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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.

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...

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.

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.

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.

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.

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.

International audience; Intercellular communication plays a crucial role in the establishment of multicellular organisms by organizing and coordinating growth, development and defence responses. In plants, cellto-cell communication takes place through nanometric membrane channels called plasmodesmata (PD). Understanding how PD dictate cellular connectivity greatly depends on a comprehensive knowledge of the molecular composition and the functional characterization of PD components. While proteomic and genetic approaches have been crucial to identify PD-associated proteins, in vivo fluorescence microscopy combined with fluorescent protein tagging is equally crucial to visualise the subcellular localisation of a protein of interest and gain knowledge about their dynamic behaviour. In this protocol we describe in detail a robust method for quantifying the degree of association of a given protein with PD, through ratiometric fluorescent intensity using confocal microscopy. Although developed for N. benthamiana and Arabidopsis, this protocol can be adapted to other plant species.

Over the past 50 years, protein complexes have been studied with techniques such as X-ray crystallography and electron microscopy, generating images which although detailed are static and homogeneous. More recently, limited application of in vivo fluorescence and other techniques has revealed that many complexes previously thought stable and compositionally uniform are dynamically variable, continually exchanging components with a freely circulating pool of "spares." Here, we consider the purpose and prevalence of protein exchange, first reviewing the ongoing story of exchange in the bacterial flagella motor, before surveying reports of exchange in complexes across all domains of life, together highlighting great diversity in timescales and functions. Finally, we put this in the context of high-throughput proteomic studies which hint that exchange might be the norm, rather than an exception.

Penicillin G acylase (PGA) is a hydrolytic enzyme, which is also industrially employed in the preparation of semisynthetic penicillin. Abuse of PGA might occur in milk products to decompose the possible excessive antibiotics. Detection of PGA, including in vivo imaging of PGA, is necessary to understand the potential function of this enzyme in living biosystems; however, a near-infrared (NIR) fluorescent probe for PGA is still lacking. On the basis of amino hemicyanine as well as the ability of PGA to hydrolyze phenylacetamide, in this work we have developed a new selective and sensitive NIR fluorescent probe for such a purpose. Combined with in vivo fluorescence imaging system, the probe has been preliminarily used to image the change of the PGA activity in mice. In addition, the NIR feature of our probe makes it of potential use in studying the function of PGA in vivo .

A rhodamine-based sensor ( 1 ) has been developed for the detection of chromium ions. Cr 3+ -induced opening of the rhodamine spirocycle in sensor ( 1 ) led to the distinct colorimetric and fluorescence responses. Among all the tested ions, only Cr 3+ generated a significant fluorescence enhancement of up to 13-fold, which indicated the high selectivity of 1 . Sensor ( 1 ) was successfully applied in the in vivo fluorescence imaging of Cr 3+ in C. elegans . The results provided solid evidences for the future estimation of Cr 3+ in environmental applications and tobacco samples.

The blood-brain barrier (BBB) plays important roles in both the physiological and pharmacological state of the brain. Transiently enhancing the permeability of the BBB may allow use of more types of medications for neuropsychiatric diseases. Several studies have demonstrated that seizures cause a transient decrease in BBB integrity. We studied the timing of BBB changes following seizures and the role of astrocytes in this process. Rats received 10 applications of electroconvulsive stimulation (ECS). They were then infused with sodium fluorescein, a fluorescent substance that rarely passes the BBB, via the inferior vena cava. After 120 min of circulation, the amount of sodium fluorescein in the brain was measured by two methods in vivo fluorescence imaging (total radiant efficiency) and the brain concentration of sodium fluorescein. To assess any changes to the BBB, we measured S100Β in serum, which is a standard marker of BBB breakdown that is expressed by astrocytes. We also examined ultrastructural changes following ECS. Total radiant efficiency and the brain concentration of sodium fluorescein were significantly increased in treated rats compared to controls when sodium fluorescein was injected immediately after ECS but not when the injection was performed more than 15 min after ECS. Astrocytic endfeet showed swelling around brain capillaries following ECS. In conclusion, ECS transiently enhances the permeability of the BBB, which may be accompanied by changes in astrocytic endfeet.

Near-infrared fluorescence imaging (NIRFI) and far-red fluorescence imaging (FRFI) were used to investigate effects of depilation-induced skin pigmentation and diet-induced background fluorescence on fluorescent signal amplitude and lymphatic contraction frequency in C57BL6 mice. Far-red fluorescent signal amplitude, but not frequency, was affected by diet-induced fluorescence, which was removed by feeding the mice an alfalfa-free diet, and skin pigmentation further impacted the amplitude measurement. NIRFI showed minimal background fluorescence; however, skin pigmentation reduced the amplitude of fluorescent signal changes. Therefore, these effects should be taken into account when imaging mice with different states of skin pigmentation and diet-induced background fluorescence in vivo.

Near-infrared (NIR)-responsive drug delivery systems have received enormous attention because of their good biocompatibility and high biological penetration. In this work, we report a novel 1-tetradecanol (TD)-controlled and indocyanine green (ICG)-loaded CuS@mSiO2 phototherapy nanoplatform (CuS@mSiO2-TD/ICG). The CuS@mSiO2 nanoparticles prepared by a facile one-pot approach can serve as drug-delivery vehicles to transport the NIR absorbing phototherapeutic agent (ICG) within the mesoporous cavities. Meanwhile a phase-change molecule (PCM), TD, is introduced as a thermosensitive gatekeeper to avoid the premature release of loaded ICG. Noticeably, the combined therapy is irradiated at an 808 nm single-light wavelength, thus performing the photothermal therapy (PTT) based on CuS@mSiO2 as well as simultaneously triggering the photodynamic (PDT)/PTT effect based on ICG. Furthermore, ICG also has the function of dual in vivo fluorescence imaging and photoacoustic (PA) imaging. This dual imaging-guided and gatekeeper-controlled nanoplatform for the single-light triggered PTT/PDT treatment holds significant promise for future cancer therapy due to their markedly improved therapeutic efficacy and decreased systemic toxicity.

Naturstoff reagent A (diphenylboric acid 2-aminoethyl ester [DPBA]) has been used historically in plant science to observe polyphenolic pigments, such as flavonoids, whose fluorescence requires enhancement to be visible by microscopy. Flavonoids are common dietary constituents and are the focus of considerable attention because of their potential as novel therapies for numerous diseases. The molecular basis of therapeutic activity is only gradually being established, and one strand of such research is making use of the social amoeba Dictyostelium discoideum. We extended the application of DPBA to flavonoid imaging in these preclinical studies, and report the first method for use of DPBA in this eukaryotic model microbe and its applicability alongside subcellular markers. This in vivo fluorescence imaging provided a useful adjunct to parallel chemical and genetic studies.

Phosphatidylserine externalization is an early molecular signature for apoptosis. In many retinal degenerative diseases, photoreceptor neurons die by apoptosis. Here, we report utility of the phosphatidylserine-binding conjugate of Bis(zinc(II)-dipicolylamine (Zn-DPA) with Texas-red (PSVue-550) in transiently labeling apoptotic photoreceptors in living pigmented or albino rats and mice with retinal degeneration. Applying PSVue-550 as eyedrop is non-toxic and eliminates need for intraocular injection. PSVue-550 fluorescence specifically and transiently labeling dying retinal photoreceptors is detectable in anesthetized animals using standard retinal or whole small animal imaging systems. Importantly, prior PSVue-550 eyedrop administration and imaging does not affect repeat testing. Altogether, our results establish PSVue-550 imaging as a completely non-invasive method that provides the opportunity to longitudinally monitor retinal photoreceptor cell death in preclinical studies.

Fluorescent carbon dots (CDs) have been extensively utilized as responsive drug nanocarriers to deliver anticancer agents, owing to their facile preparation, excellent water solubility, good photostability, and high quantum yield. Herein, we summarize the protocols for the synthesis and application of responsive CDs toward anticancer drug delivery both in vitro and in vivo. Specially, this chapter includes the preparation and structural characterization of CDs and anticancer prodrug-loaded CDs, in vitro anticancer drug release, in vitro and in vivo fluorescence imaging, and toxicity studies.

Topical drug delivery system to the posterior segment of the eye is facing many challenges, such as rapid drug elimination, low permeability, and low concentration at the targeted sites. To overcome these challenges, Multifunctional nanocomposite eye drops of dexamethasone-carboxymethyl-β-cyclodextrin@layered double hydroxides-glycylsarcosine (DEX-CM-β-CD@LDH-GS) were developed for relay drug delivery. Herein, our studies demonstrated that DEX-CM-β-CD@LDH-GS could penetrate through human conjunctival epithelial cells with an intact structure and exhibited integrity in the sclera of rabbits' eyes with in vivo fluorescence resonance energy transfer imaging. Consequently, tissue distribution indicated that DEX-CM-β-CD@LDH-GS nanocomposite eye drops could maintain the effective therapeutic concentration of DEX in choroid-retina within 3 h. As a relay drug delivery system, drug-CD@LDH nanocomposites offer an efficient strategy for drug delivery from ocular surface to the posterior segment.

Hemicyanine dyes including hemi-indocyanine and hemi-benzindocyanine have been attracted considerable attentions to develop near infrared probes for in vivo fluorescence imaging. However, hemi-benzothiazolecyanine dye (MTR) is rarely reported for detection analytes. In this work, to extend the sensing applications of MTR, we present an optimized route for facile preparation of the dye. And as a proof-of-concept, a self-immolative NIR probe MTR-HH based on hemi-benzothiazolecyanine dye was designed and synthesized for H2O2. The probe exhibited a 284-fold fluorescent enhancement to H2O2 with a detection limit of 0.32 μM over other biospecies. Moreover, MTR-HH was successfully applied for visualizing endogenous H2O2 in living cells and mice.

SummaryBackground Erythropoietic protoporphyria (EPP) is a rare genetic disease that causes severe sensitivity to visible light due to protoporphyrin IX (PpIX) accumulation in the skin. Objectives To establish a non-invasive method to measure PpIX in the skin of EPP patients and to investigate how skin PpIX relates to erythrocyte PpIX and photosensitivity. Methods Skin PpIX was measured in 25 EPP patients as the difference in PpIX fluorescence before and after complete photobleaching of PpIX by controlled illumination. Patients reported symptoms during the illumination and skin erythema was measured before and after. Confirmation of the presence of PpIX was obtained in 7 patients by measuring the in vivo fluorescence emission spectrum. The method was used to examine skin PpIX in the hours after an illumination in 7 patients. Results We established a non-invasive method to measure skin PpIX based on measurements of PpIX fluorescence before and after complete PpIX photobleaching. Patients had an average skin PpIX of 2.0 units and skin emission spectra confirmed the presence of skin PpIX (peak emission 632 nm). Skin PpIX was associated with erythrocyte PpIX (P = 0.002; R2 = 0.34), skin erythema (P = 0.0001; R2 = 0.47) and symptoms during illumination. Furthermore, skin PpIX increased in the hours after illumination. Conclusions We have developed a non-invasive method to measure skin PpIX in EPP patients. Skin PpIX is dependent on erythrocyte PpIX and light exposure of the skin. This method can be used for objective monitoring of treatment effect. This article is protected by copyright. All rights reserved.

Early diagnosis is one of the most important factors that increase the therapeutic potential of the disease. Diagnoses conducted by conventional equipment are expensive, time-consuming, burdensome to patients, and do not have high success rates. Diagnostic methods have also been investigated using nanoparticles. However, there have been no significant improvements in the early diagnosis of disease. The diagnosis technique proposed in this paper consumes less time, is more cost-effective, and more accurate. It uses a new concept-a low-intensity fluorescence molecular imaging system with a lock-in technique. This study applied the lock-in technique to basic research in contrast enhancement and optimization. This improved fluorescence distribution analysis, resulting in increased resolution of optical molecular imaging for early diagnosis of disease. An experimental lock-in fluorescence imaging system, which used a variety of fluorescent dyes, achieved signal amplification 100 times greater than that of a conventional fluorescence imaging system. The results of this study demonstrate that the lock-in technique could significantly improve optical molecular imaging technology, making it possible to achieve early diagnosis of disease.

Here, a novel latent naphthalimide bearing water-soluble nanoprobes with catechol-Fe(III) cores (Fe@LNNPs) was designed, synthesized, and evaluated for in vivo fluorescence imaging of intracellular thiols, as various diseases are associated with overexpression of cellular biothiols. The Fe@LNNPs are mainly composed of three components. The inner part constitutes pyrocatechol groups, which can coordinate with Fe(III) to form a cross-linked core for improving the stability in the complex biological environment. The naphthalimide group is linked by disulfide with the core to quench the probe fluorescence. The outer part is designed to be a hydrophilic glycol corona for prolonging blood circulation. Also, a biotin group can be easily introduced into the nanoprobe for actively targeting the HepG2 cells. The fluorescence spectra reveals that the Fe@LNNPs can be reduced explicitly by glutathione to trigger the fluorescence emission. Confocal microscopic imagings and animal experiments manifest that the Fe@LNNPs, especially with biotin groups, have much better fluorescence signal imaging compared to the reported small-molecule probe 1' both in vitro and in vivo (up to 24 h). The Fe@LNNPs thus feature great advantages such as specificity, stability, biocompatibility, and long retention time for thiol-recognition imaging and hold potential applications in clinical cancer diagnosis.

Fluorescence lifetime imaging (FLIM) is a technique that allows calculating the fluorescence lifetime at every pixel of an imaged fluorescent sample. The fluorescence lifetime is a property that characterizes each fluorophore and its environment, which makes FLIM a powerful quantitative analytical tool extensively used in a wide range of biomedical applications. In order to fully exploit the potentials of FLIM in the medical field, practical implementations that would enable fast and accurate in vivo FLIM imaging are needed. We present a handheld FLIM system capable of both acquiring and processing time-resolved fluorescence measurements at a pixel rate of at least 30 kHz. The handheld instrument provides a field of view of ~1 cm in diameter with an optical resolution of ~100 μm. Real-time FLIM processing is achieved by means of a bi-exponential model curve fitting algorithm based on a lookup table and pattern recognition techniques. The handheld FLIM system was validated by safely imaging fluorescence standards and the oral mucosa of healthy volunteers. The acquired fluorescence lifetime maps were in agreement with the fluorescence lifetime values estimated using the standard non-linear least square iterative reconvolution method (LSIR). These results demonstrated practical and accurate in vivo video rate FLIM imaging capabilities of this novel handheld FLIM implementation, which would facilitate practical FLIM applications, including clinical ones, such as clinical diagnosis and image guided interventions.

A new type of carbon dots (CD-Asp) with targeting function toward brain cancer glioma was synthesized via a straightforward pyrolysis route by using D-glucose and L-aspartic acid as starting materials. The as-prepared CD-Asp exhibits not only excellent biocompatibility and tunable full-color emission, but also significant capability of targeting C6 glioma cells without the aid of any extra targeting molecules. In vivo fluorescence images showed high-contrast biodistribution of CD-Asp 15 min after tail vein injection. A much stronger fluorescent signal was detected in the glioma site than that in normal brain, indicating their ability to freely penetrate the blood-brain barrier and precisely targeting glioma tissue. However, its counterparts, the CDs synthesized from D-glucose (CD-G), L-asparic acid (CD-A), or D-glucose and L-glutamic acid (CD-Glu) have no or low selectivity for glioma. Therefore, CD-Asp could act as a fluorescence imaging and targeting agent for noninvasive glioma diagnosis. This work highlights the potential application of CDs for constructing an intelligent nanomedicine with integration of diagnostic, targeting, and therapeutic functions.

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Knowledge of the formation of oral coatings and their influence on subsequent taste perception is necessary to understand possible taste-masking effects by oil coatings. This study investigated (a) the dynamics of the formation of oral oil coatings formed by o/w emulsions and (b) the effect of oral oil coatings on subsequent sweetness perception of sucrose solutions. In vivo fluorescence was used to quantitate the oil fraction deposited on the tongue after oral processing of oil-in-water emulsions for different times. A trained panel evaluated sweetness perception of sucrose solutions after orally processing the emulsions. The oil fraction reached its maximum value within the first 3 s of oral processing. The oil fraction did not significantly affect subsequent sweetness perception of sucrose solutions. It is suggested that the oil droplets deposited on the tongue did not form a hydrophobic barrier that is sufficient to reduce the accessibility of sucrose to taste buds.

Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1micelle) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1micelle exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1micelle in the NIR window of 800–900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1micelle, as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1micelle proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging. NRF (Natl Research Foundation, S’pore) Accepted version

Quantum dots (QDs) are thought to be a novel inorganic probe for in vivo fluorescence imaging because of their excellent fluorescence properties. Autofluorescence is generally known to be produced from various living bodies including humans, rats, and mice. However, the influence of the autofluorescence on in vivo fluorescence imaging using QDs remains poorly understood. In this article, we assessed the autofluorescence derived from a mouse body and the influence of the autofluorescence on in vivo fluorescence imaging using QDs. The dorsal and ventral autofluorescence derived from a mouse from which the hair was removed were detected under all kinds of excitation/fluorescence filter settings (blue, green, yellow, red, deep red, and NIR) using the Maestro™ in vivo imaging system. The degree of autofluorescence was found to be extremely high in the red filter condition, but transplanted ASCs labeled with QDs on the back of a mouse could be detected in the red filter condition. Moreover, the ASCs labeled with QDs could be traced for at least 5 days. We suggest that fluorescence imaging using QDs can be useful for the detection of transplanted cells.

The turbidity of biological tissue due to fundamental light-tissue interactions has been a long-standing challenge in biomedical optical technologies. Implanting fibrous optical waveguide into tissue and organ for light delivery and collection is one of the most effective way for alleviate this problem. In this manuscript, by taking advantage of the favorable designability and processibility of citrate-based synthetic polymers, two bio-elastomers with distinct optical properties but matched mechanical properties and similar biodegradation profiles were developed. Combining with an efficient two-step fabrication method, we created a new biodegradable and biocompatible step-index optical fiber. Benefited from this step-index structure and high tunability of citrate-based bio-elastomers, our optical fiber not only demonstrated outstanding optical performance (0.4dB/cm loss), but also had favorable mechanical and biodegradable properties. Apart from the fabrication and characterization of our optical fiber, we successfully demonstrated the functionalities of multimode fiber imaging, deep tissue light delivery and in vivo fluorescence detection of our newly designed optical fiber. We believe the flexible, biodegradable and low loss optical fiber designed in our work offers a valuable tool for optical applications including imaging, detecting, sensing, optogenetic stimulation, and treatment to target regions underneath deep tissue.

The conventional chemotherapeutics could not be traced in vivo and provide timely feedback on the clinical effectiveness of drugs. In this study, poly(L-γ-glutamyl-glutamine)-paclitaxel (PGG-PTX), as a model polymer, was chemically conjugated with Gd-DTPA (Gd-diethylenetriaminepentaacetic acid), a T1-contrast agent of MRI, to prepare a Gd-DTPA-conjugated PGG-PTX (PGG-PTX-DTPA-Gd) delivery system used for tumor theranostics. PGG-PTX-DTPA-Gd can be self-assembled to NPs in water with a z-average hydrodynamic diameter about 35.9 nm. The 3 T MRI results confirmed that the relaxivity of PGG-PTX-DTPA-Gd NPs (r1 = 18.98 mM−1S−1) was increased nearly 4.9 times compared with that of free Gd-DTPA (r1 = 3.87 mM−1S−1). The in vivo fluorescence imaging results showed that PGG-PTX-DTPA-Gd NPs could be accumulated in the tumor tissue of NCI-H460 lung cancer animal model by EPR effect, which was similar to PGG-PTX NPs. The MRI results showed that compared with free Gd-DTPA, PGG-PTX-DTPA-Gd NPs showed significantly enhanced and prolonged signal intensity in tumor tissue, which should be attributed to the increased relaxivity and tumor accumulation. PGG-PTX-DTPA-Gd NPs also showed effective antitumor effect in vivo. These results indicated that PGG-PTX-DTPA-Gd NPs are an effective delivery system for tumor theranostics, and should have a potential value in personalized treatment of tumor.

The investigation of protein interactions in living plant tissue has become of increasing importance in recent years. A high spatial and temporal resolution for the observation of in vivo protein interaction is needed, e.g., in order to follow changes of plant receptor kinase interactions and complex formation over time. In vivo fluorescence or Forster resonance energy transfer (FRET) measurements allow for detailed analyses of interacting proteins in their natural environment at a subcellular level. Especially FRET-FLIM (fluorescence lifetime imaging microscopy) measurements provide an extremely powerful and reliable tool meeting the demands for investigating in vivo protein interaction quantitatively and with high precision. Here, we will describe in detail how to practically perform in vivo FRET measurements of receptor kinases in plants and discuss potential pitfalls and points of consideration.

Cyanobacteria are the primary taxa responsible for freshwater harmful algal blooms (HABs), with several genera capable of producing potent intracellular toxins and off-flavor compounds. There is considerable growing interest in methods to rapidly quantify cyanobacteria in water samples. Past studies have demonstrated poor correlations between phycocyanin in vivo fluorescence and cyanobacterial cell densities. We conducted a series of laboratory experiments aimed at refining a protocol that uses benchtop fluorometry to measure the cyanobacterial pigment, phycocyanin, to accurately estimate cyanobacterial biovolume. In our study, we found strong correlations between phycocyanin concentration and cyanobacterial biovolume (but not for cell densities) both within and across ponds, which varied widely in productivity and algal diversity. Thus, benchtop fluorometry of phycocyanin is a viable method for water resource managers to quickly estimate cyanobacterial biovolume.

A novel strategy to fabricate bovine serum albumin and styrene-terminated porous silicon nanocomposites with stable near-infrared fluorescence under physiological conditions was described. And these resultant nontoxic porous silicon-based nanocomposites were administrated into nude mice via subcutaneous injection, which exhibited a potential application for in vivo fluorescence imaging.