Fluorescence Imaging Contrast Research Articles

Dye-less quantification of tissue perfusion by laser speckle contrast imaging is equivalent to quantified indocyanine green in a porcine model.

Subjective surgeon interpretation of near-infrared perfusion video is limited by low inter-observer agreement and poor correlation to clinical outcomes. In contrast, quantification of indocyanine green fluorescence video (Q-ICG) correlates with histologic level of perfusion as well as clinical outcomes. Measuring dye volume over time, however, has limitations, such as it is not on-demand, has poor spatial resolution, and is not easily repeatable. Laser speckle contrast imaging quantification (Q-LSCI) is a real-time, dye-free alternative, but further validation is needed. We hypothesize that Q-LSCI will distinguish ischemic tissue and correlate over a range of perfusion levels equivalent to Q-ICG. Nine sections of intestine in three swine were devascularized. Pairs of indocyanine green fluorescence imaging and laser speckle contrast imaging video were quantified within perfused, watershed, and ischemic regions. Q-ICG used normalized peak inflow slope. Q-LSCI methods were laser speckle perfusion units (LSPU), the base unit of laser speckle imaging, relative perfusion units (RPU), a previously described methodology which utilizes an internal control, and zero-lag normalized cross-correlation (X-Corr), to investigate if the signal deviations convey accurate perfusion information. We determine the ability to distinguish ischemic regions and correlation to Q-ICG over a perfusion gradient. All modalities distinguished ischemic from perfused regions of interest; Q-ICG values of 0.028 and 0.155 (p < 0.001); RPU values of 0.15 and 0.68 (p < 0.001); and X-corr values of 0.73 and 0.24 (p < 0.001). Over a range of perfusion levels, RPU had the best correlation with Q-ICG (r = 0.79, p < 0.001) compared with LSPU (r = 0.74, p < 0.001) and X-Corr (r = 0.46, p < 0.001). These results demonstrate that Q-LSCI discriminates ischemic from perfused tissue and represents similar perfusion information over a broad range of perfusion levels comparable to clinically validated Q-ICG. This suggests that Q-LSCI might offer clinically predictive real-time dye-free quantification of tissue perfusion. Further work should include validation in histologic studies and human clinical trials.

Fibronectin-Targeting Dual-Modal MR/NIRF Imaging Contrast Agents for Diagnosis of Gastric Cancer and Peritoneal Metastasis.

The prevalence and fatality rates of gastric cancer (GC) remain elevated, with advanced stages presenting a grim prognosis. Noninvasive diagnosis of GC cancer often proves challenging until the disease has progressed to an advanced stage or metastasized. Initially, the level of fibronectin (FN) in cancer-associated fibroblasts (CAFs) of GC was at least 3.7 times higher than that in normal fibroblasts. Herein, two FN-targeting magnetic resonance/near-infrared fluorescence (MR/NIRF) imaging contrast agents were developed to detect GC and peritoneal metastasis noninvasively. The probes CREKA-Cy7-(Gd-DOTA) and CREKA-Cy7-(Gd-DOTA)3 demonstrated significant FN-targeting capability (with dissociation constants of 1.0 and 2.1 mM) and effective MR imaging performance (with proton relaxivity values of 9.66 and 27.44 mM-1 s-1 at 9.4 T, 37 °C). In vivo imaging revealed a high signal-to-noise ratio and successful visualization of GC metastasis using NIRF imaging as well as successful tumor detection in MR imaging. Therefore, this study highlights the potential of FN-targeting probes for GC diagnosis and aids in the advancement of new diagnostic strategies for the clinical detection of GC.

Enhancing resolution and contrast in fibre bundle-based fluorescence microscopy using generative adversarial network.

Fibre bundle (FB)-based endoscopes are indispensable in biology and medical science due to their minimally invasive nature. However, resolution and contrast for fluorescence imaging are limited due to characteristic features of the FBs, such as low numerical aperture (NA) and individual fibre core sizes. In this study, we improved the resolution and contrast of sample fluorescence images acquired using in-house fabricated high-NA FBs by utilising generative adversarial networks (GANs). In order to train our deep learning model, we built an FB-based multifocal structured illumination microscope (MSIM) based on a digital micromirror device (DMD) which improves the resolution and the contrast substantially compared to basic FB-based fluorescence microscopes. After network training, the GAN model, employing image-to-image translation techniques, effectively transformed wide-field images into high-resolution MSIM images without the need for any additional optical hardware. The results demonstrated that GAN-generated outputs significantly enhanced both contrast and resolution compared to the original wide-field images. These findings highlight the potential of GAN-based models trained using MSIM data to enhance resolution and contrast in wide-field imaging for fibre bundle-based fluorescence microscopy. Lay Description: Fibre bundle (FB) endoscopes are essential in biology and medicine but suffer from limited resolution and contrast for fluorescence imaging. Here we improved these limitations using high-NA FBs and generative adversarial networks (GANs). We trained a GAN model with data from an FB-based multifocal structured illumination microscope (MSIM) to enhance resolution and contrast without additional optical hardware. Results showed significant enhancement in contrast and resolution, showcasing the potential of GAN-based models for fibre bundle-based fluorescence microscopy.

MnO2/Ce6 microbubble-mediated hypoxia modulation for enhancing sono-photodynamic therapy against triple negative breast cancer.

Sono-photodynamic therapy (SPDT) has emerged as a promising treatment modality for triple negative breast cancer (TNBC). However, the hypoxic tumor microenvironment hinders the application of SPDT. Herein, in this study, a multifunctional platform (MnO2/Ce6@MBs) was designed to address this issue. A sono-photosensitizer (Ce6) and a hypoxia modulator (MnO2) were loaded into microbubbles and precisely released within tumor tissues under ultrasound irradiation. MnO2in situ reacted with the excess H2O2 and H+ and produced O2 within the TNBC tumor, which alleviated hypoxia and augmented SPDT by increasing ROS generation. Meanwhile, the reaction product Mn2+ was able to achieve T1-weighted MRI for enhanced tumor imaging. Additionally, Ce6 and microbubbles served as a fluorescence imaging contrast agent and a contrast-enhanced ultrasound imaging agent, respectively. In in vivo anti-tumor studies, under the FL/US/MR imaging guidance, MnO2/Ce6@MBs combined with SPDT significantly reversed tumor hypoxia and inhibited tumor growth in 4T1-tumor bearing mice. This work presents a theragnostic system for reversing tumor hypoxia and enhancing TNBC treatment.

Near-Infrared Fluorescence Imaging Contrast Agents for Clinical Research: Limitations and Alternatives

Near-Infrared Fluorescence Imaging Contrast Agents for Clinical Research: Limitations and Alternatives

DeepCLEM: automated registration for correlative light and electron microscopy using deep learning

In correlative light and electron microscopy (CLEM), the fluorescent images must be registered to the EM images with high precision. Due to the different contrast of EM and fluorescence images, automated correlation-based alignment is not directly possible, and registration is often done by hand using a fluorescent stain, or semi-automatically with fiducial markers. We introduce “DeepCLEM”, a fully automated CLEM registration workflow. A convolutional neural network predicts the fluorescent signal from the EM images, which is then automatically registered to the experimentally measured chromatin signal from the sample using correlation-based alignment. The complete workflow is available as a Fiji plugin and could in principle be adapted for other imaging modalities as well as for 3D stacks.

High-Throughput, Low Background, and Wide-Field Microscopy by Flat-Field Photobleaching Imprinting Microscopy.

Wide-field photobleaching imprinting microscopy (PIM) can improve fluorescence image contrast by cleverly exploiting the fluorophores' photobleaching properties. However, as conventional wide-field PIM commonly adopts Gaussian illumination with a nonuniform lateral fluence distribution, the field-of-view (FOV) and sampling density are largely reduced. In addition, the slow axial fluence gradient of Gaussian illumination limits the signal-to-background ratio (SBR) improvement and optical sectioning capability of PIM. Here, we present flat-field photobleaching imprinting microscopy (ffPIM) with a uniform lateral excitation fluence and sharp axial intensity gradient at the focal plane. ffPIM demonstrates low background, large FOV, and thin optical section. More importantly, compared to either conventional wide-field PIM or light-sheet microscopy, ffPIM shows much better balance for FOV, sampling density, SBR, and optical sectioning capability. The performance of ffPIM is characterized by simulation and resolving multiple cellular structures. Finally, ffPIM can be easily implemented to a standard commercial wide-field microscope and, thereby, allow general laboratories to benefit from this technique.

Indocyanine green based fluorescence imaging improved by deep learning.

Intraoperative identification of malignancies using indocyanine green (ICG) based fluorescence imaging could provide real-time guidance for surgeons.Existing ICG-based fluorescence imaging mostly operates in the NIR-I (700-1,000 nm) or the NIR-IIa' windows (1,000-1,300 nm), which not optimal in terms of spatial resolution and contrast since their light scattering is higher than the NIR-IIb window (1,500-1,700 nm). It is highly desired to achieve ICG-based fluorescence imaging in the NIR-IIb window, but it is hindered by its ultra-low NIR-IIb emission tail of ICG. Herein we employ a generative adversarial network (GAN) to generate NIR-IIb ICG images directly from the acquired NIR-I ICG images. This approach was investigated by in vivo imaging of sub-surface vascular, intestine structure, and tumors, and their results demonstrated significant improvement in spatial resolution and contrast for ICG-based fluorescence imaging. It is potential for deep learning to improve ICG-based fluorescence imaging in clinical diagnostics and image-guided surgery in clinics. This article is protected by copyright. All rights reserved.

An efficient reactive oxygen species/reactive nitrogen species generator for dual imaging-guided orthotopic glioblastoma therapy through intrathecal delivery

The treatment of glioblastoma is still an enormous challenge in the clinic, and treatment based on reactive oxygen species (ROS) has exhibited good potential for its therapy. However, the weak toxicity and short lifetime (e.g., 1O2 10−6 s,·OH 10−2 s) as great obstacles of the ROS-based antitumor strategy, as well as the blood-brain barrier (BBB) obstruction of most drugs into the brain, have immensely constrained the therapeutic effect. Herein, we construct a novel theranostic nanoagent (GLIF) based on Gd2(WO4)3:Nd3+ nanoparticles loaded with ploy-L-arginine (PLA), indocyanine green (ICG), as well as lactoferrin (LF) to enable efficient low-dose drug therapy via ROS and reactive nitrogen species (RNS) using intrathecal injection. The PLA and ICG loading not only enhances GLIF’s ability to generate NO, inhibit cell migration and support ROS/RNS to kill tumors, but also produces ONOO- with greater toxicity and longer lifetime (10−2 s) than 1O2. Moreover, intrathecal injection is adopted to bypass the BBB and the modification of LF promotes accumulation in lesions, causing tremendous side effects with long blood circulation. The significant therapeutic effect of GLIF has been proved in vitro and in vivo and confirmed for the first time in glioblastoma. The excellent performances of GLIF as a second near-infrared fluorescence (NIR-II) and magnetic resonance (MR) imaging contrast agent for glioblastoma diagnosis at a lower injection dose also prove the superiority of intrathecal injection over intravenous injection. These findings show that GLIF has great potential for dual-modal imaging and effective therapy of glioblastoma by combining intrathecal injection and the new pathways of ROS/RNS.

Turmeric-derived gadolinium-doped carbon quantum dots for multifunctional fluorescence imaging and MRI contrast agent

Turmeric-derived gadolinium-doped carbon quantum dots for multifunctional fluorescence imaging and MRI contrast agent

Tumor-Targeted Polydopamine-Based Nanoparticles for Multimodal Mapping Following Photothermal Therapy of Metastatic Lymph Nodes.

PurposeLymphadenectomy with lymph node (LN) mapping is essential for surgical removal of solid tumors. Existing agents do not provide accurate multimodal mapping and antitumor therapy for metastatic LNs; therefore, we fabricated a polydopamine (PDA) nanoparticle (NP)-based tumor-targeted LN mapping agent capable of multimodal mapping and guided photothermal therapy (PTT) for metastatic LNs.Materials and MethodsPDA NPs modified with polyethylene glycol (PEG) were obtained by polymerization under alkaline conditions. The PEG-PDA NPs were loaded with the circular tripeptide Arg-Gly-Asp (cRGD) to achieve tumor-targeting capacity and with the fluorescent dye IR820 and magnetic resonance imaging (MRI) contrast Gd(NH2)2 for in situ detection. The resulting cRGD-PEG-PDA@IR820/Gd(NH2)2 (cRGD-PPIG) NPs were tested for their biosafety and metastatic LN mapping ability. They were drained specifically into LNs and selectively taken up by gastric MKN45 cells via αvβ3 integrin-mediated endocytosis.ResultsThis phenomenon enabled MR/optical/near-infrared fluorescence multimodal metastatic LN mapping, guiding the creation of accurate and highly efficient PTT for gastric cancer metastatic LNs in mice.ConclusionIn summary, we fabricated tumor-targeted cRGD-PPIG NPs with MR/optical/near-infrared fluorescence multimodal metastatic LN mapping capacity for surgery and efficient PTT guidance post-surgery.

Hollow Mesoporous CeO2-Based Nanoenzymes Fabrication for Effective Synergistic Eradication of Malignant Breast Cancer via Photothermal-Chemodynamic Therapy.

CeO2-based nanoenzymes present a very promising paradigm in cancerous therapy, as H2O2 can be effectively decomposed under the electron transmit between Ce3+ and Ce4+. However, the limitations of endogenous H2O2 and intracellular low Fenton-like reaction rate lead to single unsatisfied chemodynamic therapy (CDT) efficacy. Other therapeutic modalities combined with chemodynamic therapy are generally used to enhance the tumor eradiation efficacy. Here, we have synthesized a novel hollow pH-sensitive CeO2 nanoenzyme after a cavity is loaded with indocyanine green (ICG), as well as with surface modification of tumor targeting peptides, Arg-Gly-Asp (denoted as HCeO2@ICG-RGD), to successfully target tumor cells via αvβ3 recognition. Importantly, in comparison with single chemodynamic therapy, a large amount of reactive oxygen species in cytoplasm were induced by enhanced chemodynamic therapy with photothermal therapy (PTT). Furthermore, tumor cells were efficiently killed by a combination of photothermal and chemodynamic therapy, revealing that synergistic therapy was successfully constructed. This is mainly due to the precise delivery of ICG and release after HCeO2 decomposition in cytoplasm, in which effective hyperthermia generation was found under 808 nm laser irradiation. Meanwhile, our HCeO2@ICG-RGD can act as a fluorescent imaging contrast agent for an evaluation of tumor tissue targeting capability in vivo. Finally, we found that almost all tumors in HCeO2@ICG-RGD+laser groups were completely eradicated in breast cancer bearing mice, further proving the effective synergistic effect in vivo. Therefore, our novel CeO2-based PTT agents provide a proof-of-concept argumentation of tumor-precise multi-mode therapies in preclinical applications.

DeepCLEM: automated registration for correlative light and electron microscopy using deep learning.

In correlative light and electron microscopy (CLEM), the fluorescent images must be registered to the EM images with high precision. Due to the different contrast of EM and fluorescence images, automated correlation-based alignment is not directly possible, and registration is often done by hand using a fluorescent stain, or semi-automatically with fiducial markers. We introduce "DeepCLEM", a fully automated CLEM registration workflow. A convolutional neural network predicts the fluorescent signal from the EM images, which is then automatically registered to the experimentally measured chromatin signal from the sample using correlation-based alignment. The complete workflow is available as a Fiji plugin and could in principle be adapted for other imaging modalities as well as for 3D stacks.

A Fibrinogen-Mimicking, Activated-Platelet-Sensitive Nanocoacervate Enhances Thrombus Targeting and Penetration of Tissue Plasminogen Activator for Effective Thrombolytic Therapy.

The development of a fibrinolytic system with long circulation time, high thrombus targeting, efficient thrombus penetration, effective thrombolysis, and minimal hemorrhagic risk remains a major challenge. Herein, inspired by fibrinogen binding to activated platelets in thrombosis, this article reports a fibrinogen-mimicking, activated-platelet-sensitive nanocoacervate to enhance thrombus penetration of tissue plasminogen activator (tPA) for targeted thrombolytic therapy. This biomimetic nanothrombolytic system, denoted as RGD-Chi@tPA, is constructed by "one-pot" coacervation through electrostatic interactions between positively charged arginine-glycine-aspartic acid (RGD)-grafted chitosan (RGD-Chi) and negatively charged tPA. Flow cytometry and confocal laser scanning microscopy measurements show targeting of RGD-Chi@tPA to activated platelets. Controlled tPA release triggered by activated platelets at a thrombus site is demonstrated. Its targeted fibrinolytic and thrombolytic activities are measured in in vitro models. The pharmacokinetic profiles show that RGD-Chi@tPA can significantly prolong circulation time compared to free tPA. In a mouse tail thrombus model, RGD-Chi@tPA displays efficient thrombus targeting and penetration, enabling a complete vascular recanalization as confirmed by the fluorescence imaging, histochemical assay, and laser speckle contrast imager. Consequently, RGD-Chi@tPA induces a substantial enhancement in thrombolysis with minimal hemorrhagic risk compared to free tPA. This simple, effective, and safe platform holds great promise for the development of thrombolytic nanomedicines.

Highly stable aqueous phase black phosphorus quantum dots with enhanced fluorescence property

Black phosphorus quantum dots (BPQDs) are a kind of outstanding optical material due to the tunable band structure. However, their fluorescence property and stability are obviously weakened in the aqueous phase, which extremely restricts the development of BPQDs. Here, we propose a new method to prepare stable BPQDs in an aqueous solution directly with high absolute fluorescence quantum yield. Aqueous phase BPQDs are synthesized by liquid exfoliation and hydrothermal with NH2-polyethylene glycol (PEG)-NH2 as the modification agent to protect the BPQDs, which have high stability more than six months. In addition, NH2-PEG-NH2 is also a surface passivation agent to enhance the emission of BPQDs by forming P–N bonds, which is confirmed by an absolute fluorescent quantum yield of 11.5%. Moreover, BPQDs show excellent resistance to a strong acid environment and high ionic strengths except for Fe3+. Therefore, the BPQDs are a kind of highly selective and linear response fluorescence probe for Fe3+. Considering the good biocompatibility of BPQDs, they are employed as cell fluorescent label probes and show excellent fluorescence imaging contrast. Based on the unique structural stability and fluorescence performance in the aqueous phase, BPQDs are potential candidates for Fe3+ detection and optical bio-imaging.

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Programmable illumination smartphone microscopy (PISM): A multimodal imaging platform for biomedical applications

Microscopes on the smartphone platform are handy for a wide range of applications. We report herein a programmable illumination smartphone microscopy (PISM) as a flexible multimodal imaging platform for contrast enhancement of various samples using different low-cost consumer optoelectronics parts. A miniature (0.96 inches), programmable organic light-emitting diode (OLED) display has been used as an optical source to develop the proposed PISM system. By displaying different color and binary patterns on the OLED display, six well-established imaging modes, namely bright-field (BF), dark-field (DF), oblique illumination (OI), Fluorescence imaging (FI), Rheinberg illumination (RI), and differential phase contrast (DPC) imaging have been demonstrated on a single optical setup. Furthermore, by incorporating additional optical components such as an optical polarizer into the setup, another imaging mode-polarized imaging has been realized with the proposed PISM system. The optical resolution of the designed platform is found to be 1.7 µm and delivers an acceptably resolvable image in all modalities. The versatility of the PISM has been demonstrated through imaging several specimens that include micro-beads, human epithelial check cells (HECC), starch, fungus specimen, nerve tissue cells, and red blood cells.

Mitochondria-targeted nanoplatforms for enhanced photodynamic therapy against hypoxia tumor

BackgroundPhotodynamic therapy (PDT) is a promising therapeutic modality that can convert oxygen into cytotoxic reactive oxygen species (ROS) via photosensitizers to halt tumor growth. However, hypoxia and the unsatisfactory accumulation of photosensitizers in tumors severely diminish the therapeutic effect of PDT. In this study, a multistage nanoplatform is demonstrated to overcome these limitations by encapsulating photosensitizer IR780 and oxygen regulator 3-bromopyruvate (3BP) in poly (lactic-co-glycolic acid) (PLGA) nanocarriers.ResultsThe as-synthesized nanoplatforms penetrated deeply into the interior region of tumors and preferentially remained in mitochondria due to the intrinsic characteristics of IR780. Meanwhile, 3BP could efficiently suppress oxygen consumption of tumor cells by inhibiting mitochondrial respiratory chain to further improve the generation of ROS. Furthermore, 3BP could abolish the excessive glycolytic capacity of tumor cells and lead to the collapse of ATP production, rendering tumor cells more susceptible to PDT. Successful tumor inhibition in animal models confirmed the therapeutic precision and efficiency. In addition, these nanoplatforms could act as fluorescence (FL) and photoacoustic (PA) imaging contrast agents, effectuating imaging-guided cancer treatment.ConclusionsThis study provides an ideal strategy for cancer therapy by concurrent oxygen consumption reduction, oxygen-augmented PDT, energy supply reduction, mitochondria-targeted/deep-penetrated nanoplatforms and PA/FL dual-modal imaging guidance/monitoring. It is expected that such strategy will provide a promising alternative to maximize the performance of PDT in preclinical/clinical cancer treatment.Graphical

Preparation Fe3O4@chitosan-graphene quantum dots nanocomposites for fluorescence and magnetic resonance imaging

The recent development of nanoprobes with multiple biomolecular detection functionalities have attracted considerable interests in various biomedical imaging applications. Herein, we have designed and prepared Fe3O4@chitosan-graphene quantum dots (Fe3O4@CS-GQDs) nanocomposites for fluorescence and magnetic resonance imaging (MRI). The photoluminescent spectra of Fe3O4@CS-GQDs nanocomposites exhibit an emission peak at 434 nm under the ultraviolet excitation light of 330 nm. Fe3O4@CS-GQD nanocomposites show the excellent MRI results in aqueous solution. The cytotoxicity of Fe3O4@CS-GQDs nanocomposites is observed low cytotoxicity in Hela cells. Based on the results, Fe3O4@CS-GQDs nanocomposites are confirmed as promising fluorescence and MRI imaging contrast agents for tumor detection.

Use of zinc to increase fluorescence in an in vitro biofilm model as a tool for caries diagnosis

Artificial sound enamel and caries enamel lesions were prepared. The sound enamel did not emit fluorescence in the visible spectrum. The spectrum of Zn(II)-protoporphyrin-9 biofilm from caries enamel lesions showed the fluorescence emitted by Zn(II)-protoporphyrin-9, with two main peaks at 630 and 690 nm. The luminescence properties of protoporphyrin-9 change depended on the amount of Zn(II). The increase in fluorescence intensity as Zn(II)-protoporphyrin-9 penetrated deeper to 1.75 mm was appropriate for the diagnosis of caries enamel lesions. Fluorescence intensity was maximum when Zn(II) reached 0.0256 µM and significantly produced a high contrast of fluorescence image together with high fluorescence quantum efficiency and photostability.