Fluorescence Imaging Applications Research Articles

Self-assembled DNA nanoparticles enable cascade circuits for mRNA detection and imaging in living cells

Fluorescence molecular probes have been regarded as a valuable tool for RNA detection and imaging. However, the pivotal challenge is how to develop an efficient fluorescence imaging platform for accurate identification of RNA molecules with low expression in complicated physiological environments. Herein, we construct the DNA nanoparticles to glutathione (GSH)-responsive controllable release of hairpin reactants for catalytic hairpin assembly (CHA)-hybridization chain reaction (HCR) cascade circuits, which enables the analysis and imaging of low-abundance target mRNA in living cells. The aptamer-tethered DNA nanoparticles are constructed via the self-assembly of single-stranded DNAs (ssDNAs), exhibiting sufficient stability, cell-specific penetration, and precise controllability. Moreover, the in-depth integration of different DNA cascade circuits shows the improved sensing performance of DNA nanoparticles in live cell analysis. Therefore, through the combination of multi-amplifiers and programmable DNA nanostructure, the developed strategy enables accurately triggered release of hairpin reactants and further achieves sensitive imaging and quantitative evaluation of survivin mRNA in carcinoma cells, which provides a potential platform to facilitate RNA fluorescence imaging applications in early clinical cancer theranostics.

The Clinical Application and Future Perspective of Fluorescence Imaging in the Treatment of Lung Cancer

The Clinical Application and Future Perspective of Fluorescence Imaging in the Treatment of Lung Cancer

Application and Future Prospects of Intraoperative Fluorescence Imaging in The Treatment of Gastrointestinal Cancer

消化管癌治療における蛍光イメージングには，indocyanine green蛍光を用いた血流評価，センチネルノード（SN）の検出や，近赤外蛍光クリップ，腫瘍の蛍光ラベル等がある．血流評価は左側大腸での評価が進みつつある．SNは胃癌において時間をかけながらポジティブなな結果が積み重なってきている．さらに，近赤外蛍光樹脂を用いたクリップなどのデバイス開発や腫瘍を蛍光ラベルする試みが具体化しつつある．

Synthesis, Optical Properties, and Fluorescence Cell Imaging of Novel Mixed Fluorinated Subphthalocyanines.

Subphthalocyanines (SubPcs) are a kind of tripyrrolic macrocycle with a boron atom at their core. Incorporating different units onto the SubPc periphery can endow them with various unique properties. Herein, a series of novel fluorinated low-symmetry SubPc derivatives containing chlorine groups (F8-Cl4-SubPc, F4-Cl8-SubPc) and methoxy groups (F8-(OCH3)2-SubPc) were synthesized and characterized by spectral methods (MS, FT-IR, 1H, 13C, 11B, and 19F NMR spectroscopy), and the effect of the peripheral substituents on their electronic structure of low-symmetry macrocycle was investigated by cyclic voltammetry, theoretical calculation, electronic absorption, and emission spectroscopy. In contrast to perfluorinated SubPcs, these low-symmetry SubPcs revealed non-degenerate LUMO and LUMO + 1 orbitals, especially F8-(OCH3)2-SubPc, which was consistent with the split Q-band absorptions. The cyclic voltammetry revealed that these SubPcs exhibited two or three reduction waves and one oxidation wave, which is consistent with the reported SubPcs. Finally, an intracellular fluorescence imaging study of these compounds revealed that these compounds could enter cancer cells and be entrapped in the lysosomes, which provides a possibility of future applications in lysosome fluorescence imaging and targeting.

Self-filtering illumination source and application in fluorescence imaging

To date, fluorescence imaging systems have all relied on at least one beam splitter (BS) to ensure the separation of excitation light and fluorescence. Here, we reported SiO2/TiO2 multi-layer long pass filter integrated GaN LED. It is considered as the potential source for imaging systems. Experimental results indicate that the GaN LED shows blue emission peaked at 470.3 nm and can be used to excite dye materials. Integrating with a long pass filter (550 nm), the light source can be used to establish a real-time fluorescence detection for dyes that emit light above 550 nm. More interestingly, with this source, a real-time imaging system with signature words written with the dyes, such as ‘N J U P T’, can be converted into CCD images. This work may lead to a new strategy for integrating light sources and BS mirrors to build mini and smart fluorescence imaging systems.

A borate-based peroxynitrite fluorescent probe and its application in fluorescence imaging of living cells.

As a bioactive species with high oxidation capacity, peroxynitrite (ONOO-) plays a crucial role in the regulation of diverse pathophysiological processes, and the overproduction of ONOO- is closely associated with various physiological diseases such as liver injury, pulmonary fibrosis and so on. Herein, two borate-based fluorescent probes 3a and 3b were synthesized for monitoring ONOO- by a simple substitution reaction. The experimental results showed that 3a and 3b had high selectivity and sensitivity for ONOO-. The detection limits of 3a and 3b were 79.46 nM and 32.12 nM, respectively. Moreover, the recognition was not disturbed by other active oxygen groups and common ions. More importantly, the probes 3a and 3b had low cytotoxicity and were successfully used to detect endogenous and exogenous ONOO-. They would provide an efficient detection method for further exploring the physiological and pathological role of ONOO- in complex biological systems and related diseases.

Modification of a SOCT-ISC type triphenylamine-BODIPY photosensitizer by a multipolar dendrimer design for photodynamic therapy and two-photon fluorescence imaging.

In this study, a series of multipolar triphenylamine-BODIPY photosensitizers T-BDPn (n = 1, 2, 3) was synthesized. Compared with T-BDP1 of D-A configuration, the multipolar T-BDP3 dendrimer have higher singlet oxygen efficiency (44%), better fluorescence quantum yield (7.45%), and could be used in the simulated photodynamic therapy in A-549 cells and two-photon fluorescence imaging in zebrafish. The theoretical calculation and fs-transient absorption spectra indicated that the reason of its higher singlet oxygen efficiency was that the multipolar T-BDP3 dendrimer could generate more nearly degenerate charge transfer (CT) states and triplet states, which could further increase the possibility of spin-orbit charge-transfer intersystem crossing (SOCT-ISC) process. In the simulated photodynamic therapy of A-549 cells, T-BDP3 shows good cytocompatibility, great phototoxicity with its IC50 value of 3.17 μM, and could kill cancer cells effectively with the dosage of 5 μM under 10 min irradiation in the AO/EB double-staining experiment. In the fluorescence imaging of zebrafish, the experiment results indicate that T-BDP3 could generate superoxide radical (O2˙-) in the body of zebrafish and could be applied to the two-photon fluorescence imaging under 800 nm excitation. The above experiment results shown that the multipolar dendrimer design was an effective approach to improve the key parameters of SOCT-ISC-type BODIPY photosensitizer and was ready for further two-photon photodynamic therapy in organisms.

A new dual functional H2S donor for fluorescence imaging and anti-inflammatory application.

This study explored FL-H2S, a novel fluorescein-based H2S donor, as an anti-inflammatory agent. The results demonstrated the efficient release of H2S by FL-H2S, along with its biocompatibility, real-time intracellular H2S release and imaging capability. In vivo experiments using a rat model confirmed the anti-inflammatory effects of FL-H2S, evidenced by reduced foot swelling. We also successfully elucidated the anti-inflammatory mechanism through ELISA and WB analysis.

Application of near-infrared fluorescence imaging in theranostics of gastrointestinal tumors.

Gastrointestinal cancers have become an important cause of cancer-related death in humans. Improving the early diagnosis rate of gastrointestinal tumors and improving the effect of surgical treatment can significantly improve the survival rate of patients. The conventional diagnostic method is high-definition white-light endoscopy, which often leads to missed diagnosis. For surgical treatment, intraoperative tumor localization and post-operative anastomotic state evaluation play important roles in the effect of surgical treatment. As a new imaging method, near-infrared fluorescence imaging (NIRFI) has its unique advantages in the diagnosis and auxiliary surgical treatment of gastrointestinal tumors due to its high sensitivity and the ability to image deep tissues. In this review, we focus on the latest advances of NIRFI technology applied in early diagnosis of gastrointestinal tumors, identification of tumor margins, identification of lymph nodes, and assessment of anastomotic leakage. In addition, we summarize the advances of NIRFI systems such as macro imaging and micro imaging systems, and also clearly describe the application process of NIRFI from system to clinical application, and look into the prospect of NIRFI applied in the theranostics of gastrointestinal tumors.

Application value of indocyanine green fluorescence in sentinel lymph node biopsy for early-stage tongue cancer and oropharyngeal cancer.

Occult cervical lymph node metastasis is the most important reason for recurrence of early-stage tongue cancer and oropharyngeal cancer. Cervical sentinel lymph node (SLN) biopsy may help to identify them. Pigment dyes and radionuclide were used to label SLN. Both of them had shortage. This study aims to investigate the application and clinical value of indocyanine green fluorescence imaging in cervical SLN biopsy for patients with early-stage tongue cancer and oropharyngeal cancer. Retrospective analysis was conducted on 23 patients with early tongue cancer and oropharyngeal cancer, who received surgical treatment and used indocyanine green as a tracer to find SLN in Hunan Cancer Hospital from April to October 2021. The detection rate of SLN was calculated and the distribution of SLN in different regions of the neck was analyzed. SLN was successfully identified in 22 of 23 patients, with a detection rate of 95.65%. Among these 22 patients, 3 patients were found to have cancer metastasis, and the rate of occult lymph node metastasis was 13.63%. No pathologically positive lymph nodes were detected in SLN-negative patients, and thus the positive predictive rate was 100%. For patients with primary lesions located in the anterior 2/3 of the tongue, the constituent ratios of SLN in neck area I, II, III, and IV were 15.15%, 71.72%, 13.13%, and 0, respectively. For patients with primary lesions located in base of the tongue, the constituent ratios of SLN in neck area I, II, III, and IV were 0, 44.44%, 44.44%, and 11.12%, respectively. Indocyanine green fluorescence imaging has a high detection rate with accurate positive prediction in the anterior cervical SLN biopsy in patients with early-stage tongue cancer and oropharyngeal cancer. Meanwhile, it can also reflect the lymphatic drainage of tumors located at different primary sites, which has high clinical value.

Selective and sensitive fluorescence detection of Pd (II) in 100% water and imaging application in living cells

• A novel quinoline-benzimidazole conjugate was designed and synthesized. • Pd 2+ -selective detection in pure water was achieved based on a quinoline-based fluorescent platform. • The present senor was successfully used for Pd 2+ detection in real water samples and fluorescence imaging in living cells. A novel quinoline-based fluorescent chemosensor ( QB-3 ) for Pd 2+ was designed and achieved a good sensing performance in 100% pure water. The QB-3 showed a selective and fast fluorescence quenching response in the presence of Pd 2+ without significant competition interferences from others metal ions. The fluorescence intensity at 411nm showed a linear decrease with the addition of the Pd 2+ concentration in the range of 0–5 μM. It was found that the detection of limit was 0.089 μM (S/N = 3), lower than the threshold limit WHO recommended. Job's plot investigation revealed a 2:1 stoichiometry between the QB-3 and Pd 2+ , and the complex was confirmed by mass spectral analysis. The DFT calculation predicted that an electron transfer process toward Pd 2+ in the complex may be responsible for the fluorescence quenching of QB-3 . The practical application in real water samples and fluorescence imaging in living cells was also achieved. A quinoline-based fluorescent chemosensor for Pd 2+ -selective detection in 100% pure water was developed, and successfully applied in practical water samples analysis and fluorescence imaging in living cells.

Applications of Indocyanine Green-Guided Near-Infrared Fluorescence Imaging in Pediatric Minimally Invasive Surgery Urology: A Narrative Review.

Background: Indocyanine green (ICG) is a fluorescent dye used for several indications in adult surgery, and, more recently, adopted also in the pediatric patients. This study aimed to review the literature published on the use of ICG near-infrared fluorescence (NIRF) in pediatric urology, to address its shortcomings and disadvantages and to detect the future perspectives. Materials and Methods: An electronic literature search of PubMed on all studies reporting use of ICG-NIRF in pediatrics was performed. We included only studies reporting ICG-NIRF application in minimally invasive surgery (MIS) for pediatric urology indications. Results: Forty-two articles reporting MIS procedures performed using ICG-NIRF in children were obtained, but only 15 studies that focused on urological applications of ICG-NIRF in children were included in this review. The included studies described use of ICG-NIRF for kidney malformations such as duplex system, kidney tumors, renal cysts, ureteral pathology, bladder malformations, varicocele, and lymph node sampling in tumors. The pediatric urological applications in which ICG-NIRF provided significant advantages included partial nephrectomy, lymphatics sparing varicocele repair, and oncological procedures. The ICG-NIRF use was clinically safe, without reported adverse systemic reactions in all pediatric series. The main drawback of this technology is the need of specific laparoscopic equipment such as camera system, light sources, and telescopes or the da Vinci Xi Robot, with the software for ICG-NIRF, Firefly®, already integrated within. Conclusions: ICG-enhanced fluorescence-guided surgery is gaining growing popularity among pediatric surgeons due to the excellent results that have been published until now. ICG-NIRF technology has proven to be safe, easy to use, not time-consuming, cheap, and very effective to improve intraoperative view and surgical ability. Nonetheless, further evidence, including larger series, longer follow-up, and more specific assessments, is necessary to confirm the preliminary results and enlarge the applications.

Progress of BODIPY dyes with aggregation-induced emission

<p indent="0mm">With the development of organic optoelectronic materials and bioimaging technology, the investigation of organic light-emitting materials with aggregation-induced emission (AIE) properties has gradually become a hot spot in material science field. The fluoroborondipyrromethenes (BODIPY) dyes have become one of the research objects of AIE materials due to their excellent luminescence properties. This paper reviews the recent advances in the design and synthesis of AIE-type BODIPY derivatives, the mechanism of AIE properties and their promising applications in fluorescence imaging, gas sensing, photodynamic therapy, and mechanofluorochromic materials. It aims to explore the structure-activity relationship of AIE-type BODIPY fluorescent molecules, and provides ideas for the further design, synthesis and potential applications of BODIPY-type aggregated luminescent materials through classification, analysis and summary.

Dual-modality hyperspectral microscopy for transmission and fluorescence imaging

Conventional color imaging provides information in the red, green, and blue (RGB) channels with limited resolution in the spectral domain. This could lead to poor sensitivity in imaging biological samples. In fluorescence imaging, the use of multiple fluorophores is often necessary to label proteins and DNAs for in vivo experiments. Crosstalk between fluorophores can be troublesome because it is challenging to differentiate each fluorophore when their emission spectra are overlapped. To help address these issues, we developed a dual-modality hyperspectral microscopy system that combines hyperspectral imaging and microscopy imaging to provide spatial and spectral information of the samples. The dual-modality feature allows us to study biological samples and fluorescent samples using the same system. We show that applications of the system enable: the identification of different tissue and cell structures; identification of each quantum dot fluorophore, calculation of their relative proportions at each location, and elucidation of their spatial distributions in a mixture sample containing multiple types of quantum dot fluorophores. The results give a brief showcase of how hyperspectral imaging can be useful for biomedical imaging and fluorescence imaging applications.

Application of near-infrared fluorescence imaging in the accurate assessment of surgical margins during breast-conserving surgery

ObjectiveTo evaluate the feasibility and accuracy of near-infrared fluorescence imaging technology for assessing margins during breast-conserving surgery for breast cancer.MethodsForty-three breast cancer patients who received surgical treatment at Yijishan Hospital of Wannan Medical College were selected. Before the operation, the patients were administered with an indocyanine green injection of 0.5 mg/kg intravenously 2 h before operation. During and after the operation, all patients underwent surgical margin monitoring with the near-infrared fluorescence imaging system for fluorescence imaging and acquisition of images and quantitative fluorescence intensity. During the operation, the patients’ tissue specimens were collected on the upper, lower, inner, outer, apical, and basal sides of the fluorescence boundary of the isolated lesions for pathological examination.ResultsFluorescence was detected in the primary tumor in all patients. The average fluorescence intensities of tumor tissue, peritumoral tissue, and normal tissue were 219.41 ± 32.81, 143.35 ± 17.37, and 105.77 ± 17.79 arbitrary units, respectively (P < 0.05, t test). The signal-to-background ratio of tumor to peritumor tissue and normal tissue was 1.54 ± 0.20 and 2.14 ± 0.60, respectively (P < 0.05, t test). Abnormal indocyanine green fluorescence was detected in 11.6% patients (5/43), including 3 patients with residual infiltrating carcinoma and 2 patients with adenosis with ductal dilatation.ConclusionThis study confirms the high sensitivity and specificity of near-infrared fluorescence imaging technology for breast-conserving surgery margin assessment. Near-infrared fluorescence imaging technology can be used as an intraoperative diagnosis and treatment tool to accurately determine the surgical margin and is of important guiding value in breast-conserving surgery for breast cancer.

New and effective EGFR-targeted fluorescence imaging technology for intraoperative rapid determination of lung cancer in freshly isolated tissue.

During lung cancer surgery, it is very important to define tumor boundaries and determine the surgical margin distance. In previous research, systemically application of fluorescent probes can help medical professionals determine the boundaries of tumors and find small tumors and metastases, thereby improving the accuracy of surgical resection. However, there are very few safe and effective probes that can be applied to clinical trials up to now, which limits the clinical application of fluorescence imaging. Here we developed a new technology that can quickly identify the tumor area in the resected lung tissue during the operation and distinguish the tumor boundary and metastatic lymph nodes. For animal studies, a PDX model of lung cancer was established. The tumors, lungs, and peritumoral muscle tissues of tumor-bearing mice were surgically removed and incubated with a probe targeting epidermal growth factor receptor (EGFR) for 20min, and then imaged by a closed-field near-infrared two-zone (NIR-II) fluorescence imaging system. For clinical samples, ten surgically removed lung tissues and 60 lymph nodes from 10 lung cancer patients undergoing radical resection were incubated with the targeting probe immediately after intraoperative resection and imaged to identify the tumor area and distinguish the tumor boundary and metastatic lymph nodes. The accuracy of fluorescence imaging was confirmed by HE staining and immunohistochemistry. The ex vivo animal imaging experiments showed a fluorescence enhancement of tumor tissue. For clinical samples, our results showed that this new technology yielded more than 85.7% sensitivity and 100% specificity in identifying the tumor area in the resected lung tissue. The average fluorescence tumor-to-background ratio was 2.5 ± 1.3. Furthermore, we also used this technique to image metastatic lymph nodes intraoperatively and showed that metastatic lymph nodes have brighter fluorescence than normal lymph nodes, as the average fluorescence tumor-to-background signal ratio was 2.7 ± 1.1. Calculations on the results of the fluorescence signal in relation to the number of metastatic lymph nodes yielded values of 77.8% for sensitivity and 92.1% for specificity. We expect this new technology to be a useful diagnostic tool for rapid intraoperative pathological detection and margin determination. By using fluorescently labeled anti-human EGFR recombinant antibody scFv fragment to incubate freshly isolated tissues during surgery, the probes can quickly accumulate in lung cancer tissues, which can be used to quickly identify tumor areas in the resected lung tissues and distinguish tumor boundaries and find metastases in lymph nodes. This technology is expected to be used for rapid intraoperative pathological detection and margin determination.

Applications of indocyanine green fluorescence imaging in colorectal surgery: a narrative review

Background and Objective: The need to optimize visualization systems in minimally invasive surgery has given rise to a growing number of applications of indocyanine green (ICG) fluorescence. This narrative review aims to summarize the existing literature about the use of this fluorophore in colon and rectal surgery.

Fluorescein-derived carbon dots with chitin-targeting for ultrafast and superstable fluorescent imaging of fungi

Abstract Fluorescence microscopy based on fluorochrome has been rapidly developed as the candidate for morphological identification of pathogenic fungi over recent years, offering superior rapidity and efficacy over traditional culture methods. However, the intrinsic quenching properties of fluorescein limit the clinical application of fluorescence imaging. Herein, we report a nano-strategy by converting a commercial fluorescein dye, fluorescent brightener-33 (FB-33), into carbon dots (FB-CDs) through a one-pot hydrothermal method. FB-CDs exhibit a chitin-targeting capacity allowing the selective recognition and ultrafast imaging of fungi within 30 s. The fluorescence quantum yield of FB-CDs is 51.6% which is 8.6-fold higher than that of commercial dye, FB-33. Moreover, FB-CDs also display superstable fluorescence signals under continuous intense light irradiation for 2 h and long-term storage for more than 2 months. The significantly improved photobleaching resistance meets the prolonged fluorescence observation and quantitative analysis of microbial samples. This work offers a novel nanoconversion strategy of commercial dyes for point-of-care testing of pathogenic organisms.

Application of near-infrared fluorescence imaging in lingual lymph node screening and drainage pattern observation for tongue cancer.

Objective: Lingual lymph node (LLN) metastasis is regarded as an indicator of unfavorable prognosis and a crucial sign of the high degree of primary tumor aggressiveness. However, detecting LLN metastasis is an important but frequently overlooked aspect of diagnosis and surgical treatment planning. The study aims to identify LLNs by intraoperative near-infrared (NIR) fluorescence imaging with indocyanine green absorbed into human serum albumin (ICG: HSA) and describe the presence of lymphatic drainage channels from the floor of the mouth in patients with tongue carcinoma. Materials and Methods: 21 patients diagnosed with cT1-T4 squamous cell carcinoma (SCC) of the tongue margin and scheduled to undergo tumor resection and unilateral neck dissection were enrolled. After exposing the neck, the patients were injected with 0.3 ml of ICG: HSA (500 μM) in three quadrants around the tumor, excluding the mucous membrane of the basal region cavity. Employing a near-infrared fluorescence imaging system, the fluorescence of levels I, II, III, and IV was measured during neck dissection. Results: LLNs were detected in four patients and were identified as metastatic LLNs in all 21 patients. The near-infrared fluorescence imaging system showed the existence of lymphatic drainage channels in the floor of the mouth. In patients receiving peritumoral injection of ICG: HSA, the mean fluorescence intensity (MFI)of metastatic lymph nodes (LNs) (178.4 ± 64.39, mean ± SD) was higher than that in non-metastatic LNs (132.0 ± 76.5, mean ± SD) (p < 0.05). Conclusion: NIR fluorescence imaging with ICG: HSA could be used for intraoperative identification of LLNs and assist in the determination of metastatic lymph nodes for tongue carcinoma patients. Additionally, this finding demonstrates the feasibility of near-infrared fluorescence imaging in defining lymphatic drainage channels in the head and neck.

Construction of MPDA@IR780 nano drug carriers and photothermal therapy of tumor cells

The combination of chemotherapy and photothermal therapy can provide better tumor treatment effect than monotherapy, while the combination of imaging technology can achieve diagnostic effect during the treatment. In this paper, biocompatible mesoporous polydopamine (MPDA) were combined with the near-infrared fluorescent dye IR780 under alkaline condition to form an integrated drug delivery platform for tumor diagnosis and photothermal co-therapy. The particles size and mesoporous pore size of the nanospheres were adjusted by changing the preparation conditions of MPDA, so the platform had strong permeability and large drug loading capacity in tumor cells as drug carriers. The loading and release results of doxorubicin hydrochloride (DOX) proved that the carrier had a high drug loading and sustained release rate, as well as a faster dependent release under acidic and lighting conditions. Photothermal experiments showed that the carrier had high photothermal conversion efficiency, which was very critical in its application in photothermal therapy and fluorescence imaging of tumors. The vitro cytotoxicity test evaluated the survival rates of LX-2 cells and HepG2 cells after drug loading. The results showed that the MPDA@IR780-DOX + NIR carrier had a significant inhibitory effect on HepG2 cells (12.57%), but had little effect on LX-2 cells, which was beneficial for its application in integration of biological diagnosis and treatment.