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Background The freshwater algae and cyanobacteria growth inhibition test (OECD test guideline 201) is frequently used to assess the ecotoxicity of chemicals or particles. A central issue is the measurement of algal growth by quantifying algal biomass over time. Chlorophyll fluorescence measurements are recommended for the testing of particles. The analysis of in vivo fluorescence is the simplest and fastest approach, but is only suitable if there is no interference with the materials. Therefore, in vitro fluorescence analysis is often preferred. We carried out a comprehensive comparison of chlorophyll fluorescence measurements in vitro and in vivo to evaluate the suitability of rapid in vivo testing for the determination of Raphidocelis subcapitata biomass in the presence of diverse particles. Results For the in vitro measurement, we applied a method that separates particles from chlorophyll using locust bean gum. We tested inorganic and organic particles (including alloys and polymers), ion-releasing and non-releasing materials, and particle sizes in the nanometer to micrometer range with a variety of shapes (spherical, flaky and fibrous). Some of the materials were nontoxic, whereas others showed varying degrees of toxicity (ErC50 = 0.2–100 mg/L in both methods). There were only minor differences between the methods in ErC50 values and the percent inhibition at various test concentrations, but the confidence intervals for the ErC50 values in vivo were narrower and were covered by the range observed in vitro. The in vivo approach showed no limitations, whereas the validity criteria listed in OECD test guideline 201 were not always fulfilled by the in vitro measurements. Conclusion The in vivo approach was a suitable and time-saving method for a wide range of particles, although we cannot completely exclude the possibility that some particles may interfere with fluorescence measurement. To avoid false assessments, pre-tests with simple measurements are therefore recommended. Graphical abstract

Bio-optical imaging can noninvasively describe specific biochemical reaction events in small animals using endogenous or exogenous imaging reagents to label cells, proteins, or DNA. The fluorescence optical bio-imaging system excites the fluorescent group to a high energy state by excitation light and then generates emission light. However, many substances in the organism will also emit fluorescence after being excited by the excitation light, and the nonspecific fluorescence generated will affect the detection sensitivity. This paper designs and develops a set of high-level biosafety in vivo fluorescence imaging system for small animals suitable for virology research and proposes a target area extraction algorithm for fluorescence images. The fluorescence image target extraction algorithm first maps the nonlinear separation data in the low-dimensional space to the high-dimensional space. Then, based on the analysis of the characteristics of the fluorescent region, a method for discriminating the target fluorescent region based on the two-step entropy function is proposed, and the real target fluorescent region is obtained according to the set connected region. Based on the experiment of collecting and analyzing the in vivo fluorescent images of mice, it is verified that the proposed algorithm can automatically extract the target fluorescent region better than the classical linear model. It shows that the proposed algorithm is less affected by background fluorescence, and the estimated separated spectrum based on this method is closer to the real target spectrum.

Herein, we develop a novel tripartite DNA probe to transport phosphorothioated substrate hairpins and an aptamer for the intramolecular CHA circuit, which achieves detection of a low amount of specific mRNA in living cells and mice. Our study provides an improved strategy to promote in vivo fluorescence imaging applications in early-stage clinical diagnosis.

Near-infrared (NIR, 700–1700 nm) bioimaging has gained considerable attention in recent years owing to its high imaging resolution and increasing penetration depth of tissues. NIR emissive lanthanide(III) complexes have emerged as alluring candidates for the design of NIR molecular probes in clinical imaging and diagnosis because of their desirable temporal–spatial resolution, negligible photobleaching effect, long lifetime and low biotoxicity. In this mini-review, we summarize our efforts in the molecular design of NIR luminescent lanthanide porphyrinoids and their biological applications in bioimaging and further clinical diagnosis and treatment. Remarkably, β-fluorinated porphyrin Yb3+ probes exhibit high luminescence (5–13% quantum yields in H2O), long decay lifetimes (>100 μs) and enable in vivo fluorescence intensity imaging and time-resolved fluorescence lifetime imaging (FLIM). Finally, the main concerns and challenges for NIR emissive Ln biolabels such as the diversity of the NIR Ln3+ optical toolbox and their biocompatibility and targeting capability in biological media are discussed so as to facilitate the long-term development of lanthanide chemical biology.

Personalized approaches for continuous monitoring of chloride levels are potentially valuable for evaluating the efficacy of new treatments of genetic disorders such as cystic fibrosis. In this report, we validated optode-based nanosensors for real-time chloride monitoring in the interstitial fluid of living animals. These nanosensors take advantage of a ratiometric sensing scheme which demonstrates reversible and selective chloride detection in the physiological range. We further investigate how skin pigmentation affects the sensor performance during in vivo fluorescence imaging. We successfully monitored endogenous chloride changes using nanosensors during pharmacological treatment in a cystic fibrosis mouse model. We believe this platform is a valuable tool for chloride detection which could assess the efficacy of new treatments for cystic fibrosis.

The use of multi-wavelength spectrofluorometers for the fast detection of algal taxa, based on chlorophyll a (Chl-a) emission spectra, has become a common practice in freshwater water management, although concerns about their accuracy have been raised. Here, inter-laboratory comparisons using monoalgal cultures have been performed to assess the reliability of different spectrofluorometer models, alongside Chl-a extraction methods. Higher Chl-a concentrations were obtained when using the spectrofluorometers than extraction methods, likely due to the poor extraction efficiencies of solvents, highlighting that traditional extraction methods could underestimate algal or cyanobacterial biomass. Spectrofluorometers correctly assigned species to the respective taxonomic group, with low and constant percent attribution errors (Chlorophyta and Euglenophyceae 6–8%, Cyanobacteria 0–3%, and Bacillariophyta 10–16%), suggesting that functioning limitations can be overcome by spectrofluorometer re-calibration with fresh cultures. The monitoring of a natural phytoplankton assemblage dominated by Chlorophyta and Cyanobacteria gave consistent results among spectrofluorometers and with microscopic observations, especially when cell biovolume rather than cell density was considered. In conclusion, multi-wavelength spectrofluorometers were confirmed as valid tools for freshwater monitoring, whereas a major focus on intercalibration procedures is encouraged to improve their reliability and broaden their use as fast monitoring tools to prevent environmental and public health issues related to the presence of harmful cyanobacteria.

This chapters provides an overview of the recent applications of tetrapyrrolic macrocycles for in vivo fluorescence imaging. Recently, porphyrinic compounds have been used as theranostic agents for photodynamic therapy (PDT) or photothermal therapy. They have also been used as multimodal imaging agents, a way to combine fluorescence photoacoustic imaging, positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound imaging. The simple porphyrin derivatives typically possess low fluorescence brightness and short absorption wavelengths. However, structural modifications allow for a great improvement of these properties and tailor them for deep tissue applications. Thus, benzoporphyrins, strongly conjugated hydroporphyrin arrays, as well as hydroporphyrins (i.e., partially saturated porphyrin derivatives) show optical properties adequate for in vivo fluorescence imaging. Specifically, hydroporphyrins have been broadly used for simultaneous cancer treatment and multimodal imaging. Synthetic bacteriochlorins can be used as fluorescent probes for multicolor fluorescence-guided surgery. Benzoporphyrins and lanthanide porphyrinic complexes have been applied for in vivo oxygen sensing and as theranostic agents for PDT and multimodal imaging, respectively. Considerable efforts have been devoted to formulation of nanostructures containing hydroporphyrins. Nanoscience enables new modes for delivering, targeting, and activating fluorescent hydroporphyrins, as well as more efficient combination of fluorescence with other imaging and therapeutic modalities.

The interaction between light and tissue such as absorption and scattering limits the penetration depth and spatiotemporal resolution of in vivo fluorescence bioimaging in a noninvasive manner. The...

In vivo fluorescence imaging within the second near-infrared region (NIR-II, 1000–1700 nm) has advantages of a higher signal-to-background ratio (SBR), spatial resolution, and deeper tissue penetra...

Despite the recognition of the lethality of cancer metastasis and the importance of developing specific anti-metastasis therapies directed at the cancer metastatic cascade, the dynamics of cancer metastasis remains poorly understood. In this study, we examined the dynamics of circulating tumor cell (CTC) survival in the bloodstream using experimental mouse models. CTCs were arrested in the capillaries by adhesion to vascular endothelium within a few minutes after injection into the bloodstream. The loss of CTCs from the circulation followed a bi-phasic decay pattern, with the number of CTCs in the bloodstream being closely associated with the number of blood circulation cycles. The calculated in vivo Vd (apparent volume of distribution) of the CTC revealed organ specific binding of the CTCs. Moreover, confocal microscopy, in vivo fluorescence imaging in syngeneic mouse metastatic models and analysis of blood circulation patterns support the notion of organ-specific tumor metastasis. The present study suggests that organ-specific tumor metastasis is influenced by cooperation between blood circulation patterns and 'seed-soil' compatibility factors. These new findings provide further insights for optimized cancer metastatic prevention strategies such as by creating a hostile circulation microenvironment and targeting the organ-specific 'seed-soil' compatibility factors.

Current therapeutics for glioblastoma multiforme (GBM) treatment are unsatisfactory due to their limited ability to control the progression from tumour margins. In this work, organic nanoparticles (NPs) are synthesized by co-encapsulating a fluorogen with aggregation-induced emission to generate a bright red emission for imaging and a semiconducting polymer to offer NIR absorption for photothermal therapy. The NPs are further modified with different ratios of two targeting ligands, folate and cRGD peptide. The best ratio that performs specific and efficient GBM targeting is screened out through in vitro and ex vivo fluorescence imaging analysis. The NPs with an FA to cRGD ratio of 25 : 75 exhibit superior ability to target GBM cells in vitro and also show efficient accumulation at the GBM margin and in the tumour interior after in vivo administration. The progression of GBM can be greatly suppressed through photothermal therapy, which provides a simple but promising strategy for GBM treatment.

BackgroundIntracranial photometry through chronically implanted optical fibers is a widely adopted technique for measuring signals from fluorescent probes in deep-brain structures. The recent proliferation of bright, photo-stable, and specific genetically-encoded fluorescent reporters for calcium and for other neuromodulators has greatly increased the utility and popularity of this technique.New MethodHere we describe an open-source, cost-effective, microcontroller-based solution for controlling optical components in an intracranial photometry system and processing the resulting signal.ResultsWe show proof-of-principle that this system supports high quality intracranial photometry recordings from dorsal striatum in freely moving mice. A single system supports simultaneous fluorescence measurements in two independent color channels, but multiple systems can be integrated together if additional fluorescence channels are required. This system is designed to work in combination with either commercially available or custom-built optical components. Parts can be purchased for less than one tenth the cost of commercially available alternatives and complete assembly takes less than one day for an inexperienced user.Comparison with Existing Method(s)Currently available hardware draws on a variety of commercial, custom-built, or hybrid elements for both optical and electronic components. Many of these hardware systems are either specialized and inflexible, or over-engineered and expensive.ConclusionsThis open-source system increases experimental flexibility while reducing cost relative to current commercially available components. All software and firmware are open-source and customizable, affording a degree of experimental flexibility that is not available in current commercial systems.

Rod-shape nanoplatform have received tremendous attention owing to their enhanced ability for cell internalization and high capacity for drug loading. MoS2, widely used in electronic devices, electrocatalysis, sensor and energy-storage, has been studied as photothermal agents over the years. However, the efficacy of rod-shape MoS2 based photothermal agents for photothermal therapy has not been studied before. Here, a near-infrared (NIR) light-absorbing MoS2 nanosheets coated mesoporous silica nanorods with human serum albumin (HSA) modifying and Ce6 loading (MSNR@MoS2-HSA/Ce6) were constructed for combined photothermal and photodynamic therapy. Methods: The near-infrared (NIR) light was used to trigger the synergistic anti-tumor therapy. In addition, breast cancer cell line was applied to evaluate the in vitro anti-tumor activity. The multi-modal imaging capacity and tumor-killing efficiency of the designed nanocomposites in vivo was also demonstrated with the 4T1 tumor-bearing mouse model. Results: These nanocomposites could not only perform NIR light triggered photodynamic therapy (PDT) and photothermal therapy (PTT), but also achieve in vivo fluorescence (FL) /multispectral optical tomography (MSOT)/X-ray computed tomography (CT) triple-model bioimaging. What's more, the rod-shape nanoplatform could be endowed with better anti-tumor ability based on the EPR effect and HSA-mediated active tumor targeting. At the same time, the hyperthermia generated by MoS2 could synergistically improve the PDT effect with the acceleration of the blood flow, leading to the increase of the oxygen level in tumor tissue. Conclusion: MSNR@MoS2-HSA/Ce6 proves to be a promising multi-functional nanoplatform for effective treatment of tumor.

Colorectal cancer (CRC) is one of the most frequently lethal forms of cancer. Intramucosal injection allows development of better mouse models of CRC, as orthotopic xenografts allow development of adenocarcinoma in the submucosa of the mouse colon wall. In this paper, a method of orthotopic injection is monitored longitudinally using cellular-resolution real-time in vivo fluorescence microendoscopy, following the injection of three different cell lines: 3T3-GFP to confirm immunosuppression and HCT116-RFP cells to model CRC. Adenoma formation is first observable after 7 to 10 days, and by use of 33 G needles a tumor induction rate of greater than 85% is documented. An additional experiment on the injection of rapamycin reveals drug efficacy and localization between 24 and 48 hours, and suggests the promise of real-time cellular-resolution fluorescence micro-endoscopy for developing longitudinal therapy regimes in mural models of CRC.

In vivo fluorescence imaging in the second near-infrared window (NIR-II) has been considered as a promising technique for visualizing mammals. However, the definition of the NIR-II region and the mechanism accounting for the excellent performance still need to be perfected. Herein, we simulate the photon propagation in the NIR region (to 2340 nm), confirm the positive contribution of moderate light absorption by water in intravital imaging and perfect the NIR-II window as 900–1880 nm, where 1400–1500 and 1700–1880 nm are defined as NIR-IIx and NIR-IIc regions, respectively. Moreover, 2080–2340 nm is newly proposed as the third near-infrared (NIR-III) window, which is believed to provide the best imaging quality. The wide-field fluorescence microscopy in the brain is performed around the NIR-IIx region, with excellent optical sectioning strength and the largest imaging depth of intravital NIR-II fluorescence microscopy to date. We also propose 1400 nm long-pass detection in off-peak NIR-II imaging whose performance exceeds that of NIR-IIb imaging, using bright fluorophores with short emission wavelength., Moderate light absorption by bio-tissue is conducive to the imaging performance. The second near-infrared window is perfected as 900–1880 nm, and 2080–2340 nm is proposed as the third near-infrared window.

In vivo fluorescence imaging in the second near-infrared window (NIR-II) has been considered as a promising technique for visualizing the mammals. However, the definition of the NIR-II region and the mechanism accounting for the excellent performance still need to be perfected. Herein, we simulated bioimaging in the NIR spectral range (to 2340 nm), confirmed the positive contribution of moderate light absorption by water in intravital imaging and perfected the NIR-II window as 900-1880 nm, where the 1400-1500 nm was defined as NIR-IIx region and the 1700-1880 nm was defined as NIR-IIc region, respectively. Moreover, the 2080-2340 nm was newly proposed as the third near-infrared (NIR-III) window, which was believed to provide the best imaging quality. The wide-field fluorescence microscopy in brain, in addition, was performed around NIR-IIx region with excellent optical sectioning strength and the largest imaging depth of in vivo NIR-II fluorescence microscopy to date. We also proposed 1400 nm long-pass detection in off-peak NIR-II imaging whose profits exceeded those of NIR-IIb imaging, using bright fluorophores with short peak emission wavelength.

BackgroundMesenchymal cell has been frequently used in clinical studies. Mesenchymal stem cells (MSCs) are self-renewing, multipotent stem cells with the potential to differentiate into multiple mesoderm lineages. But MSC have limitation in clinical application for treating human diseases because they can differentiate several types of cell but not all types. PSL (Pluripotent Stem cell Like cell) are newly developed pluripotent stem cells from human mesenchymal stem cells (hMSC) induced by small molecule compounds. These cells have potential advantages for clinical cell treatment compared with ESCs and iPSCs.MethodsWe induced pluripotency from MSC using small molecules. It has tried to trace MSC and PSL in mice using bioluminescent techniques, which can detect visible light emitted from cells labeled with miRNA conjugated fluorescent molecules. ResultsMSCs predominantly migrate into the brain and testis. They also migrate to the liver, omentum, mesentery, kidneys and spleen. Migration of PSL is similar to MSCs, in that they go to the brain, testis and other intraperitoneal organs. Fluorescent images of explanted organs show that the intensity of brain images is higher in the PSL mouse group than the MSC mouse group. However, testis, image intensity is higher in MSC mouse group than the PSL mouse group. In PSL but not MSC mice, fluorescence persisted at the injection site in the tail.ConclusionsIn this study, injected MSCs and PSL predominantly migrated to the brain and testis. But, PSL migration was more than MSC migration in Brain. Both cell types had a similar migration pattern except for persistent fluorescence at injection site in the tail vein of PSL mice. We expect these cell therapy may have many potentials for clinical studies on these notable treatments.

Hepatic glutathione depletion, a crucial pathophysiological event, can now be readily monitored for early disease remedy., Hepatic glutathione plays a key role in regulating redox potential of the entire body, and its depletion is known to increase susceptibility to oxidative stress involved in many diseases. However, this crucial pathophysiological event can only be detected noninvasively with high-end instrumentation or invasively with surgical biopsy, limiting both preclinical research and clinical prevention of oxidative stress–related diseases. Here, we report that both in vivo fluorescence imaging and blood testing (the first-line detection in the clinics) can be used for noninvasive and consecutive monitoring of hepatic glutathione depletion at high specificity and accuracy with assistance of a body-clearable nanoprobe, of which emission and surface chemistries are selectively activated and transformed by hepatic glutathione in the liver sinusoids. These findings open a new avenue to designing exogenous blood markers that can carry information of local disease through specific nanobiochemical interactions back to the bloodstream for facile and rapid disease detection.

BACKGROUND: Photobiomodulation (PBM) by far-red (FR) to near-infrared (NIR) light has been demonstrated to accelerate diabetic wound healing in preclinical and clinical studies. Mitochondrial dysfunction and oxidative stress play key roles in impaired diabetic wound healing, and the effect of PBM on the metabolic state of diabetic wounds remains to be elucidated. METHODS: In this study, a custom-designed in vivo fluorescence imaging technique was used to quantitatively assess the effect of FR-PBM on the mitochondrial bioenergetics of diabetic wounds. The intrinsic fluorescence of two mitochondrial co-enzymes, nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), was monitored to quantify the redox ratio (RR) (NADH/FAD) of wounds over time. RESULTS: Using an excisional model of wound healing, we demonstrated that 670 nm (FR) PBM improved mitochondrial bioenergetics and stimulated the rate of wound healing in diabetic db/db mice. Wound closure and the RR of diabetic wounds in response to 670 nm PBM (4.5 J/cm(2), 60 mW/cm(2) for 90 s per day, 5 days/week) were compared to the sham-treated group. At day 9 of post-wounding, we observed a 43% decrease in the wound area and a 75% increase in RR in FR-treated diabetic mice compared to sham-treated diabetic mice. CONCLUSIONS: We conclude that the increase in mitochondrial RR and the related decrease in oxidative stress may be an important factor in FR-PBM mediated acceleration of wound healing in diabetic mice.

Lifetime multiplexed imaging refers to the simultaneous labeling of different structures with fluorescent probes that present identical photoluminescence spectra and distinct fluorescence lifetimes. This technique allows extracting quantitative information from multichannel in vivo fluorescence imaging. In vivo lifetime multiplexed imaging requires fluorophores with excitation and emission bands in the near-infrared (NIR) and tunable fluorescence lifetimes, plus an imaging system capable of time-resolved image acquisition and analysis.

A portable sensor for fluorescence spectroscopy of liquids has been developed. This sensor exploits the autofluorescence produced by organic chemical compounds when exposed to ultraviolet or visible light. In particular, the autofluorescence produced by pigments present in cyanobacteria and microalgae allow their in vivo detection. Liquid jet waveguide approach has been chosen in order to provide efficient fluorescence light collection. A mini-spectrophotometer is used as a detector and a mini-PC manages all the instrumentation. The whole sensor is housed inside a weather-proof plastic enclosure which allows it to be transported as hand luggage. The sensor can be equipped with three different LED sources that can be easily replaced according to the needs due to the type of substances intended to detect. Several sources at different wavelengths have been taken into consideration and characterized for this purpose. The excitation at multiple wavelengths provides the possibility of discrimination between different substances or phytoplankton groups. Preliminary measurements, using excitation wavelength at 275, 405 and 590 nm, show the possibility of detecting the presence of cyanobacteria in water collected at different pickup points in an aquaponics plant.

The exciting advancements that we are currently witnessing in terms of novel materials and synthesis approaches are leading to the development of colloidal nanoparticles (NPs) with increasingly greater tunable properties. We have now reached a point where it is possible to synthesize colloidal NPs with functionalities tailored to specific societal demands. The impact of this new wave of colloidal NPs has been especially important in the field of biomedicine. In that vein, luminescent NPs with improved brightness and near-infrared working capabilities have turned out to be optimal optical probes that are capable of fast and high-resolution in vivo imaging. However, luminescent NPs have thus far only reached a limited portion of their potential. Although we believe that the best is yet to come, the future might not be as bright as some of us think (and have hoped!). In particular, translation of NP-based fluorescence imaging from preclinical studies to clinics is not straightforward. In this Perspective, we provide a critical assessment and highlight promising research avenues based on the latest advances in the fields of luminescent NPs and imaging technologies. The disillusioned outlook we proffer herein might sound pessimistic at first, but we consider it necessary to avoid pursuing "pipe dreams"and redirect the efforts toward achievable - yet ambitious - goals, This work has been cofinanced by European Structural and Investment Fund and by the European Union’s Horizon 2020 FET Open program under grant agreement no. 801305 (NanoTBTech). E.X. is grateful for a Juan de la Cierva Formacion scholarship (FJC2018-036734-I). A.B. acknowl- ́ edges funding from Comunidad de Madrid through TALENTO grant ref. 2019-T1/IND-14014. D.J

A new family of α, α directly linked bisBODIPYs was developed through a MoCl5-mediated intramolecular oxidative reaction. Due to the coplanar structure of the two conformationally locked BODIPY units, these bisBODIPYs showed well-extended conjugations and gave strong near-infrared absorptions and emissions with maxima around 760 and 780 nm, respectively, with high fluorescence quantum yields of ≤0.84. These dyes were successfully applied for in vitro and in vivo fluorescence imaging by taking advantage of their beneficial photophysical properties.

We have developed a compact optical endoscopic probe for in vivo fluorescence optical imaging device. We obtained fluorescence image for the colon tissue of a mouse using a compacted optical endoscopic probe with a designed endoscopic optical lens. In order to demonstrate endoscopic fluorescence imaging for the colon cancer, we have manufactured a compacted optical endoscopic probe to pass through the biopsy channel of electric flexible endoscope. The compacted optical endoscopic probe with maximum outside diameter of 2.8mm consists of fiber-optic illumination part and imaging part. The imaging part consists of a fiber-optic imaging bundle linked to an endoscopic optical lens and focus assembly. We considered a compact structure, sensitivity, and FOV for the design of the endoscopic optical lens. We have suggested an endoscopic optical lens with an FOV of 90 ° and DOF of 3 – 80mm. The optical system consists of glass-based aspheric lenses. The total track is less than 2.5 mm, and the diameter is limited to less than 1.5 mm to obtain a compact system. We have presented the compacted optical endoscopic probe for cancer imaging of a mouse. The proposed endoscopic optical lens showed sufficient sensitivity and a wide field of view for obtaining fluorescence imaging. We demonstrated endoscopic ex vivo fluorescence imaging for the colon cancer of a mouse using the compacted optical endoscopic probe.

Fluorescence imaging over large areas of the brain in freely behaving animals would allow researchers to better understand the relationship between brain activity and behavior; however, traditional microscopes capable of high spatial resolution and large fields of view (FOVs) require large and heavy lenses that restrict animal movement. While lensless imaging has the potential to achieve both high spatial resolution and large FOV with a thin lightweight device, lensless imaging has yet to be achieved in vivo due to two principal challenges: (a) biological tissue typically has lower contrast than resolution targets, and (b) illumination and filtering must be integrated into this non-traditional device architecture. Here, we show that in vivo fluorescence imaging is possible with a thin lensless microscope by optimizing the phase mask and computational reconstruction algorithms, and integrating fiber optic illumination and thin-film color filters. The result is a flat, lensless imager that achieves better than 10 μm spatial resolution and a FOV that is 30× larger than other cellular resolution miniature microscopes.

Recently, near-infrared (NIR) fluorescent dyes such as indocyanine green (ICG) have received tremendous interest as contrast agents for use in fluorescence-guided, intraoperative cancer resection surgery. However, despite showing great promise, ICG has many shortcomings such as rapid clearance and poor tumor accumulation. To improve the selective accumulation of ICG within tumors, numerous groups have formulated ICG into nanoparticles, but these approaches can suffer from rapid leakage of ICG, use of materials that exhibit poor or incomplete excretion, or complex chemistries that are not easily amenable to scale up for clinical use. Here, we developed a simple one-step method to prepare ICG-based fluorescent micelles that are composed solely of unmodified ICG and polycaprolactone (PCL), two clinically used materials with well-characterized safety profiles. The ICG-PCL micelles are prepared via oil-in-water emulsions, and the resulting micelles exhibit a uniform size, good reproducibility, and high loading efficiency. In vivo fluorescence imaging demonstrated that the ICG-PCL micelles led to a significant improvement in the accumulation and retention of ICG, in four different tumor models, compared with free dye, making them an attractive option for image-guided surgery.

The development of nanoparticles for NIR imaging and diagnostics is an area of considerable interest. Among the different imaging modalities, optics emerged as an interesting technique since it is a non-invasive, cheap imaging technique allowing real-time imaging. In vitro, this technique is very useful; however, in vivo fluorescence imaging suffers from suboptimal signal-to-noise ratio, which is caused by the strong tissue autofluorescence under constant external excitation. To address this limitation, novel types of optical nanoprobes are actually being developed in the deep red/near infrared (NIR) range and among them, persistent luminescence nanoparticles (PLNPs), with long-lasting near-infrared luminescence capability. These NPs allow optical imaging to be performed in an excitation-free and consequently autofluorescence-free manner. This chapter will first introduce the physical phenomenon associated to the long luminescence delay of such nanoprobes, from minutes to hours after ceasing the excitation, and will then highlight the tools used in physicochemistry laboratories to characterize these nanoparticles with a focus on the ZnGa2O4:Cr3+ nanoparticles which emit persistent luminescence in the first biological window in the deep red range and which are widely studied over the world. Then their biocompatibility will be mentioned and finally the evaluation in term of new advances for in vivo bioimaging theranostics nanoprobes will be presented. We will conclude this chapter by envisioning perspectives for such nanomaterials.

Two new cyanine-based fluorescent probes 1 and 2 have been developed. Probe 1 bears two cyanine units in a single molecule, and probe 2 contains a bis(trifluoromethyl)benzenethiol moiety. Both are non-fluorescent. The addition of intracellular glutathione (GSH) significantly enhanced the NIR fluorescence of the two probes. Both probes were used to image varying amounts of GSH in living cells. In tumor bearing mice, the in vivo fluorescence intensity of both probes was higher in tumors, where GSH is overexpressed, than in normal tissues. These results suggest that these new fluorogenic probes have potential for GSH-targeting diagnostic imaging.

Fluorescence molecular imaging is a promising tool for molecular tracking, and thus can visualize the vascular structure of small animal. However, with the strong scattering in biological tissues, traditional fluorescence molecular imaging in the visible spectrum or the first near-infrared spectrum (NIR-I, 700-900 nm) has a limitation for high-resolution vascular imaging. Recently, the novel in vivo fluorescence molecular imaging in the longer second near-infrared window (NIR-IIb, 1500-1700 nm) is successfully developed for small animal imaging. The NIR-IIb window affords high imaging resolution and deep tissue penetration because of the diminished photon scattering effect, which is suitable for vascular imaging. However, the clinical applications of NIR-IIb fluorescence molecular imaging have been severely limited for lack of the clinical fluorophores. Here, we show that the clinically available dye, indocyanine green (ICG), can also emit the NIR-IIb signal, which provides high-resolution vascular imaging of small animal. We construct a novel imaging system for NIR-I and NIR-IIb imaging simultaneously and perform two vascular imaging experiments. The results demonstrate that the NIR-IIb imaging using ICG shows great superiority for high-resolution vascular imaging of small animal compared with NIR-I imaging. It is believed that this study will facilitate the preclinical and clinical applications of NIR-IIb molecular imaging using ICG in the future.

Currently, there are no non-invasive tools to accurately diagnose wound and surgical site infections before they become systemic or cause significant anatomical damage. Fluorescence and photoacoustic imaging are cost-effective imaging modalities that can be used to noninvasively diagnose bacterial infections when paired with a molecularly targeted infection imaging agent. Here, we develop a fluorescent derivative of maltotriose (Cy7-1-maltotriose), which is shown to be taken up in a variety of gram-positive and gram-negative bacterial strains in vitro. In vivo fluorescence and photoacoustic imaging studies highlight the ability of this probe to detect infection, assess infection burden, and visualize the effectiveness of antibiotic treatment in E. coli-induced myositis and a clinically relevant S. aureus wound infection murine model. In addition, we show that maltotriose is an ideal scaffold for infection imaging agents encompassing better pharmacokinetic properties and in vivo stability than other maltodextrins (e.g. maltohexose)., Sensitive diagnostic tools for bacterial infections of wounds and surgical sites are necessary to enable early detection and determine optimal means of treatment. Here, the authors develop a fluorescent and optoacoustic probe based on a maltotriose scaffold, which is selectively taken up by gram-positive and gram-negative bacteria.

Perihilar cholangiocarcinoma (PHCC) presents a formidable challenge due to its occult anatomic location, aggressive growth, insensitivity to conventional chemotherapy, and poor prognosis. Herein, we engineered a human epidermal growth factor receptor 2 (HER2) affibody to the surface of cell membrane nanovesicles (A-NVs) in a ligand-oriented manner and loaded them with indocyanine green (ICG) as precision theranostics for PHCC treatment. The A-NVs@ICG were prepared and exhibited satisfactory targeting effects in HER2-overexpressing PHCC cells. In vivo fluorescence and photoacoustic imaging demonstrated that A-NVs@ICG promoted the accumulation of ICG in PHCC tissue, leading to enhanced tumor regression and improved anti-cancer effects when combined with photoirradiation. Therefore, bio-engineered A-NVs@ICG represent a promising nanotheranostic agent for PHCC with potential for clinical translation.

In the past decade, biological imaging in the second near infrared (NIR-II) window has emerged as a promising imaging method to visualize deep-tissue anatomical structures and profile internal physiological status. Owing to advantages in reduced light absorption, suppressed photon scattering, and minimized interference from tissue autofluorescence, NIR-II imaging presents advantages on improved penetration depth and high spatial resolution, opening up wide opportunities to unmask the underlying mechanisms of various physiological processes. An ideal NIR-II fluorophore for in vivo fluorescence imaging should have high quantum yields, red-shifted emission wavelengths as well as favorable pharmacokinetic properties in order to afford high imaging quality, monitor dynamic physiological process in real time, and mitigate safety concerns. Here, in this chapter, we summarize recent advances in rational design and optimization of NIR-II fluorescence imaging agents with superior properties as mentioned above, present their applications, and offer our opinions on the unique potentials of these imaging agents for future clinical translation.

Using vertical profiles of temperature and in vivo fluorescence sampled between Tanhoy and Listvyanka, we show that temperature and fluorescence fluctuations exhibit specific levels of internal structuration that are independent of the overall vertical structure of the water column, which suggest a previously untapped small-scale biophysical coupling.