Despite intracellular molecular dynamics being fundamental to understand pathological, biomechanical or biochemical events, several processes are still not clear because of the difficulty of monitoring and measuring these phenomena. To engineer an effective fluorescent tool useful to improve protein intracellular tracking studies, we fused a supernegative green fluorescent protein, (−30)GFP, to a myogenic transcription factor, MyoD. The (−30)GFP-MyoD was able to pass the plasma membrane when complexed with cationic lipids. Fluorescence confocal microscopy showed the protein delivery in just 3 hours with high levels of protein transduction efficiency. Confocal acquisitions also confirmed the maintenance of the MyoD nuclear localization. To examine how the supernegative GFP influenced MyoD activity, we did gene expression analyses, which showed an inhibitory effect of (−30)GFP on transcription factor function. This negative effect was possibly due to a charge-driven interference mechanism, as suggested by further investigations by molecular dynamics simulations. Summarizing these results, despite the functional limitations related to the charge structural characteristics that specifically affected MyoD function, we found (−30)GFP is a suitable fluorescent label for improving protein intracellular tracking studies, such as nucleocytoplasmic transport in mechanotransduction.

An eco-friendly fluorescence polymer nanoparticle based on carbon quantum dots and poly(methyl methacrylate) nanoparticles is successfully fabricated to detect sulfadiazine. By making use of the abundant functional group of carbon quantum dots and poly(methyl methacrylate) nanoparticles, without any extra modification, the synthetic process of the fluorescence nanoparticles is reduced and the unnecessary chemical molecules are avoided being brought into the reaction system. The investigation of the fluorescence property of carbon quantum dots shows that the prepared carbon quantum dots are the excitation independent. In addition, the morphology of the synthesized fluorescence polymer nanoparticle is tested by the scanning electron microscope and shows that the fluorescence sensor possesses a good spherical core–shell structure. Moreover, under the optimized condition, the prepared fluorescence polymer nanoparticle possesses a good selectivity in the detection of sulfadiazine under a mixture solution. Moreover, the limit of detection is 4 μmol.l−1 within the detective range from 10 μmol.l−1 to 60 μmol.l−1. Meanwhile, the fluorescence quenching mechanism is considered with the photoinduced electron transfer mechanism. Finally, the practical research on the detection of sulfadiazine in tap water shows that the recovery range and relative standard deviation are 97.5% − 105.1% and 2.1%–4.5%, respectively.

To better understand pH-dependence of endogenous fluorescence of algae, we employed spectroscopy and microscopy methods, including advanced time-resolved fluorescence imaging microscopy (FLIM), using green algae Chlorella sp. as a model system. Absorption spectra confirmed two peaks, at 400–420 nm and 670 nm. Emission was maximal at 680 nm, with smaller peaks between 520 and 540 nm. Acidification led to a gradual decrease in the red fluorescence intensity with the maximum at 680 nm when excited by 450 nm laser. FLIM measurements, performed using 475 nm picoseconds excitation, uncovered that this effect is accompanied by a shortening of the tau1 fluorescence lifetime. Under severe acidification, we also noted an increase in the green fluorescence with a maximum between 520–540 nm and a shift toward 690–700 nm of the red fluorescence, accompanied by prolongation of the tau2 fluorescence lifetime. Gathered data increase our knowledge on the responsiveness of algae to acidification and indicate that endogenous fluorescence derived from chlorophylls can potentially serve as a biosensing tool for monitoring pH change in its natural environment.

Photochemical stability is one of the most important parameters that determine the usefulness of organic dyes in different applications. This Review addresses key factors that determine the dye photostability. It is shown that photodegradation can follow different oxygen-dependent and oxygen-independent mechanisms and may involve both 1S1–3T1 and higher-energy 1Sn–3Tn excited states. Their involvement and contribution depends on dye structure, medium conditions, irradiation power. Fluorescein, rhodamine, BODIPY and cyanine dyes, as well as conjugated polymers are discussed as selected examples illustrating photobleaching mechanisms. The strategies for modulating and improving the photostability are overviewed. They include the improvement of fluorophore design, particularly by attaching protective and anti-fading groups, creating proper medium conditions in liquid, solid and nanoscale environments. The special conditions for biological labeling, sensing and imaging are outlined.

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.

Due to the concern over food safety, it is important to detect the pesticides residues in agricultural products. Here, a highly sensitive and low background fluorescent strategy for the detection of pesticides residues has been developed. The fluorescence intensity of N-methyl mesoporphyrin IX (NMM) binding G-quadruplex could be turn off because of inhibiting effect of the pesticides on the acetylcholinesterase (AChE) activity. For that, four single-stranded DNAs (named linker, trigger, H1 and H2, respectively) are rational designed and T-Hg-T mismatches duplex DNAs as a recognizer combined with the separation of magnetic beads. The design of hybridization chain reaction (HCR) amplification strategy assisted by magnetic separation has been adopted to improve the detection sensitivity. In the presence of pesticides, the amount of the thiol group generated by hydrolysis reaction of acetylcholine (ACh) is reduced, lead to release of less trigger DNA. Therefor subsequent HCR process is retarded with decreased fluorescence intensity. The reduced fluorescence intensity has a quantitative relationship with the pesticide concentration. The limit of detection of chlorpyrifos was estimated to be 2.0 ng ml−1. It has been applied to detect the pesticides residues in real samples.

In this perspective, we aim to present an overview of some important physical and chemical aspects of lanthanide-doped upconverting nanoparticle research to be considered, from synthesis considerations to bioapplications. To this end, we have reviewed several practical considerations and prepared several straightforward recommendations toward improved cohesion in the field, based on observed trends over the last decade of research.

Hydrogen sulfide is an important gasotransmitter that exhibits various functions in physiological processes. We present a ratiometric fluorescence probe (SN-N3) for H2S by functionalizing naphthalene imide with 4-(azidomethyl)benzene as a H2S recognition moiety and morpholine moiety as a lysosomal targeting unit. After reaction with H2S, the azido moiety is reduced to amine group, and the probe releases the self-immolative linker and regenerates the fluorophore with internal charge transfer effect. SN-N3 is responsive to H2S in a ratiometric mode, exhibiting excellent sensitivity and high selectivity. The probe is demonstrated to be localized in lysosomes with high specificity. More importantly, SN-N3 is successfully demonstrated to image lysosomal H2S in a ratiometric manner. Our design provides a novel tool to image H2S in living cells that would hold great potential in exploring various H2S-related physiological and pathological cellular processes.

Among epigenetic modifications of DNA, methylation of cytosine at its carbon 5 is the most common mark that is usually associated with gene silencing in human. Determining whether a particular DNA molecule is methylated or not, is an indispensable task in many epigenetic investigations. Presenting detection methods with less labor-intensive and time-consuming procedures has substantial value. Here a facile method based on gold nanocluster (AuNCs) fluorescence enhancement is presented. Target sequences were selected from Sept9 promoter region as its hypermethylation is demonstrated as a reliable biomarker of colorectal cancer. DNA probe was complementary to a 25 nucleotide of the target region and possessed 9 additional cytosines in the middle to allow the formation of AuNCs. Probe-AuNCs strands were first hybridized with methylated and non-methylated targets separately, and then their fluorescence intensities were recorded. Fluorescence intensity was higher with methylated targets than non-methylated one. Applying silver ions reversed the situation and fluorescence intensities of non-methylated DNA got higher than methylated one.

Upon direct excitation with green light (522 nm), Er3+ ion doped nanoparticles feature a number of radiative and non-radiative decay pathways, leading to distinct and sharp emission lines in the visible and near-infrared (NIR) range. Here we apply, in addition to continuous 522 nm irradiation, a modulated NIR irradiation (1143 nm) to actively control and modulate the red emission intensity (around 650 nm). The modulation of red Er3+ ion emission at a chosen frequency allows us to reconstruct fluorescence images from the Fourier transform amplitude at this particular frequency. Since only the emission from the Er3+ ion is modulated, it allows to selectively recover the lanthanide specific signal, removing any non-modulated auto-fluorescence or background emission resulting from the continuous 522 nm excitation. The modulated emission of specific lanthanides can open up new detection opportunities for selective signal recovery.