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Nanobodies have been extensively demonstrated in biomedical imaging and therapy. However, nanobody probes often suffer from rapid renal clearance due to its small size. Herein, we reported a recombinant nanobody with a 200 amino-acid polypeptide chain consisting of Pro, Ala, and Ser (PAS) at the C-terminal, which can be easily expressed in Escherichia coli with a high yield. The PASylated nanobody was functionalized with a fluorescent dye and the cell labeling properties were characterized by flow cytometry and confocal microscopy. In vivo fluorescence imaging indicated that the PASylated nanobody showed comparable blood circulation time, but $\sim 1.5$ times higher tumor targeting ability as compared to the PEGylated nanobody of comparable molecular weight. Our findings demonstrate that nanobody PASylation is a promising approach to produce long-circulating nanobody probes for imaging and therapeutic applications.

$N$-methyl mesoporphyrin IX (NMM) is a water-soluble, non-symmetric porphyrin with excellent optical properties and unparalleled selectivity for G-quadruplex (GQ) DNA. G-quadruplexes are non-canonical DNA structures formed by guanine-rich sequences. They are implicated in genomic stability, longevity, and cancer. The ability of NMM to selectively recognize GQ structures makes it a valuable scaffold for designing novel GQ binders. In this review, we survey the literature describing the GQ-binding properties of NMM as well as its wide utility in chemistry and biology. We start with the discovery of the GQ-binding properties of NMM and the development of NMM-binding aptamers. We then discuss the optical properties of NMM, focusing on the light-switch effect — high fluorescence of NMM induced upon its binding to GQ DNA. Additionally, we examine the affinity and selectivity of NMM for GQs, as well as its ability to stabilize GQ structures and favor parallel GQ conformations. Furthermore, a portion of the review is dedicated to the applications of NMM-GQ complexes as biosensors for heavy metals, small molecules ($e.g.$ ATP and pesticides), DNA, and proteins. Finally and importantly, we discuss the utility of NMM as a probe to investigate the roles of GQs in biological processes.

We have developed a two-photon fluorescence microscope capable of imaging up to 4mm in turbid media with micron resolution. The key feature of this instrument is the innovative detector, capable of collecting emission photons from a wider surface area of the sample than detectors in traditional two-photon microscopes. This detection scheme is extremely efficient in the collection of emitted photons scattered by turbid media which allows eight fold increase in the imaging depth when compared with conventional two-photon microscopes. Furthermore, this system also has in-depth fluorescence lifetime imaging microscopy (FLIM) imaging capability which increases image contrast. The detection scheme captures emission light in a transmission configuration, making it extremely efficient for the detection of second harmonic generation (SHG) signals, which is generally forward propagating. Here we present imaging experiments of tissue phantoms and in vivo and ex vivo biological tissue performed with this microscope.

Protein-directed fluorescent Au nanoclusters have been widely studied owing to their potential applications in sensing, imaging, and drug and gene delivery. However, the use of nanoclusters in drug delivery is limited by low cellular uptake. In this study, human serum albumin-directed Au nanoclusters served as building blocks to obtain protein nanoparticles by desolvation. The nanoparticles had a decent quantum yield (QY), high colloidal stability and low cytotoxicity, and they could be readily conjugated with biological molecules. The cellular uptake of the Au nanoclusters and nanocluster-loaded protein nanoparticles were studied by confocal fluorescence microscopy. Agglomeration of the protein-directed Au nanoclusters into 50–150-nm nanoparticles dramatically increased the cellular uptake.

In this paper, the mathematical model of distribution of the injected compound in biological liquid flow has been described. It is considered that biological liquid contains a few phases such as water, peptides and cells. The injected compound (for example, photosensitizer) can interact with peptides and cells. At the time, viscosity of the biological liquid depends on pathology present in organism. The obtained distribution of the compound connects on changes of its fluorescence spectra which are registered during fluorescent diagnostics of tumors. It is obtained that the curves do not have monotonic nature. There is a sharp curves decline in the first few seconds after injection. Intensivity of curves rises after decreasing. It is especially pronounced for wavelength 590nm and 580nm (near the “transparency window” of biological tissues). Time of inflection point shifts from 8.4s to 6.9s for longer wavelength. However, difference between curves is little for different viscosity means of the biological liquid. Thus, additional pathology present in organism does not impact to the results of in vivo biomedical investigations.

High resolution optical endoscopes are increasingly used in diagnosis of various medical conditions of internal organs, such as the cervix and gastrointestinal (GI) tracts, but they are too expensive for use in resource-poor settings. On the other hand, smartphones with high resolution cameras and Internet access have become more affordable, enabling them to diffuse into most rural areas and developing countries in the past decade. In this paper, we describe a smartphone microendoscope that can take fluorescence images with a spatial resolution of 3.1 $\mu$m. Images collected from ex vivo, in vitro and in vivo samples using the device are also presented. The compact and cost-effective smartphone microendoscope may be envisaged as a powerful tool for detecting pre-cancerous lesions of internal organs in low and middle-income countries (LMICs).

The fluorescence-based in vivo flow cytometry (IVFC) is an emerging tool to monitor circulating cells in vivo. As a noninvasive and real-time diagnostic technology, the fluorescence-based IVFC allows long-term monitoring of circulating cells without changing their native biological environment. It has been applied for various biological applications (e.g., monitoring circulating tumor cells). In this work, we will review our recent works on fluorescence-based IVFC. The operation principle and typical biological applications will be introduced. In addition, the recent advances in IVFC flow cytometry based on photoacoustic effects and other label-free detection methods such as imaging-based methods, diffuse-light methods, hybrid multimodality methods and multispectral methods are also summarized.

Time-gated (TG) fluorescence imaging (TGFI) has attracted increasing attention within the biological imaging community, especially during the past decade. With rapid development of light sources, image devices, and a variety of approaches for TG implementation, TGFI has demonstrated numerous biological applications ranging from molecules to tissues. The paper presents inclusive TG implementation mainly based on optical choppers and electronic units for synchronization of fluorescence excitation and emission, which also serves as guidelines for researchers to build suited TGFI systems for selected applications. Note that a special focus will be put on TG implementation based on optical choppers for TGFI of long-lived probes (lifetime range from microseconds to milliseconds). Biological applications by TG imaging of recently developed luminescent probes are described.

Short wave near-infrared (SWIR, 900–1700nm) fluorescence imaging has attracted extensive research interest from scientists due to its high imaging quality. However, the variety of SWIR fluorescence imaging agents are quite limited and the corresponding quantum efficiency is relatively low. In this work, a novel conjugated polymer PDTSDTBT was reported, consisting of a donor unit with a tetrahedral Si (sp3) named DTS and an acceptor unit named DTBT with branched side chains. The design approach of endowing the donor–acceptor structure with the branched side chains successfully increase the fluorescence quantum efficiency. The polymer was prepared into nanoparticles by nanoprecipitation. The PDTSDTBT nanoparticles showed an absorption peak of 626nm and fluorescence emission peak of 924nm. The quantum efficiency of the nanoparticles is 0.53%, which is higher than that of nanotube fluorophores (0.4%). The nanoparticles also demonstrate a photothermal effect, the temperature of nanoparticles solution could reach $45^{\circ }$C under excitation by 660nm laser. Therefore, the PDTSDTBT nanoparticles is an excellent fluorescent imaging agent with potential photothermal applications.

The absorption, scattering, and autofluorescence of biological tissues in short-wave infrared region (SWIR, 900–1700nm) are relatively low, so SWIR fluorescence usually has deeper penetration into living tissues, and can show a higher signal-to-noise ratio when used for imaging in vivo. However, there are few types of organic SWIR fluorescent materials currently. In this work, $p$-azaquinodimethane ($p$-AQM) with a quinoid structure is used as the acceptor unit, and carbazole or fluorene with sp3 hybridization are used as the donor units, two conjugated polymers were synthesized. The quinone structure is conducive to the redshift of absorption and fluorescence spectra, and the sp3 hybridization structure is conducive to weakening the aggregation quenching of polymer fluorescence. PF and PCz exhibited absorption peaks of 492nm and 508nm, respectively. The emission peaks of the two polymers are 920nm and 950nm, respectively, both in the short-wave near infrared region. The quantum yield (QY) of PF and PCz is 0.4% and 0.3%, respectively.

Parkinson’s disease (PD) is closely related to the oxidative stress induced by excess hydrogen peroxide (H₂O₂) in organisms. Developing an efficient method for noninvasive and real-time H₂O₂ detection is beneficial to investigate the role played by H₂O₂ in PD. In this work, a novel fluorogenic probe (CBH) for living organisms H₂O₂ detection has been designed, synthesized and characterized. The emission of CBH in PBS solution is very weak. However, when H₂O₂ was added, the fluorescence of CBH solution was sharply increased for 12-fold, accompanied by the emission peak blue-shifted from 600 to 530 nm. Moreover, the response of CBH to H₂O₂ is highly sensitive and selective and is not affected by various ROS/RNS, anions, cations, and amino acids. Based on the good performance of CBH for H₂O₂ detection, it has been successfully applied to visualizing the H₂O₂ concentration in living cells, Zebrafish and $C$. elegans PD models.

Intensity-based quantitative fluorescence resonance energy transfer (FRET) is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit. This widely used technique needs complicated experimental process and manual image analyses to obtain precise results, which take a long time and restrict the application of quantitative FRET especially in living cells. In this paper, a simplified and automatic quantitative FRET (saqFRET) method with high efficiency is presented. In saqFRET, photoactivatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein (EGFP) are used to simplify FRET crosstalk elimination. Traditional manual image analyses are time consuming when the dataset is large. The proposed automatic image analyses based on deep learning can analyze 100 samples within 30s and demonstrate the same precision as manual image analyses.

Reduced nicotinamide adenine dinucleotide (NADH) plays a crucial role in many biochemical reactions in human metabolism. In this work, a flow-mediated skin fluorescence (FMSF)–post-occlusion reactive hyperaemia (PORH) system was developed for noninvasive and in vivo measurement of NADH fluorescence and its real-time dynamical changes in human skin tissue. The real-time dynamical changes of NADH fluorescence were analyzed with the changes of skin blood flow measured by laser speckle contrast imaging (LSCI) experiments simultaneously with FMSF-PORH measurements, which suggests that the dynamical changes of NADH fluorescence would be mainly correlated with the intrinsic changes of NADH level in the skin tissue. In addition, Monte Carlo simulations were applied to understand the impact of optical property changes on the dynamical changes of NADH fluorescence during the PORH process, which further supports that the dynamical changes of NADH fluorescence measured in our system would be intrinsic changes of NADH level in the skin tissue.

Bioprobe based on fluorescence is widely used in biological and medical research due to its high sensitivity and selectivity. Yet, its quantification in vivo is complicated and often compromised by the interaction between the fluorophore with the environmental factors, as well as the optical scattering and absorption by the tissue. A high florescence quantum yield and minimal interference by the environment are key requirements for designing an effective bioprobe, and the pre-requisitions severely limit the available options. We propose that a comprehensive evaluation of potential bioprobe can be achieved by simultaneously measuring both radiative and non-radiative transitions, the two fundamental and complementary pathways for the energy de-excitation. This approach will not only improve the accuracy of the quantification by catching the information from a broader spectrum of the energy, but also provide additional information of the probe environment that often impacts the balance between the two forms of the energy transition. This work first analyzes the underlying mechanism of the hypothesis. The practical feasibility is then tested by means of simultaneous measurements of photoacoustic signal for the non-radiative and fluorescence for the radiative energy processes, respectively. It is demonstrated that the systematic evaluation of the probe energy de-excitation results in an improved quantitative tracing of a bioprobe in complex environment.

We present results of on-sky tests performed in the summer of 2013 to characterize the performance of a prototype high-power pulsed laser for adaptive optics. The laser operates at a pulse repetition rate (PRR) of 600–800Hz, with a 6% duty cycle. Its coupling efficiency was found to be, in the best test case (using 18W of transmitted power), $231\pm 14$ photons s${}^{-1}$ sr${}^{-1}$ atom${}^{-1}$ W${}^{-1}$ m² when circular polarization was employed and $167\pm 17$ photons s${}^{-1}$ sr${}^{-1}$ atom${}^{-1}$ W${}^{-1}$ m² with linear polarization. No improvement was seen when D${}_{2b}$ repumping was used, but this is likely due to the relatively large laser guide star (LGS) diameter, typically 10 arcsec or more, which resulted in low irradiance levels. Strong relaxation oscillations were present in the laser output, which have the effect of reducing the coupling efficiency. To better understand the results, a physical modeling was performed using the measured pulse profiles and parameters specific to these tests. The model results, for a 10 arcsec angular size LGS spot, agree well with the observations. When extrapolating the physical model for a sub-arcsecond angular size LGS (typical of what is needed for a successful astronomical guide star), the model predicts that this laser would have a coupling efficiency of 130 photons s${}^{-1}$ sr${}^{-1}$ atom${}^{-1}$ W${}^{-1}$ m², using circular polarization and D${}_{2b}$ repumping, for a LGS diameter of 0.6 arcsec Full Width at Half Maximum (FWHM), and free of relaxation oscillations in the 589 nm laser light.

N-methyl mesoporphyrin IX (NMM) is a water-soluble, non-symmetric porphyrin with excellent optical properties and unparalleled selectivity for G-quadruplex (GQ) DNA. G-quadruplexes are non-canonical DNA structures formed by guanine-rich sequences. They are implicated in genomic stability, longevity, and cancer. The ability of NMM to selectively recognize GQ structures makes it a valuable scaffold for designing novel GQ binders. In this review, we survey the literature describing the GQ-binding properties of NMM as well as its wide utility in chemistry and biology. We start with the discovery of the GQ-binding properties of NMM and the development of NMM-binding aptamers. We then discuss the optical properties of NMM, focusing on the light-switch effect — high fluorescence of NMM induced upon its binding to GQ DNA. Additionally, we examine the affinity and selectivity of NMM for GQs, as well as its ability to stabilize GQ structures and favor parallel GQ conformations. Furthermore, a portion of the review is dedicated to the applications of NMM-GQ complexes as biosensors for heavy metals, small molecules (e.g. ATP and pesticides), DNA, and proteins. Finally and importantly, we discuss the utility of NMM as a probe to investigate the roles of GQs in biological processes.