biopsy (SLNB) to stage axilla in breast cancer. However due to cost & infrastructural demands of nuclear medicine department most of the oncology centers are not doing slnb or are doing SLNB with blue dye which is not a standard of care. Indocyanine green (ICG) has recently been used as a method of identifying sentinel lymph nodes. Studies have shown that ICG fluorescence imaging alone or in combination with the blue dye method or the radionuclide method is a safe and easy technique. The objective of the present study was to assess the diagnostic performance of sentinel lymph node (SLN) biopsy using the indocyanine green (ICG) fluorescence method compared with that using the conventional method in detection of sentinel lymph nodes. Material & Methods: 60 patients diagnosed with early breast cancer underwent the SLNB procedure using technetium-99m radio colloid (R), methylene blue dye (MB), and ICG. Fluorescence imaging was done using an indigenously designed , very economical fluorescence imaging system, Irilic.nm fluorescence imaging along with Indocyanine green. All SLNs that were removed during surgery were labelled as hot, blue or/and fluorescent and sent for pathological examination. The detection rate of SLNs and positive SLNs, and the number of SLNs of ICG, MB+ R, ICG + MB, ICG + R were compared. Injection safety of ICG and MB was evaluated. Results: Sentinel Lymph Node was identified in all 60 cases. Total Sentinel lymph nodes removed was 145 (Mean=2, Range 2-5), ICG was able to identify more nodes than the dual dye technique. The identification rate with the dual dye technique was 95%, with blue dye alone 93.6% and with radioisotope alone 96.8% whereas with ICG alone was 100%, with ICG + MB was 96.6% & ICG + R was 96.6 %. 28(46.6%) out of 60 patients had positive nodes which was identified by both dual dye & ICG. None of the patients had any local or systemic reaction with ICG, 3 patients with blue dye had tattooing & staining of skin. Conclusion: ICG is as effective as the dual dye for SLNB. ICG is safe & reliable. In addition, as a near-infrared dye, it has the advantages of real-time visualization, lower cost, and wider availability. It can be a boon for developing countries & second tier referral centers of developed country where there is limited access to nuclear medicine department & radiocolloid and even if its accesible the cost involved is too high which comes with added radiation exposure to medical personnel handling them. A combination of blue dye and ICG is useful dual approach when radioisotope is unavailable. ICG verus Conventional Dye Clinical Profile ICGRadio-colloid+ B;ue DyeDetection Rate100%95%Sensitivity100% (CI 83.16% to 100.00%)100% (CI 83.16% to 100.00%)Positive Predictive Value100%100%Accuracy100%100% Citation Format: Somashekhar S, Rohit Kumar C, Zaveri S, Rajgopal A, Rakshit S, Ali SH. A prospective two arm comparative study of indo-cyanine green (ICG) enhanced fluorescence imaging vs conventional methods (blue dye and radiocolloid/hand held gamma probe) for sentinel lymph node detection in breast cancer - Going beyond the horizon [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-03-40.

Self-assembly of Fluorescent Dehydroberberine Enhances Mitochondria-Dependent Antitumor Efficacy.

Selective imaging and inducing mitochondrial dysfunction in tumor cells using mitochondria-targeting probes has become as a promising approach for cancer diagnosis and therapy. Here, we report the design of a fluorescent berberine analog, dehydroberberine (DH-BBR), as a new mitochondria-targeting probe capable of self-assembling into monodisperse organic nanoparticles (DTNPs) upon integration with a lipophilic counter anion, allowing for enhanced fluorescence imaging and treatment of tumors in living mice. X-ray crystallography revealed that the self-assembly process was attributed to a synergy of different molecular interactions, including π-π stacking, O⋅⋅⋅π interaction and electrostatic interaction between DH-BBR and counter anions. We demonstrated that DTNPs could efficiently enter tumor tissue following intravenous injection and enhance mitochondrial delivery of DH-BBR via an electrostatic interaction driven anion exchange process. Selective accumulation in the mitochondria capable of emitting strong fluorescence and causing mitochondrial dysfunction was achieved, enabling efficient inhibition of tumor growth in living mice. This study demonstrates promise for applying lipophilic anions to control molecular self-assembly and tune antitumor activity of mitochondria-targeting probes, which can facilitate to improve cancer treatment in vivo.

Photonic crystal enhanced fluorescence immunoassay on diatom biosilica.

Fluorescence biosensing is one of the most established biosensing methods, particularly fluorescence spectroscopy and microscopy. These are two highly sensitive techniques but require high-grade electronics and optics to achieve the desired sensitivity. Efforts have been made to implement these methods using consumer grade electronics and simple optical setups for applications such as point-of-care diagnostics, but the sensitivity inherently suffers. Sensing substrates, capable of enhancing fluorescence are thus needed to achieve high sensitivity for such applications. In this paper, we demonstrate a photonic crystal-enhanced fluorescence immunoassay biosensor using diatom biosilica, which consists of silica frustules with sub-100 nm periodic pores. Utilizing the enhanced local optical field, the Purcell effect and increased surface area from the diatom photonic crystals, we create ultrasensitive immunoassay biosensors that can significantly enhance fluorescence spectroscopy as well as fluorescence imaging. Using standard antibody-antigen-labeled antibody immunoassay protocol, we experimentally achieved 100× and 10× better detection limit with fluorescence spectroscopy and fluorescence imaging respectively. The limit of detection of the mouse IgG goes down to 10-16 M (14 fg/mL) and 10-15 M (140 fg/mL) for the two respective detection modalities, virtually sensing a single mouse IgG molecule on each diatom frustule. The effectively enhanced fluorescence imaging in conjunction with the simple hot-spot counting analysis method used in this paper proves the great potential of diatom fluorescence immunoassay for point-of-care biosensing.

Monitoring individual cell-signaling activity using combined metal-clad waveguide and surface-enhanced fluorescence imaging.

Evanescent field based biosensing systems such as surface plasmon resonance (SPR), diffraction gratings, or metal-clad waveguides (MCWGs) are powerful tools for label-free real-time monitoring of signaling activity of living cells exposed to hormones, pharmacological agents, and toxins. In particular, MCWG-based imaging is well suited for studying relatively thick objects such as cells due to its greater depth of penetration into the sensing medium, compared to SPR. Label-free methods, however, provide only indirect measurements in that the measured signal arises from local changes in material properties rather than from specific biomolecular targets. In the case of cells, the situation is especially complex as the measured label-free signal may result from a combination of very diverse sources: morphological changes, intra-cellular reorganization, cascaded molecular events, protein expression etc. Consequently, deconvolving the contributions of specific sources to a particular cell response profile can be challenging. In the following, we present a cell imaging platform that combines two distinct sensing modalities, namely label-free MCWG imaging and label-based surface enhanced fluorescence (SEF), designed to facilitate the identification of the underlying molecular and structural contributions to the label-free MCWG images. We demonstrate the bimodal capabilities of this imaging platform in experiments designed to visualize actin cytoskeleton organization in vascular smooth muscle cells. We then monitored the real-time response of HEK293 cells expressing the Angiotensin 1 receptor (AT1R), when stimulated by the receptor agonist Angiotensin II (AngII). The analysis of the simultaneous label-free signal obtained by MCWG and the intracellular calcium signal resulting form AT1R activation, measured by SEF, allows relating label-free signal features to specific markers of receptor activation. Our results show that the intracellular calcium levels normally observed following AT1R activation are not required for the initial burst of cellular activity observed in the MCWG signal but rather indicates signaling activity involving the intracellular kinase ROCK.

Strong Surface Enhanced Florescence of Carbon Dot Labeled Bacteria Cells Observed with High Contrast on Gold Film.

Strong surface (metal) enhanced fluorescence (SEF or MEF) is observed from clusters and single E coli bacteria cells labeled with Carbon nanodots (CDs), which were synthesized from date pits. The enhancement factor (EF) for SEF of the cell clusters were close to 50 for both 533 and 633nm laser excitation wavelength. Those EFs are ratios of emission peak areas from CD labeled cell clusters on gold film to the peak areas of the same batch cell clusters on glass substrate. SEF with 633nm excitation performed better than SEF with 532nm excitation, achieving higher fluorescence intensity and much higher contrast. The contrast as high as 66 for cell clusters on gold film is a ratio of fluorescent emission peak area measured at the CD labeled cell clusters to the fluorescent peak area measured at unlabeled cell clusters (autofluorescence) on the same substrate. The contrast with the background (S/N) or the ratio of fluorescent peak area measured at bacteria cells to area measured at bare substrate was as high as 200. This report may pave a way for the broader application of surface enhanced fluorescence and especially metal enhanced fluorescence imaging of CD labeled cells and other biological objects. Graphical abstract Carbon dots, synthesized from dates, are used for direct staining of E coli cells. Emission fluorescent spectroscopy of those CD labelled cells on gold film and glass, demonstrated enhancement factor about 50 for emission on gold as compared to glass, Excitation at 633nm appears far superior to excitation at 532nm in terms of contrast (up to 67) with unlabeled cells /control due to decrease in auto fluorescence of cells. Maximum Signal to noise ratio is 200.

The use of ICG enhanced fluorescence for the evaluation of parathyroid gland preservation.

Indocyanine green (ICG) enhanced fluorescence imaging is recent innovation as the "real-time intraoperative imaging" technique. Many clinical studies have been reported in the literature which use different devices and techniques that employ various doses and usages of ICG as a non-specific contrast agent. Several groups have performed studies in endocrine surgery, especially with regards to parathyroid-related outcomes after thyroid and parathyroid surgery. However, there is no consensus on the technical details that should be applied. With this study, we aimed to review the current literature on potential use of intraoperative ICG angiography for evaluating parathyroid gland (PTG) preservation.

Doxorubicin and Indocyanine Green Loaded Hybrid Bicelles for Fluorescence Imaging Guided Synergetic Chemo/Photothermal Therapy.

Hybrid bicelles have been demonstrated to have great potential for hydrophobic drug delivery. Herein, we report a near-infrared light-driven, temperature-sensitive hybrid bicelles co-encapsulating hydrophobic doxorubicin (DOX) and indocyanine green (ICG) (DOX/ICG@HBs). Encapsulation of ICG into the lipid bilayer membrane of DOX/ICG@HBs results in higher photostability than free ICG. DOX/ICG@HBs exhibited temperature-regulated drug release behavior and significant photothermal cytotoxicity. After tail vein injection, such discotic nanoparticles of DOX/ICG@HBs were found to accumulate selectively at the tumor site and act as an efficient probe to enhance fluorescence imaging greatly. The in vivo experiments showed that the DOX/ICG@HBs-mediated chemo- and photothermal combination therapy was more cytotoxic to tumor cells than the photothermal treatment or the chemotherapy alone due to the synergistic effect, reducing the occurrence of tumor metastasis. Therefore, DOX/ICG@HBs can act as a powerful nanotheranostic agent for chemo/photothermal therapy of cancer under the guidance of near-infrared fluorescence imaging.

Poly (m-chloroaniline) and poly (1-naphthylamine) based conjugated polymer for enhanced fluorescence imaging in diverse cell types

Fluorescent probes, based on doped polyaniline, poly(m-chloroaniline) and poly(1-naphthylamine) have been tailored for in vitro cell imaging application. Its structure was characterized by FTIR, UV–visible, fluorescence, atomic force microscopy and BET surface area analysis. The intensity of fluorescence in poly(m-chloroaniline) and poly(1-naphthylamine) was much stronger compared to polyaniline alone. The optimal emitting properties of poly(m-chloroaniline), poly(1-naphthylamine) and polyaniline were related to both structure and shape of the particle and behave according to concentration dependent manner. The poly(m-chloroaniline) and polyaniline emits greenish blue color while poly(1-naphthylamine) exibits intense red and greenish blue color emission respectively. Poly(m-chloroaniline) and poly(1-naphthylamine) demonstrate highly enhanced signals for fluorescence based cell imaging in K-562, U2-OS and MCF-7 cells. Above compounds are cytocompatible, and with no significant loss of viability in K-562, U2-OS and MCF-7 cells following treatment. Higher concentration of the material however, causes moderate loss in viability of the cell besides inducing cytotoxicity. Luminescent micro particles of doped poly(m-cholroaniline) and poly(1-naphthylamine) showed significant potential for biological and diagnostic application.

Intracellular Messenger RNA Triggered Catalytic Hairpin Assembly for Fluorescence Imaging Guided Photothermal Therapy.

We show a theranostic nanoplatform for messenger RNA (mRNA) triggered enhanced fluorescence imaging guided therapy. Catalytic hairpin assembly (CHA) and gold nanorods (AuNRs) are employed to fabricate the theranostic nanoplatform. Two hairpin DNAs and Cy5 labeled duplex DNA are integrated into the CHA for mRNA triggered fluorescence signal amplification via hybridization and displacement with mRNA. The AuNRs act both as the fluorescence quencher and the photothermal therapy (PTT) agent. The nanoplatform not only enables sensitive and specific imaging of target mRNA in living cells and good differentiating of the survivin mRNA expression levels in different cell lines but also offers excellent photothermal conversion efficiency for PTT. The developed nanoplatform has great potential for sensitive and specific intracellular mRNA imaging guided PTT.

Cover Picture: Enhanced Fluorescence Imaging and Photodynamic Cancer Therapy Using Hollow Mesoporous Nanocontainers (Chem. Asian J. 14/2017)

Cover Picture: Enhanced Fluorescence Imaging and Photodynamic Cancer Therapy Using Hollow Mesoporous Nanocontainers (Chem. Asian J. 14/2017)

Enhanced Fluorescence Imaging and Photodynamic Cancer Therapy Using Hollow Mesoporous Nanocontainers.

Here we show that "off-on" type of photodynamic therapy agents could be developed using hollow mesoporous silica nanoparticles (HMSNPs), which can be used not only for enhancing delivery of photosensitizers to cancer cells but also for enabling switchable optical properties of the photosensitizers. Fluorescence and singlet oxygen generation of the photosensitizer-loaded HMSNP are turned off in its native state. In vitro cell studies showed that this HMSNP-based "off-on" agent may have potential utility in selective fluorescence detection and photodynamic therapy of cancers.

Enhanced fluorescence microscopy with the Bull's eye-plasmonic chip.

A Bull's eye-plasmonic chip composed of concentric circles was applied to enhanced fluorescence microscopy. Among one dimensional (1-D), 2-D, and Bull's eye periodic structures, the Bull's eye-plasmonic chip provided the most enhanced fluorescence intensity under the epi-fluorescence microscope, because incident light through the objective lens with all azimuthal angles can be effectively applied to the surface plasmon resonance- field (excitation field) and the plasmon-enhanced emission was also effectively collected. In the fluorescence observation of a single nanoparticle, the enhanced fluorescence images for a microsphere with ϕ 2 μm and a nanosphere with ϕ 200 nm were observed. For the nanospheres with ϕ 40 and 20 nm, the fluorescence image, which was undetectable on a glass slide, was observed in a spatial resolution of roughly diffraction limit on the Bull's eye-plasmonic chip. Furthermore, the use of an appropriate pinhole at the aperture stop in the incident optical system improved the fluorescence enhancement. The applicability of a Bull's eye-plasmonic chip to fluorescence imaging was demonstrated.

Visualization of Sweat Fingerprints on Various Surfaces Using a Conjugated Polyelectrolyte.

A conformation-variable conjugated polyelectrolyte responding to oppositely charged biomolecules was examined as an imaging agent for the detection of latent fingerprints (LFPs). Sulfonated poly(diphenylacetylene) (SPDPA) produces high-resolution fluorescence (FL) LFP images by simple wetting of the target objects with the polymer solution without any additional treatment. SPDPA readily interacts with LFP sweat components (especially amino acids) via electrostatic interactions, leading to significantly enhanced FL images in a "turn-on" mode. The FL emission enhancement was examined in a model reaction between SPDPA and an amino acid standard. Visualization with SPDPA is effective on various surfaces, including both rough (paper) and smooth (glass and plastic) ones. Moreover, SPDPA readily interacts with extremely thin sweat LFPs, especially on smooth glass surfaces.

(Near-Infrared) Fluorescence-Guided Surgery Under Ambient Light Conditions: A Next Step to Embedment of the Technology in Clinical Routine.

Background and Purpose In open surgery procedures, after temporarily dimming the lights in the operation theatre, the Photo Dynamic Eye (PDE) fluorescence camera has, amongst others, been used for fluorescence-guided sentinel node (SN) biopsy procedures. To improve the clinical utility and logistics of fluorescence-guided surgery, we developed and evaluated a prototype modified PDE (m-PDE) fluorescence camera system.MethodsThe m-PDE works under ambient light conditions and includes a white light mode and a pseudo-green-colored fluorescence mode (including a gray-scaled anatomical background). Twenty-seven patients scheduled for SN biopsy for (head and neck) melanoma (n = 16), oral cavity (n = 6), or penile (n = 5) cancer were included. The number and location of SNs were determined following an indocyanine green-99mTc-nanocolloid injection and preoperative imaging. Intraoperatively, fluorescence guidance was used to visualize the SNs. The m-PDE and conventional PDE were compared head-to-head in a phantom study, and in seven patients. In the remaining 20 patients, only the m-PDE was evaluated.ResultsPhantom study: The m-PDE was superior over the conventional PDE, with a detection sensitivity of 1.20 × 10−11 M (vs. 3.08 × 10−9 M) ICG in human serum albumin. In the head-to-head clinical comparison (n = 7), the m-PDE was also superior: (i) SN visualization: 100 versus 81.4 %; (ii) transcutaneous SN visualization: 40.7 versus 22.2 %; and (iii) lymphatic duct visualization: 7.4 versus 0 %. Findings were further underlined in the 20 additionally included patients.ConclusionThe m-PDE enhanced fluorescence imaging properties compared with its predecessor, and provides a next step towards routine integration of real-time fluorescence guidance in open surgery.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-016-5186-3) contains supplementary material, which is available to authorized users.

Prussian Blue Modified PLA Microcapsules Containing R6G for Ultrasonic/Fluorescent Bimodal Imaging Guided Photothermal Tumor Therapy.

A theranostic agent has been successfully constructed for fluorescence/ultrasound dual-modal imaging guided photothermal therapy by loading the fluorescent dye R6G into polylactide microcapsules (PLA MCs) followed by deposition of Prussian blue nanoparticles (PB NPs) into the surface of PLA MCs. It was proved that the obtained microcapsules of R6G@PLA/PB MCs could serve as an efficient probe to simultaneously enhance fluorescence imaging and ultrasound imaging greatly in vivo. R6G@PLA/PB MCs exhibited significant photothermal cytotoxicity. Cancer cells could be killed efficiently through photothermal effects of R6G@PLA/PB MCs due to the strong absorption of PB NPs in the near infrared region under laser irradiation. In a word, R6G@PLA/PB MCs integrate multiple capabilities for effective tumor imaging and therapy. Such a single agent provides us a possibility to interpret accurately the obtained images, identify the size and location of the tumor, as well as guide and monitor the photothermal therapy.

Hyaluronic Acid Modified Tantalum Oxide Nanoparticles Conjugating Doxorubicin for Targeted Cancer Theranostics.

Theranostic tantalum oxide nanoparticles (TaOxNPs) of about 40 nm were successfully developed by conjugating functional molecules including polyethylene glycol (PEG), near-infrared (NIR) fluorescent dye, doxorubicin (DOX), and hyaluronic acid (HA) onto the surface of the nanoparticles (TaOx@Cy7-DOX-PEG-HA NPs) for actively targeting delivery, pH-responsive drug release, and NIR fluorescence/X-ray CT bimodal imaging. The obtained nanoagent exhibits good biocompatibility, high cumulative release rate in the acidic microenvironments, long blood circulation time, and superior tumor-targeting ability. Both in vitro and in vivo experiments show that it can serve as an excellent contrast agent to simultaneously enhance fluorescence imaging and CT imaging greatly. Most importantly, such a nanoagent could enhance the therapeutic efficacy of the tumor greatly and the tumor growth inhibition was evaluated to be 87.5%. In a word, multifunctional TaOx@Cy7-DOX-PEG-HA NPs can serve as a theranostic nanomedicine for fluorescence/X-ray CT bimodal imaging, remote-controlled therapeutics, enabling personalized detection, and treatment of cancer with high efficacy.

Enhanced fluorescence imaging guided photodynamic therapy of sinoporphyrin sodium loaded graphene oxide

Extensive research indicates that graphene oxide (GO) can effectively deliver photosensitives (PSs) by π–π stacking for photodynamic therapy (PDT). However, due to the tight complexes of GO and PSs, the fluorescence of PSs are often drastically quenched via an energy/charge transfer process, which limits GO-PS systems for photodiagnostics especially in fluorescence imaging. To solve this problem, we herein strategically designed and prepared a novel photo-theranostic agent based on sinoporphyrin sodium (DVDMS) loaded PEGylated GO (GO-PEG-DVDMS) with improved fluorescence property for enhanced optical imaging guided PDT. The fluorescence of loaded DVDMS is drastically enhanced via intramolecular charge transfer. Meanwhile, the GO-PEG vehicles can significantly increase the tumor accumulation efficiency of DVDMS and lead to an improved PDT efficacy as compared to DVDMS alone. The cancer theranostic capability of the as-prepared GO-PEG-DVDMS was carefully investigated both in vitro and in vivo. Most intriguingly, 100% in vivo tumor elimination was achieved by intravenous injection of GO-PEG-DVDMS (2 mg/kg of DVDMS, 50 J) without tumor recurrence, loss of body weight or other noticeable toxicity. This novel GO-PEG-DVDMS theranostics is well suited for enhanced fluorescence imaging guided PDT.

Enhanced fluorescence imaging performance of hydrophobic colloidal ZnO nanoparticles by a facile method

A facile synthesis method for the formation of ZnO nanoparticles by using a double-phase reaction was demonstrated in this paper. The morphology of the synthesized ZnO nanoparticles shows a flower-shape. Hydrogen peroxide was used as a unique oxygenic source to promote the formation of ZnO in the presence of organic zinc precursor. The as-synthesized ZnO nanoparticles also show a stability and solubility in the aqueous environment. The structure and properties of ZnO nanoparticles were investigated by the transmission electron microscopy (TEM) and X-ray diffraction (XRD) as well as UV–vis and photoluminescence spectroscopy. The as-prepared hydrophobic colloidal ZnO nanoparticles could be modified to become water-soluble via ligand exchange with amineothanethiol⋅HCl while retaining the photoluminescence properties. In addition, the potential application for biological label of water-soluble ZnO nanoparticles were also demonstrated. These results not only have applications towards using colloidal ZnO nanoparticles effectively in biological fluorescence imaging, but also promote its application in the field of targeted drug delivery.