Search

The usefulness of incorporating near-infrared autofluorescence into the surgical workflow of endocrine surgeons is unclear. Our aim was to develop a prospective registry and gather expert opinion on appropriate use of this technology.This was a prospective multicenter collaborative study of patients undergoing thyroidectomy and parathyroidectomy at 7 academic centers. A questionnaire was disseminated among 24 participating surgeons.Overall, 827 thyroidectomy and parathyroidectomy procedures were entered into registry: 42% of surgeons found near-infrared autofluorescence useful in identifying parathyroid glands before they became apparent; 67% correlated near-infrared autofluorescence pattern to normal and abnormal glands; 38% of surgeons used near-infrared autofluorescence, rather than frozen section, to confirm parathyroid tissue; and 87% and 78% of surgeons reported near-infrared autofluorescence did not improve the success rate after parathyroidectomy or the ability to find ectopic glands, respectively. During thyroidectomy, 66% of surgeons routinely used near-infrared autofluorescence to rule out inadvertent parathyroidectomy. However, only 36% and 45% felt near-infrared autofluorescence decreased inadvertent parathyroidectomy rates and improved ability to preserve parathyroid glands during central neck dissections, respectively.This survey study identified areas of greatest potential use for near-infrared autofluorescence, which can form the basis of future objective trials to document the usefulness of this technology.

A biotin-guided near-infrared fluorescent probe could be transported into cancer cells selectively and could further detect H2S.

Near-infrared autofluorescence and indocyanine green fluorescence are 2 recent tools introduced to improve postoperative parathyroid function during thyroid surgery.We conducted a randomized prospective study. Patients undergoing total thyroidectomy were randomly assigned either to the fluorescence group, in which near-infrared autofluorescence and indocyanine green fluorescence were used, or to the control group. The primary outcomes of the study were the rate of postoperative transient and symptomatic hypocalcemia.A significantly higher number of parathyroid glands were identified in the fluorescence group (3.83 vs 3.64, P = .028). The rate of postoperative symptomatic hypocalcemia was significantly lower in the fluorescence group (6% vs 17%, P = .015), as was the dosage (1.53 vs 1.91 g, P = .007) and the duration of calcium therapy (32.30 vs 45.66 days, P = .003). Having at least 2 well-vascularized parathyroid glands correlates to lower rates of transient hypocalcemia (7.4% vs 21.9%, P = .037) as well as to higher serum calcium (8.70 vs 8.42 mg/dL, P = .027) and parathyroid hormone levels (19.15 vs 11.4 pg/mL, P = .0002) on postoperative day 1.Near-infrared autofluorescence and indocyanine green fluorescence are novel tools that may support the endocrine surgeon in preserving and predicting post-thyroidectomy parathyroid gland function.

There is a recent proliferation of clinical studies about the minimally invasive scarless thyroid surgery. The transoral endoscopic thyroidectomy vestibular approach (TOETVA) carries a great potential for being scarless surgery via a short dissection flap. However, TOETVA has limitations in extracting larger thyroid tumours via the transoral vestibular incision and due to its potential damage to the branches of the mental nerve. The rapidly evolving surgical innovations have now introduced transoral and submental thyroidectomy (TOaST) approach that allows extraction of large thyroid tumours with less flap dissection and minimal postoperative pain. We present a 39-year-old man with a large multinodular goitre. The patient was euthyroid with moderate to severe compression symptoms of difficulty in breathing and swallowing. We performed a TOaST procedure using intraoperative neuromonitoring and indocyanin green fluorescence imaging with an uneventful recovery. This is a first case report from the middle east region that will pave the way to large clinical trials to determine the efficacy and safety of TOaST.

Here, we describe a new synchrotron X-ray Fluorescence (XRF) imaging instrument with an integrated High Energy Fluorescence Detection X-ray Absorption Spectroscopy (HERFD-XAS) spectrometer at the Stanford Synchrotron Radiation Lightsource at beamline 6-2. The X-ray beam size on the sample can be defined via a range of pinhole apertures or focusing optics. XRF imaging is performed using a continuous rapid scan system with sample stages covering a travel range of 250 × 200 mm2, allowing for multiple samples and/or large samples to be mounted. The HERFD spectrometer is a Johann-type with seven spherically bent 100 mm diameter crystals arranged on intersecting Rowland circles of 1 m diameter with a total solid angle of about 0.44% of 4π sr. A wide range of emission lines can be studied with the available Bragg angle range of ∼64.5°–82.6°. With this instrument, elements in a sample can be rapidly mapped via XRF and then selected features targeted for HERFD-XAS analysis. Furthermore, utilizing the higher spectral resolution of HERFD for XRF imaging provides better separation of interfering emission lines, and it can be used to select a much narrower emission bandwidth, resulting in increased image contrast for imaging specific element species, i.e., sparse excitation energy XAS imaging. This combination of features and characteristics provides a highly adaptable and valuable tool in the study of a wide range of materials.

The refractive index points to the interplay between light and objects, which is rarely studied down to micronano scale. Herein, we demonstrated a conventional bright-field imaging technique to determine the local refractive index of single particles combined with a series of refractive index standard solutions. This intrinsic optical property is independent with the particle size and surface roughness with a single chemical component. Furthermore, we accurately tuned refractive index of homemade core-shell nanoparticles by adjusting the ratio of core-to-shell geometry. This simple and effective strategy reveals extensive applications in exploring, designing and optimizing the physical and optical characterizations of composite photonic crystals with high precision. It also indicates potentials in the field of reflective displays, optical identification, and encryption.

Phototheranostics, a local non-invasive approach that integrates light-based diagnostics and therapeutics, enables precise treatment using nanotheranostic agents with minimal damage to normal tissues. However, ensuring high-efficiency ablation of cancer cells using phototheranostics for one time irradiation is highly challenging. Herein, we designed and synthesized a single-walled carbon nanohorns-based nanotheranostic agent, HA-IR808-SWNHs, by loading IR808, a photosensitizer, conjugated hyaluronic acid (HA) with an amide bond on the surface of single-walled carbon nanohorns (SWNHs) through noncovalent π-π interaction by the sonication method. The HA in HA-IR808-SWNHs improves the water dispersibility of SWNHs and endows SWNHs with targeting capabilities. Importantly, overexpressed endogenous hyaluronidase in cancer cells actively disassembles HA-IR808-SWNHs, forming small HA-IR808 fragments. The fragments exhibit a strong fluorescence signal and can be used to guide programmed photodynamic therapy for sequentially eliminating the residual living cancer cells. The current study confirms that HA-IR808-SWNHs is an endogenous enzyme-responsive nanotheranostic agent that can be employed to precisely track and ablate residual cancer cells in a spatiotemporal manner. The results strengthen the understanding of SWNH functionalization and expand its potential biomedical application, especially in cancer theranostics.

BACKGROUND: Published empirical data have increasingly suggested that using near-infrared fluorescence cholangiography during laparoscopic cholecystectomy markedly increases biliary anatomy visualization. The technology is rapidly evolving, and different equipment and doses may be used. We aimed to identify areas of consensus and nonconsensus in the use of incisionless near-infrared fluorescent cholangiography during laparoscopic cholecystectomy.METHODS: A 2-round Delphi survey was conducted among 28 international experts in minimally invasive surgery and near-infrared fluorescent cholangiography in 2020, during which respondents voted on 62 statements on patient preparation and contraindications (n = 12); on indocyanine green administration (n = 14); on potential advantages and uses of near-infrared fluorescent cholangiography (n = 18); comparing near-infrared fluorescent cholangiography with intraoperative x-ray cholangiography (n = 7); and on potential disadvantages of and required training for near-infrared fluorescent cholangiography (n = 11).RESULTS: Expert consensus strongly supports near-infrared fluorescent cholangiography superiority over white light for the visualization of biliary structures and reduction of laparoscopic cholecystectomy risks. It also offers other advantages like enhancing anatomic visualization in obese patients and those with moderate to severe inflammation. Regarding indocyanine green administration, consensus was reached that dosing should be on a milligrams/kilogram basis, rather than as an absolute dose, and that doses >0.05 mg/kg are necessary. Although there is no consensus on the optimum preoperative timing of indocyanine green injections, the majority of participants consider it important to administer indocyanine green at least 45 minutes before the procedure to decrease the light intensity of the liver.CONCLUSION: Near-infrared fluorescent cholangiography experts strongly agree on its effectiveness and safety during laparoscopic cholecystectomy and that it should be used routinely, but further research is necessary to establish optimum timing and doses for indocyanine green.

In recent years, fluorescence imaging-relying both on parathyroid gland autofluorescence under near-infrared light and angiography using the fluorescent dye indocyanine green-has been used to reduce risk of iatrogenic parathyroid injury during thyroid and parathyroid resections, but no published guidelines exist regarding its use. In this study, orchestrated by the International Society for Fluorescence Guided Surgery, areas of consensus and nonconsensus were examined among international experts to facilitate future drafting of such guidelines.A 2-round, online Delphi survey was conducted of 10 international experts in fluorescence imaging use during endocrine surgery, asking them to vote on 75 statements divided into 5 modules: 1 = patient preparation and contraindications to fluorescence imaging (n = 11 statements); 2 = technical logistics (n = 16); 3 = indications (n = 21); 4 = potential advantages and disadvantages of fluorescence imaging (n = 20); and 5 = training and research (n = 7). Several methodological steps were taken to minimize voter bias.Overall, parathyroid autofluorescence was considered better than indocyanine green angiography for localizing parathyroid glands, whereas indocyanine green angiography was deemed superior assessing parathyroid perfusion. Additional surgical scenarios where indocyanine green angiography was thought to facilitate surgery are (1) when1 parathyroid gland requires resection; (2) during redo surgeries, (3) facilitating parathyroid autoimplantation; and (4) for the predissection visualization of abnormal glands. Both parathyroid autofluorescence and indocyanine green angiography can be used during the same procedure and employing the same imaging equipment. However, further research is needed to optimize the dose and timing of indocyanine green administration.Though further research remains necessary, using fluorescence imaging appears to have uses during thyroid and parathyroid surgery.

To introduce a novel method of quantifying retinal displacement in three dimensions (3D) using ultra-widefield fundus autofluorescence (UWF-FAF).Prospective clinical cohort study.Patients with primary macula-off rhegmatogenous retinal detachment (RRD) treated with pneumatic retinopexy (PnR) or vitrectomy were included. Masked graders selected corresponding points on retinal vasculature and retinal vessel printings (RVPs) within Zone 1, a circular region centered on the fovea with a radius extending to the optic disc (OD) center. Two-dimensional (2D) UWF-FAF was projected to 3D using OD and foveal coordinates. Vertical, horizontal, and diagonal distances between corresponding vessel and RVPs were calculated. Vector displacement of the RVPs to vessels were averaged.A total of 170 retinal vessel-RVP pairs were identified from 54 UWF-FAF images. Mean displacement in Zone 1 was 0.36 ± 0.38 mm, with an absolute mean angle of displacement of 94.93 ± 41.48° and directionality of 191.84 ± 97.39°. Mean Zone 1 displacement was 0.44 ± 0.42mm and 0.21 ± 0.27 mm in vitrectomy (n = 35) and PnR (n = 19) eyes, respectively (P = .041), with no differences in mean angle of displacement/directionality.A novel method of quantifying the magnitude and direction of retinal displacement is presented, accounting for the spherical 3D curvature of the eye by using corresponding points on retinal vessels and RVPs using UWF-FAF. This provides a 3D vector of displacement agnostic of a singular fixation point such as the optic disc. Accurately quantifying the magnitude and angle/direction of displacement may allow for a better understanding of the mechanisms involved in retinal displacement in various surgical techniques, and a better assessment of the association with functional outcomes.

BACKGROUND: Despite exponentially growing evidence supporting the use of intraoperative fluorescence imaging + indocyanine green dye, considerable variability exists in how and when it is used, and no published consensus guidelines exist. We have conducted Delphi surveys of international experts in the use of intraoperative fluorescence imaging covering 6 distinct surgical scenarios: laparoscopic cholecystectomy; colorectal, lymphedema, gastric cancer, and plastic surgery; and thyroid and parathyroid resections. Although each survey asked experts to vote on field-specific consensus statements, they also had 29 shared statements to permit some analysis spanning the 6 specialties. This article summarizes these results.METHODS: Data on the 29 shared statements from 6 two-round Delphi consensus surveys were compiled to identify areas of overall consensus and compare the different specialties. As with the individual surveys, consensus was defined as ≥70% intervoter agreement.RESULTS: Among 140 participating experts, overall consensus was achieved on 16 statements, including strong agreement that using indocyanine green is extremely safe, that it can be used even when informed written consent cannot be provided, that it significantly enhances anatomical visualization and impacts how procedures are performed, and that it significantly reduces overall procedural risk. However, indocyanine green dosing and timing are procedure-specific, with considerable variability persisting for some applications, and the overall consensus is that further research is necessary to optimize this facet of intraoperative fluorescence imaging.CONCLUSION: Fluorescence imaging is gaining traction across multiple surgical specialties as an invaluable intraoperative tool. Its use in clinical practice and research seems destined to increase.

The second near-infrared IIa window (NIR-IIa, 1300nm∼1400nm) enables high-resolution imaging and deep-tissue tumor treatment due to its unique low tissue scattering and autofluorescence, high temporal-spatial resolution, and deep tissue penetration. Therefore, NIR-IIa fluorescence imaging-guided phototherapy is of specific interest. However, organic dyes and their nanoparticles for NIR-IIa phototheranostics are still scarce. Here, we have synthesized a Br- and piperazine-modified cyanine dye (FN) and its nanomicelles encapsulated by an amphiphilic polypeptide with sidechains of tertiary amine (PEA). The J-aggregates of P@FN

Fluorescence imaging in the second near-infrared (NIR-II, 1000-1700 nm) region holds great promise for in vivo bioimaging. However, it is challenging to develop a brilliant donor-acceptor-donor (D-A-D) type NIR-II fluorophore with maximal absorption beyond 1000 nm in aqueous solution. Herein, we report a bright D-A-D type BOIMPY-based NIR-II dye (NK1143) with peak absorption/emission at 1005/1143 nm for in vivo bioimaging. Co-assembly of NK1143, SC12 (intermolecular steric hindrance modulator), and DSPE-PEG2000 effectively inhibits H-aggregation of NK1143 in aqueous solution and enhances the brightness simultaneously up to 53-fold by leveraging synergistic steric regulation strategy. Notably, this strategy allows for deep optical penetration of 8 mm and high-resolution blood vessels imaging in vivo, displaying high signal-to-background ratio of 7.8/1 under 980 nm excitation. More importantly, the BOIMPY-based nanoprobe can passively target and clearly visualize broad types of tumor xenografts, further improving intraoperative NIR-II fluorescence-guided resection of tiny metastases of less than 1 mm. This work provides an effective strategy for the development of BOIMPY-based NIR-II organic fluorophores with broad applications.

Detection and imaging of highly reactive oxygen species (hROS) in biological systems using fluorescent probes are critical for the study of physiological and pathological processes induced by hROS. Herein, we report a redox-active luminescent metal-organic framework (MOF), which incorporates a hydroquinone moiety that can undergo a reversible transformation from the hydroquinone to the quinone by hROS like •OH and ClO

Aberrant cerebral glucose metabolism is related to many brain diseases, especially brain tumor. However, it remains challenging to measure the dynamic changes in cerebral glucose. Here, we developed a near-infrared (NIR) optical transducer to sensitively monitor the glucose variations in cerebrospinal fluid in vivo. The transducer consists of an oxygen-sensitive nanoparticle combined with glucose oxidase (GOx), yielding highly sensitive NIR phosphorescence in response to blood glucose change. We demonstrated long-term continuous glucose monitoring by using the NIR transducer. After subcutaneous implantation, the glucose transducer provides a strong luminescence signal that can continuously monitor blood glucose fluctuations for weeks. By using the NIR emission of the transducer, we further observed abnormal dynamic changes in cerebrospinal fluid glucose and quantitatively assessed cerebral glucose uptake rates in transgenic mice bearing brain tumors. This study provides a promising method for the diagnosis of various metabolic diseases with altered glucose metabolism.

The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, cytosol, or nucleus could be accurately distinguished using FLIM and phasor analysis. Next, we successfully resolved and quantified three probes that were all targeted to cell surface biomarkers. Finally, we demonstrated that lifetime-based quantitation accuracy can be improved through intensity matching of various probe-biomarker combinations, which will expand the utility of this technique. Importantly, we reconstructed images for each individual probe, as well as an overlay of all three probes, from a single FLIM image. Our results demonstrate that FLIM and phasor analysis can be leveraged as a powerful tool for simultaneous detection of multiple biomarkers with high sensitivity and accuracy.

Jiao Song,* Huixia Ye,* Senwei Jiang, Yuebo Yang, Xiaomao Li Gynaecology Department, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510630, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yuebo Yang; Xiaomao Li, Gynaecology Department, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510630, People’s Republic of China, Tel/Fax +86 20-85252259 ; +86 20-85253289, Email yangyueb@mail.sysu.edu.cn; Lixmao@mail.sysu.edu.cnIntroduction: Complete resection of all visible disease (R0 resection) is critical for the treatment of ovarian cancer patients, and accurate real-time guidance provided by intraoperative near-infrared (NIR) fluorescence images is beneficial for achieving complete resection of all visible disease.Methods: Based on the optical properties of IR780 and the characteristics of the acidic tumor microenvironment, we develop a new smart nanoparticle (eg, FA-IR780&PFOB-SNPs) by using the pH response nano framework (FA-PEG-PLGA-PEOz) and adjusting the amount of IR780. The FA-IR780&PFOB-SNPs was characterized for morphology, microstructure, particle size, pH-response, drug-loading efficiency and biological safety. The ultraclear fluorescence Navigation Endoscopy System was applied to evaluate the tumor recognition of FA-IR780&PFOB-SNPs in vivo.Results: The structure of FA-IR780&PFOB-SNPs was stable in a neutral environment, and the near-infrared (NIR) fluorescence was turned off, while the structure of FA-IR780&PFOB-SNPs was degraded in the acidic tumour microenvironment, and the NIR fluorescence was turned on. Through the ovarian subcutaneous xenograft tumour and ovarian intraperitoneal xenograft tumour models, it was confirmed that FA-IR780&PFOB-SNPs could clearly display the boundaries of abdominal micron-sized tumours through near-infrared fluorescence imaging, with a TBR greater than 5.Conclusion: The FA-IR780&PFOB-SNPs have the potential to provide to ovarian cancer intraoperative near infrared fluorescence navigation during precision tumour resection to achieve R0 and improve the prognosis of ovarian cancer patients.Keywords: near-infrared fluorescence imaging, IR780 iodide, epithelial ovarian cancer, tumour-activatable nanoparticles

Phototheranostics that integrates real-time optical imaging and light-controlled therapy has recently emerged as a promising paradigm for cancer theranostics. Herein, a new small molecule dye DPP-BT-TPA with strong emission above 1000 nm and a redox-responsive prodrug camptothecin-combretastatin A4 (CPT-CA4) were designed and successfully synthesized. A multifunctional phototheranostic nanoplatform was then fabricated by encapsulating them within an amphiphilic polymer. The presence of DPP-BT-TPA enabled high-resolution imaging in the second near-infrared window (NIR-II) and efficient photothermal therapy. The prodrug was cleaved by the overexpressed glutathione (GSH) in the tumor microenvironment to release the chemotherapeutic drug CPT and the angiogenesis inhibitor CA4. Because this process can be accelerated with elevated temperature, laser-induced hyperthermia was utilized to control the drug release and enhance the therapeutic effect. Tumors in living mice were observed through NIR-II imaging after intravenous injection of the obtained nanoparticles. Improved antitumor efficacy by photothermal/chemo/antiangiogenic combination therapy was achieved with a NIR laser both in vitro and in vivo. This work provides a promising strategy for developing tumor microenvironment responsive and light-controlled theranostic platforms. STATEMENT OF SIGNIFICANCE: Fluorescence imaging in the second near-infrared (NIR-II, 1000-1700 nm) window and near-infrared light-controlled drug release have been recognized as efficient strategies for cancer theranostics. Herein, we present a phototheranostic platform fabricated with a biocompatible NIR-II emissive dye DPP-BT-TPA and a redox-responsive prodrug camptothecin-combretastatin A4 (CPT-CA4). DPP-BT-TPA not only provides high-resolution NIR-II imaging in vivo but also enables efficient photothermal therapy. In addition, the photothermal effect largely accelerates the release of the chemotherapeutic drug CPT and the angiogenesis inhibitor CA4 in the glutathione-overexpressed tumor microenvironment. Thus, the designed phototheranostic platform can be used for NIR-II imaging-guided photothermal/chemo/antiangiogenic combination therapy for tumors with a single laser.

Bioluminescence (BL) imaging has emerged to tackle the potential challenges of fluorescence (FL) imaging including the autofluorescence background, inhomogeneous illumination over a wide imaging field, and the light-induced overheating effect. Taking advantage of the bioluminescence resonance energy transfer (BRET) mechanism between a conventional luciferin compound and a suitable acceptor, the visible light of the former can be extended to photons with longer wavelengths emitting from the latter. Although BRET-based self-illuminating imaging probes have already been prepared, employing potentially cytotoxic elements as the acceptor with the emission wavelengths which hardly reach the first near-infrared (NIR-I) window, has limited their applications as safe and high performance

Edible crops are important in terms of food security and sustainable agriculture. Heavy-metal-ion contamination of water/soil has deleterious impacts on the growth of edible crops. Among the heavy metals, cadmium (Cd) is toxic to plants, people, and animals, as it is widely used in industry; it has become the most important metal ion in the soil/water pollution. Once the toxic Cd ion enters edible crops via the water/soil in which the crops grow, it will induce oxidative stress (overproduction of reactive oxygen species with H

Amyloid precursor protein (APP) is a major contributor to the pathology of Alzheimer's and other neurodegenerative diseases through the accumulation of extracellular plaques. Here, we have studied changes in APP translation and aggregation of the APP intracellular domain when the Gαq/PLCβ signaling system is activated by neurotransmitters. Using RT-PCR and a molecular beacon that follows APP mRNA in live cells, we find that Gαq activation sequesters APP mRNA similar to the stress granule response found in heat shock and hypo-osmotic shock thereby shutting down the production of APP. Following the intracellular domain of eGFP-APP, we find that Gαq stimulation increases aggregation as followed by number and brightness (NB) analysis of single molecule fluorescence time series. Additionally, we show that APP aggregation is affected by changes in the levels of PLCβ1 and its cytosolic binding partners. Our studies show the neurotransmitter activation of Gαq/PLCβ reduces translation of APP and increases aggregation of its intracellular domain. These studies better establish a link between APP production and complexation and Gαq stimulation.

Ferroptosis, as a new regulated mode of cell death, is mainly characterized by iron-dependent accumulation of lipid peroxides related to reactive oxygen species (ROS). However, the changes of peroxynitrite (ONOO

Epigenome contains a lot of information about cell state. Epigenetic analysis includes primarily sequence-based methods, which provide detailed data on distribution of modifications along the genome, but are poorly applicable for screenings. Specific fluorescence labeling and imaging of epigenetic modifications is an attractive complementary approach. It is currently based mainly on histone modifications study. We expect that inclusion of DNA modifications into imaging-based study would empower the method. In this review we discuss methods for fluorescence imaging of DNA modifications (mainly 5-methylcytosine). It opens an easy way to single cell analysis and high-throughput screening. Moreover, tracking epigenome changes in live cells becomes possible with genetically encoded probes.

Lipid droplets (LDs), which are ubiquitous organelles existing in almost all eukaryotic cells, have attracted a lot of attention in the field of cell biology over the last decade. For the biological study of LDs via fluorescence imaging, the superior LD fluorescent probes with environmental polarity-sensitive character are highly desired and powerful but are very scarce. Herein, we have newly developed such a kind of fluorescent probe named LDs-Red which enables us to visualize LDs and to further reveal their polarity information. This fluorescent probe displays the advantages of intense red/near-infrared emission, high LD staining specificity, and good photostability; thus, it would be very useful for LD fluorescence imaging application. As a result, the three-dimensional confocal imaging to visualize spatial distribution of LDs and the multicolor confocal imaging to simultaneously observe LDs and other cellular organelles have been realized using this new LD fluorescent probe. Furthermore, the polarity-sensitive emission character of this probe enables us to quantitatively determine the LD polarity via spectral scan imaging. Consequently, the cancer cells (HepG2, HeLa, and Panc02) displaying lower polarity of LDs than the normal cells (L929, U251, and HT22) have been systematically demonstrated. In addition, this polarity-sensitive probe displaying shorter fluorescence wavelengths in cancer cells than in normal cells has an important and potential ability to distinguish them.

Excessive oxidative stress is the main cause of neurotransmitter metabolism disorder in the brain with depression. Lysosomal hypobromic acid (HOBr) is an important reactive oxygen species produced in oxidative stress. Its abnormal content can lead to macromolecular damage and neurodegenerative diseases. However, due to the high reactivity and low concentration of HOBr and the lack of in situ imaging methods, the role of HOBr in depression is not clear. Herein, based on the HOBr-initiated aromatic substitution of a tertiary amine, we developed a novel two-photon (TP) fluorescence probe (

Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) holds great potential for the accurate visualization of deeply located biological structures

The development of three-photon fluorophores with 1700 nm excitation is pressingly desirable for

Lymphatic flow mapping using near-infrared fluorescence (NIR) imaging with indocyanine green (ICG) has been used for intraoperative diagnosis of lymph node metastasis (LNM) in various cancers. Accurate prediction of LNM intraoperatively may allow minimization of the extent of lymphadenectomy. However, a consistent method and diagnostic ability, allowing application of NIR-guided lymphatic flow mapping to esophageal cancer (EC), have not been established due to the multidirectional and complex characteristics of lymphatic flow in the esophagus. Herein, we present a novel NIR-guided surgical technique for predicting lymph node stations potentially containing LNM in EC with high diagnostic accuracy derived from appropriately adjusting the ICG injection setting.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) using small-molecule dyes is highly potential for clinical use. However, many NIR-II dyes suffer from the emission quenching effect and extremely low quantum yields (QYs) in the practical usage forms. The AIE strategy has been successfully utilized to develop NIR-II dyes with donor-acceptor (D-A) structures with acceptable QYs in the aggregate state, but there is still a large room for QY improvement. Here, we rationally designed NIR-II emissive dye named TPE-BBT and its derivative (TPEO-BBT) by changing the electron-donating triphenylamine unit to tetraphenylethlene (TPE). Their nanoparticles exhibited ultrahigh relative QYs of 31.5% and 23.9% in water, respectively. By using an integrating sphere, the absolute QY of TPE-BBT nanoparticles was measured to be 1.8% in water. Its crystals showed an absolute QY of 10.4%, which is the highest value among organ-ic small molecules reported so far. The optimized D-A interaction and the higher rigidity of TPE-BBT in the aggregate state are believed to be the two key factors for its ultrahigh QY. Finally, we utilized TPE-BBT for NIR-II photoluminescence (PL) and chemiluminescence (CL) bioimaging through successive CL resonance energy transfer and Förster resonance energy transfer processes. The ultrahigh QY of TPE-BBT realized an excellent PL imaging quality in mouse blood vessels, and an excellent CL imaging quality in the local arthrosis inflammation in mice with a high signal-to-background ratio of 130. Thus, the design strategy presented here brings new possibilities for the development of bright NIR-II dyes and NIR-II bioimaging technologies.

Emissive excimers, which are formed by planar polycyclic aromatic fluorophores (e.g., coumarin), enable high contrast tumor imaging. However, it is still challenging to "turn on" excimer fluorescence in physiological dilute solutions. The biocompatible CBT-Cys click condensation reaction enables both intra- and intermolecular aggregations of the as-loaded fluorophores on the probe molecules, which may promote the generation of emissive excimers in a synergistic manner. As a proof-of-concept, we herein design a fluorescence probe Cbz-Gly-Pro-Cys(StBu)-Lys(coumarin)-CBT (

Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.

During sleep, the brain undergoes transitions of activity states and reorganization of neural circuits. Recent in vivo optical imaging and manipulation techniques are revealing spatiotemporal and multiscale brain dynamics during sleep and their functional roles in awake state cognition, including learning and memory. First, along with electrophysiological recordings, Ca

Fluorescence imaging is a noninvasive and dynamic real-time imaging technique; however, it exhibits poor spatial resolution in centimeter-deep tissues because biological tissues are highly scattering media for optical radiation. The recently developed ultrasound-controlled fluorescence (UCF) imaging is a novel imaging technique that can overcome this bottleneck. Previous studies suggested that the effective contrast agent and sensitive imaging system are the two pivotal factors for generating high-resolution UCF images ex vivo and/or in vivo. Here, this review highlights the recent advances (2015–2020) in the design and synthesis of contrast agents and the improvement of imaging systems to realize high-resolution UCF imaging of deep tissues. The imaging performances of various UCF systems, including the signal-to-noise ratio, imaging resolution, and imaging depth, are specifically discussed. In addition, the challenges and prospects are highlighted. With continuously increasing research interest in this field and emerging multidisciplinary applications, UCF imaging with higher spatial resolution and larger imaging depth may be developed shortly, which is expected to have a far-reaching impact on disease surveillance and/or therapy.

The near-infrared window II (NIR-II, 1000-1700 nm) imaging, including NIR-IIa (1300-1400 mm) and NIR-IIb (1500-1700 mm), outperforms the near-infrared window I (NIR-I, 700-900 nm) imaging in biological researches. However, the advantages of NIR-IIa/IIb imaging in human study are ambiguous. This study aims to apply the NIR-IIa/IIb imaging to glioma resection and evaluate their performance by using the developed imaging instrument and intraoperative image fusion method.A multispectral fluorescence imaging instrument that integrated NIR-I/II/IIa/IIb fluorescence imaging and an intraoperative image fusion method have been developed. Seven patients with grade III/IV glioma have been enrolled. NIR-I/II images of the tumor and NIR-I/II/IIa/IIb images of cerebral vessels were acquired with the administration of indocyanine green. Images were fused using the specialized fusion method to synchronously provide the distribution of the vessels and the surgical boundaries.The NIR-IIa/IIb imaging was successfully applied to the clinic. High imaging resolution and contrast have been attained in the NIR-IIa/IIb spectra. Besides, capillaries with an apparent diameter as small as 182 μm were acquired using NIR-IIb imaging. Tumor-feeding arteries were precisely blocked and tumors were excised to the maximum extent for all patients. The blood loss volume during surgery was significantly reduced compared with the control group.The multispectral fluorescence imaging showed high performance, which led to a significant reduction in blood loss volume.The novel multispectral fluorescence imaging technology can assist surgeons in other vascular surgeries in the future.

Near-infrared window IIb (NIR-IIb, 1500-1700 nm) fluorescence imaging demonstrates attractive properties including low scattering, low absorption, and deep tissue penetration, and photothermal therapy (PTT) is also a promising modality for cancer treatment. However, until now, there is no report on theranostic systems based on small organic molecules combining fluorescence imaging in the NIR-IIb and PTT, highlighting the challenge and strong need for development of such agents. Herein, we report a novel small molecule NIR-IIb dye IT-TQF with a D-A-D structure, which exhibited high fluorescence intensity in the NIR-IIb window. To further translate IT-TQF into an effective theranostic agent, IT-TQF was encapsulated into DSPE-PEG

The surgery of glioblastoma (GBM) requires a maximal resection of the tumor when it is safe and feasible. The infiltrating growth property of the GBM makes it a challenge for neurosurgeons to identify the tumor tissue even with the assistance of the surgical microscope. This highlights the urgent requirement for imaging techniques that can differentiate tumor tissues during surgery in real time. Fluorescence image-guided surgery of GBM has been investigated using several non-specific fluorescent probes that emit light in the visible and the first near-infrared window (NIR-I, 700-900 nm), which limit the detection accuracy because of the non-specific targeting mechanism and spectral characteristics. Targeted NIR-II (1000-1700 nm) fluorescent probes for GBM are thus highly desired. The folate receptor (FR) has been reported to be upregulated in GBM, which renders it to be a promising target for specific tumor imaging.In this study, the folic acid (FA) that can target the FR was conjugated with the clinically approved indocyanine green (ICG) dye and DOTA chelator for radiolabeling withsup64/supCu to achieve targeted positron emission tomography (PET) and fluorescence imaging of GBM.Surprisingly it was found that the resulted bioconjugate, DOTA-FA-ICG and non-radioactivesupnat/supCu-DOTA-FA-ICG, were both self-assembled into nanoparticles with NIR-II emission signal. The radiolabeled DOTA-FA-ICG,sup64/supCu-DOTA-FA-ICG, was found to specifically accumulate in the orthotopic GBM models using in vivo PET, NIR-II, and NIR-I fluorescence imaging. The best time window of fluorescence imaging was demonstrated to be 24 h after DOTA-FA-ICG injection. NIR-II fluorescence image-guided surgery was successfully conducted in the orthotopic GBM models using DOTA-FA-ICG. All the fluorescent tissue was removed and proved to be GBM by the Hamp;E examination.Overall, our study demonstrates that the probes,sup64/supCu-DOTA-FA-ICG and DOTA-FA-ICG, hold promise for preoperative PET examination and intraoperative NIR-II fluorescence image-guided surgery of GBM, respectively.

This study shows a one-pot preparation of carbon dots by a solvothermal method in ethylene glycol. The carbon dots show yellow-colored fluorescence emission in water. The carbon dots showed distinct preference to be present in the hydrophobic environment which was evident from their efficient transfer from aqueous phase to organic phase. They were also found to locate themselves in the vesicle bilayer and micelle core. This inherent lipophilic character of these carbon dots has been successfully utilized for the selective imaging of lipid droplets inside the living cells. The selective imaging of lipid droplets was confirmed by similar staining patterns with other staining dyes and the starvation study.

Tumor marker-responsive drug delivery systems have been developed for cancer imaging and chemotherapy. However, improving their ability of controlled drug release remains a challenge. In this study, we have developed an adenosine triphosphate (ATP)-responsive DNA nanohydrogel for specifically activated fluorescence imaging and chemotherapy in cancer cells. Acrylamide and acrydite-modified DNAs were polymerized to obtain DNA-grafted polyacrylamide copolymers. Then, the copolymers acted as the backbone of the nanohydrogel and were assembled by base complementation with ATP aptamer linkers to construct an ATP-responsive nanohydrogel. Meanwhile, the chemotherapeutic drug doxorubicin (DOX) was added and loaded into the ATP-responsive nanohydrogel during the assembly process. After endocytosis by cancer cells and response to a high intracellular ATP level, the DOX-loaded nanohydrogel disassembled due to the formation of aptamer/ATP complexes. Subsequently, the released DOX played a role in fluorescence imaging and chemotherapy of cancer cells. Through the ATP-responsive property and satisfying drug delivery capability, this nanohydrogel realized fluorescence imaging and specific cancer cell killing capabilities due to different intracellular ATP levels in normal and cancer cell lines. In summary, this study has provided a novel strategy of constructing a tumor microenvironment-responsive drug delivery system triggered by the tumor markers for tumor intracellular imaging and chemotherapy.

Ferroptosis, a new regulatory cell death modality, underlies the pathogenesis of a broad range of disorders. Although much efforts have been made to uncover the molecular mechanisms, some mechanistic details of ferroptosis still remain poorly understood. Particularly, the functional relevance of mitochondrial reactive oxygen species (ROS) in ferroptosis is still highly controversial, which is partially due to the fact that it still remains puzzled how the mitochondrial ROS level varies during ferroptosis. The conventional mitochondria-targeted probes may react with cytosolic ROS and show fluorescence variation before entering mitochondria, thus probably giving a false result on the mitochondrial ROS level and leading to the misjudgment on its biofunction. To circumvent this issue, we rationally designed a photocontrollable and mitochondria-targeted fluorescent probe to in situ visualize the mitochondrial peroxynitrite (ONOO

This meta-analysis aimed to assess the position of near-infrared autofluorescence (NIRAF) imaging in the recognition and protection of the parathyroid gland (PG) during thyroidectomy.The PubMed, MEDLINE, EMBASE, Web of Science, and Cochrane Library databases were searched up to June 2021. The primary outcome was to evaluate the rates of postoperative hypocalcemia, inadvertent PG resection, and autotransplantation of PG when adopting NIRAF imaging compared with standard naked-eye (N-E) surgery.Eight studies with 2,889 patients were enrolled in the analysis. Our analysis showed that the incidence of transient hypocalcemia was 7.11% (60/844) in the NIRAF group and 22.40% (458/2045) in the N-E group (The application of NIRAF imaging in thyroidectomy can help lower the incidence of inadvertent PG resection and reduce the risk of postoperative hypocalcemia and hypoparathyroidism compared with N-E recognition.

SignificancePerturbation and differential Monte Carlo (pMC/dMC) methods, used in conjunction with nonlinear optimization methods, have been successfully applied to solve inverse problems in diffuse optics. Application of pMC to systems over a large range of optical properties requires optimal "placement" of baseline conventional Monte Carlo (cMC) simulations to minimize the pMC variance. The inability to predict the growth in pMC solution uncertainty with perturbation size limits the application of pMC, especially for multispectral datasets where the variation of optical properties can be substantial.AimWe aim to predict the variation of pMC variance with perturbation size without explicit computation of perturbed photon weights. Our proposed method can be used to determine the range of optical properties over which pMC predictions provide sufficient accuracy. This method can be used to specify the optical properties for the reference cMC simulations that pMC utilizes to provide accurate predictions over a desired optical property range.ApproachWe utilize a conventional error propagation methodology to calculate changes in pMC relative error for Monte Carlo simulations. We demonstrate this methodology for spatially resolved diffuse reflectance measurements with ±20% scattering perturbations. We examine the performance of our method for reference simulations spanning a broad range of optical properties relevant for diffuse optical imaging of biological tissues. Our predictions are computed using the variance, covariance, and skewness of the photon weight, path length, and collision distributions generated by the reference simulation.ResultsWe find that our methodology performs best when used in conjunction with reference cMC simulations that utilize Russian Roulette (RR) method. Specifically, we demonstrate that for a proximal detector placed immediately adjacent to the source, we can estimate the pMC relative error within 5% of the true value for scattering perturbations in the range of [-15%,+20%]. For a distal detector placed at ∼3 transport mean free paths relative to the source, our method provides relative error estimates within 20% for scattering perturbations in the range of [-8%,+15%]. Moreover, reference simulations performed at lower (μs'/μa) values showed better performance for both proximal and distal detectors.ConclusionsThese findings indicate that reference simulations utilizing continuous absorption weighting (CAW) with the Russian Roulette method and executed using optical properties with a low (μs'/μa) ratio spanning the desired range of μs values, are highly advantageous for the deployment of pMC to obtain radiative transport estimates over a wide range of optical properties.

In this paper, an on-chip optical circulator on the InP-membrane-on-Si (IMOS) platform is demonstrated. The circulator is composed of two multi-mode interferometers (MMIs), four polarization converters (PCs), and a Cerium-doped Yttrium Iron Garnet (Ce:YIG) die. The Ce:YIG die is adhesively bonded on the InP membrane via a 125-nm thin bonding layer. Nonreciprocal phase shift (NRPS) is employed in the presence of a transverse magnetic field. The device works as a 4-port optical circulator with a maximum optical isolation of 27.0 dB and a minimum optical isolation of 18.6 dB for the TE mode. The measured optical isolation bandwidth is 0.12 nm. Two methods are proposed in order to improve the optical isolation bandwidth.

Cancer associated fibroblasts (CAFs) are one of the predominant cell types of non-small cell lung cancer (NSCLC) tumour stroma. This thesis will investigate fibroblast heterogeneity in NSCLC; defining their phenotype by expression of CAF markers, phenotypic changes in culture and develop optical imaging probes for imaging fibroblasts in-situ. In the first results chapter, CAFs have been isolated from NSCLC patient resections and their phenotype characterised using known CAF markers by flow cytometry. This data led to the identification of five CAF subsets, CAFS1-S5. By comparing CAFs isolated from tumour tissue and fibroblasts isolated from tumour adjacent non-cancerous lung (NCL), it was identified that two of these subsets, CAF-S2 and CAF-S3, were phenotypically similar to NCL fibroblasts, whereas the three subsets, CAF-S1, CAF-S4 and CAF-S5, were predominant in cancerous tissue. The presence of these subsets was then investigated by multiplex immunofluorescence staining of a tumour microarray, which contained a cohort of 163 NSCLC patient tumours. It was found that the presence of CAF-S1 and CAF-S5, both subsets where CAFs expressed fibroblast activation protein (FAP) and podoplanin, with CAF-S1 also expressing alpha-smooth muscle actin, were indicative of poorer recurrence free survival outcome. Then in the second results chapter, the effects on cell phenotype of culturing CAFs and NCL fibroblasts was investigated. This revealed that when cultured by standard tissue culture methods, fibroblast phenotype converged to a CAF-S3 phenotype, regardless of cancerous or non-cancerous origin. To try and overcome the limitations of standard tissue culture methods, a NSCLC tumour organoid model was then developed, which allowed for the culture of other cell types, predominantly epithelial cells, alongside fibroblasts. Fibroblast activation protein (FAP) was expressed on the CAF subsets present predominantly in tumour tissue, and expression of FAP was low on NCL fibroblasts. In the final results chapter, FAP was considered as a target for optical imaging probes. The iterative development of a FAP probe which is specific over similar peptidases to FAP is described. These optical imaging probes could be used to highlight the tumour stroma and allow disease-monitoring, prognostication and potentially stratify treatment therapies. It is shown that a FAP probe can be designed to be selective over closely related peptidases, compatible with physiological levels of FAP expressed on primary CAFs, and the probe can be used to image NSCLC samples on a clinically compatible fibre-based imaging system. In conclusion, this thesis describes the CAF signatures in human NSCLC, changes over time in culture, the development of an organoid model and an optical imaging strategy for FAP imaging.

Quantitative oblique back-illumination microscopy (qOBM) is an emerging label-free optical imaging technology that enables 3D, tomographic quantitative phase imaging (QPI) with epi-illumination in thick scattering samples. In this work, we present a robust optimization of a flexible, fiber-optic-based qOBM system. Our approach enables in silico optimization of the phase signal-to-noise-ratio over a wide parameter space and obviates the need for tedious experimental optimization which could easily miss optimal conditions. Experimental validations of the simulations are also presented and sensitivity limits for the probe are assessed. The optimized probe is light-weight (∼40g) and compact (8mm in diameter) and achieves a 2µm lateral resolution, 6µm axial resolution, and a 300µm field of view, with near video-rate operation (10Hz, limited by the camera). The phase sensitivity is

The working distance is an essential parameter of the laser metal deposition (LMD) process which has to be kept at a constant level for successful applications. Especially when applying LMD in additive manufacturing (AM) to generate large-scale components, distance control is crucial as these parts can take multiple hours to complete. If over time the working distance runs out of the processing limits, the process efficiency will decrease, and this can lead to a process break down destroying the component build. The development approach is to integrate an enhanced distance measurement system based on optical coherence tomography (OCT) into a commercial LMD powder processing head. By deflecting an elliptic measuring track around the processing area, the OCT system can detect the working distance as well as topological information (2D + d) compared to a standard 1D distance measurement (1D + d). By continuously collecting data when moving the processing head, a 3D-scan of the surface can be recorded online. This technology is beneficial for AM as well as LMD repair applications as the system allows the scanning of unknown surfaces prior to LMD processing. In a next development step, the position data of the laser-cell (robot or CNC-Machine) is linked to the 2D + d data gaining full 3D information with high spatial and temporal resolution in real-time. Based on these monitoring capabilities, a closed loop control can be set up by implementing stabilization methods. By adapting LMD process parameters like the powder feed rate or the processing speed, a work distance and LMD track height control can be achieved.

Laser metal deposition is an additive manufacturing process that allows the production of near net shape structures. Moreover, the process can also be applied for the addition of material to an existing component for repair. To obtain structures with reproducible and good characteristics, it is necessary to understand the powder particle flow and the corresponding melt pool flow better. One of the critical parameters in this process is the convection within the melt pool. This convection is directly impacted and driven by flow phenomena occurring at the melt pool surface. The convection influences the geometry of the melt pool including its shape, aspect ratio, and can provoke defects such as lack of fusion, porosities, and unstable dilution levels. The surface flow can also have a direct impact on the formation of surface ripples. In the pursuance of a better understanding of the melt pool surface flow behaviour a high-speed camera was applied at a framerate of 40000 frames per second with a specific illumination system. In this paper an optical set-up with a high-speed camera and illumination system will be described. The images were obtained during the laser metal deposition process of single tracks from stainless steel (316L). The set-up optimization and employed image processing techniques allowed the detection of floating (316L) powder particles on the melt-pool. The visualization of their trajectories on the melt pool surface will be presented and discussed. In literature this kind experimental evaluations typically apply tracer particles to simplify the detection of the particles. In the future the obtained experience can be used to generate more data, which can support the validation numerical simulation model results and support the development of monitoring solutions or controlling systems.

Optoacoustic imaging (OAI) enables microscale imaging of endogenous chromophores such as hemoglobin at significantly higher penetration depths compared to other optical imaging technologies. Raster-scanning optoacoustic mesoscopy (RSOM) has recently been shown to identify superficial microvascular changes associated with human skin pathologies. In animal models, the imaging depth afforded by RSOM can enable entirely new capabilities for noninvasive imaging of vascular structures in the gastrointestinal tract, but exact localization of intra-abdominal organs is still elusive. Herein the development and application of a novel transrectal absorber guide for RSOM (TAG-RSOM) is presented to enable accurate transabdominal localization and assessment of colonic vascular networks in vivo. The potential of TAG-RSOM is demonstrated through application during mild and severe acute colitis in mice. TAG-RSOM enables visualization of transmural vascular networks, with changes in colon wall thickness, blood volume, and OAI signal intensities corresponding to colitis-associated inflammatory changes. These findings suggest TAG-RSOM can provide a novel monitoring tool in preclinical IBD models, refining animal procedures and underlines the capabilities of such technologies to address inflammatory bowel diseases in humans.