The phasor approach to fluorescence lifetime imaging microscopy (FLIM) is used to identify different types of tissues from hematoxylin and eosin (H&E) stained basal cell carcinoma (BCC) sections. The results suggest that working directly on the phasor space with the clustering assignment achieves immunofluorescence like simultaneous five or six-color imaging by using multiplexed fluorescence lifetimes of H&E. The phase approach is of particular effectiveness for enhanced visualization of the abnormal morphology of a suspected nidus. Moreover, the phasor approach to H&E FLIM data can determine the actual paths or the infiltrating trajectories of basophils and immune cells associated with the preneoplastic or neoplastic skin lesions. The integration of the phasor approach with routine histology proved its available value for skin cancer prevention and early detection. We therefore believe that the phasor analysis of H&E tissue sections is an enhanced visualization tool with the potential to simplify the preparation process of special staining and serve as color contrast aided imaging in clinical pathological examination.

Atomically thin transition metal dichalcogenide nanomaterials have shown superior optical and electronic properties in the two-dimensional (2D) scale. They are considered as promising alternative materials to graphene. Here, we have precisely engineered a plasmonic sensing substrate with four types of two-dimensional transition metal dichalcogenide nanomaterial to achieve significant phase sensitivity improvement. Phase modulation is currently the most sensitive interrogation method among all the plasmonic detection approaches. The tuning of the substrate thickness in an atomic scale with a step less than 1 nm allows the efficient modulation of phase signals. More importantly, the optical absorption rate for each of these nanomaterials is different and can be tuned by changing the number of 2D layers, where perfect absorption and interrogation of the plasmonic signal can be obtained. Through systematically optimizing the parameters of the transition metal dichalcogenide structured plasmonic substrate, we can balance the optical absorption efficiencies and the electron losses at the plasmonic resonance condition. All of the calculations were based on the transfer matrix method and Fresnel equations. A very low minimum reflectivity of 3.2560 × 10–8 was demonstrated with an excitation wavelength of 1024 nm, showing a complete transfer (∼100%) of the light energy into the plasmon resonance energy. The ultradark singularity at the resonance dip leads to an ultrahigh plasmonic sensitivity of 1.1 × 107 deg/RIU, which is 3 orders of magnitude higher than those with bare metallic sensing substrates used in commercial plasmonic sensors. The resolution is also improved by at least 3 orders of magnitude compared with conventional substrates.

The aim of this study was to distinguish basal cell carcinoma (BCC) from actinic keratosis (AK) and Bowen’s disease (BD) by fluorescence lifetimes of hematoxylin and eosin (H&E) and phasor analysis. Pseudocolor images of average fluorescence lifetime (τm) exhibited more contrast than conventional bright field and/or fluorescence images of H&E-stained sections. The mean values (μ) of τm distribution (τmμ) in three layers of skin were first explored for comparison with the corresponding layers of AK, BD, and BCC. Moreover, analysis of the H&E fluorescence lifetimes in the phasor space was performed by observing clusters in specific regions of the phasor plot. Various structures in the skin were distinguished. Comparisons of phase distributions from the corresponding layers of skin resulted in quantitative separation and calculation of distinctive parameters including coordinate values, diagonal slopes, and phasor areas. The combination of fluorescence lifetime imaging microscopy (FLIM) and phasor approach (phasor–FLIM) provides a simple method for histopathology analysis and can significantly improve the accuracy of bright field H&E diagnosis. We therefore believe that phasor–FLIM is an aided tool with the potential to provide rapid confirmation of diagnostic criteria and classification of histological types of skin neoplasms.

The aim of this study was to distinguish basal cell carcinoma (BCC) from actinic keratosis (AK) and Bowen’s disease (BD) by fluorescence lifetimes of hematoxylin and eosin (H&E) and phasor analysis. Pseudocolor images of average fluorescence lifetime (τm) exhibited more contrast than conventional bright field and/or fluorescence images of H&E-stained sections. The mean values (μ) of τm distribution (τmμ) in three layers of skin were first explored for comparison with the corresponding layers of AK, BD, and BCC. Moreover, analysis of the H&E fluorescence lifetimes in the phasor space was performed by observing clusters in specific regions of the phasor plot. Various structures in the skin were distinguished. Comparisons of phase distributions from the corresponding layers of skin resulted in quantitative separation and calculation of distinctive parameters including coordinate values, diagonal slopes, and phasor areas. The combination of fluorescence lifetime imaging microscopy (FLIM) and phasor approach (phasor–FLIM) provides a simple method for histopathology analysis and can significantly improve the accuracy of bright field H&E diagnosis. We therefore believe that phasor–FLIM is an aided tool with the potential to provide rapid confirmation of diagnostic criteria and classification of histological types of skin neoplasms.

CsPbBr3@Ag hybrid nanocrystals are synthesized for the first time by reacting CsPbBr3 nanocrystals with AgX (X = Cl, Br, or I) powders in hexane. The morphologies of the hybrid nanocrystals showed that 2–5 nm Ag nanoparticles were nucleated randomly outside the CsPbBr3 nanocrystals, which indicated that Ag+ ions released by AgX powders could be reduced to Ag0 by surfactant ligands of CsPbBr3 nanocrystals and aggregated to Ag nanoparticles. Significant enhancement of the photoluminescence intensity of CsPbBr3@Ag hybrid nanocrystals was observed compared with that of pure CsPbBr3 nanocrystals under the excitation of 400 nm light, which was mainly attributed to the enhanced absorbance of ultraviolet or blue light by the Ag induced plasmonic near-field effect. Numerical simulations showed that CsPbBr3@Ag hybrid nanocrystals provided intense local field amplification at the perimeter of Ag nanoparticles. However, Ag adhesion can also cause the deteriorating surface quality of CsPbBr3 nanocrystals, and in turn reduce photoluminescence quantum yield. Therefore, Ag adhesion has two completely different influences on the photoluminescence of hybrid perovskite quantum dots. To achieve an enhanced photoluminescence, we have to optimize CsPbClxBr3−x@Ag hybrid nanocrystals so that the effect of plasmonic resonance enhancement occupied a significantly advantageous position. The surface trap states served as a nonradiative relaxation pathway for the photogenerated charge carriers and decreased the emission quantum yield of the hybrid nanocrystals. This suggests that a potential issue concerning the performance of CsPbBr3@Ag hybrid nanocrystals was a trade-off between enhanced light absorbance of CsPbBr3@Ag hybrid nanocrystals and reduced photoluminescence due to energy transfer from CsPbBr3 nanocrystals to Ag nanoparticles. This simple method to construct CsPbBr3@Ag hybrid nanocrystals offers new opportunities to enhance optical properties and expand the application of perovskite quantum dots for light emitting diodes and other optoelectronic devices.

Herein, we report rational design, synthesis, and application of a two-photon fluorescent probe (Tyro-1) for tracking intracellular tyrosinase activity. The chemoselective detection of tyrosinase is precluded from interference of other competitive omnipresent oxidizing entities in cellular milieu. The probe showed 12.5-fold fluorescence enhancement at λem = 450 nm in the presence of tyrosinase. The nontoxic probe Tyro-1 provides information about H2O2-mediated upregulation of tyrosinase through cellular imaging. Its two-photon imaging ability makes it a noninvasive tool for validating the expression of tyrosinase in the live cells.

Herein, we report rational design, synthesis, and application of a two-photon fluorescent probe (Tyro-1) for tracking intracellular tyrosinase activity. The chemoselective detection of tyrosinase is precluded from interference of other competitive omnipresent oxidizing entities in cellular milieu. The probe showed 12.5-fold fluorescence enhancement at λem = 450 nm in the presence of tyrosinase. The nontoxic probe Tyro-1 provides information about H2O2-mediated upregulation of tyrosinase through cellular imaging. Its two-photon imaging ability makes it a noninvasive tool for validating the expression of tyrosinase in the live cells.

Two-photon photodynamic therapy (PDT) is able to offer precise 3D manipulation of treatment volumes, providing a target level that is unattainable with current therapeutic techniques. The advancement of this technique is greatly hampered by the availability of photosensitizers with large two-photon absorption (TPA) cross section, high reactive-oxygen-species (ROS) generation efficiency, and bright two-photon fluorescence. Here, an effective photosensitizer with aggregation-induced emission (AIE) characteristics is synthesized, characterized, and encapsulated into an amphiphilic block copolymer to form organic dots for two-photon PDT applications. The AIE dots possess large TPA cross section, high ROS generation efficiency, and excellent photostability and biocompatibility, which overcomes the limitations of many conventional two-photon photosensitizers. Outstanding therapeutic performance of the AIE dots in two-photon PDT is demonstrated using in vitro cancer cell ablation and in vivo brain-blood-vessel closure as examples. This shows therapy precision up to 5 µm under two-photon excitation.

•The working mechanism and practical applications of conventional far-field optical trapping-assisted SERS are discussed.•The working mechanism and practical applications of emerging near-field optical trapping-assisted SERS are presented.•Some important strategies to suppress the plasmonic heating effect are outlined.•New trends in designing next generation of optical trapping-based SERS sensing platform are also discussed.Useful approaches to tackle Brownian motion of micro/nanoparticles and overcome the poor reproducibility of surface-enhanced Raman scattering (SERS) events are highly desirable, especially for performing SERS sensing in ultra-low concentration of analytes. When integrated with the SERS measurement system, optical trapping is a versatile approach for manipulating particles and thereby improving the SERS performance. A review of the recent research advancements in optical trapping-assisted SERS platform can provide critical inputs for optimizing SERS-based sensing methods for real-life applications. In this paper, we present an in-depth review on the systematic classification of optical trapping-assisted (e.g., far-field and near-field optical tweezers) SERS sensing platform and discuss its latest practical applications in biosensing, bioimaging, chemical monitoring, particle manipulation, single cell analysis, etc. Also, we summarize some important strategies to suppress the plasmonic heating effect which hinders the stability of optical tweezers. Furthermore, we also propose non-optical trapping approaches for manipulating nanoparticles/molecules that are promising for prospective SERS sensing. For example, plasmonic heating is not completely deleterious to particle manipulation. Particularly, plasmon-enhanced thermophoresis technique is a useful non-optical approach for trapping particles/molecules and incorporating with SERS detection. Finally, we conclude with future perspectives for designing the new generation of optical tweezers.

NaMnF3: Yb,Er upconversion nanoparticles (UCNPs) have received considerable attention due to their single-band emission. In this paper, a modified thermal decomposition method to synthesize single or core/shell structured lanthanide-doped NaMnF3 nanoparticles is proposed. The NaMnF3: 20% Yb3+, 2% Er3+ nanoparticles displayed pure red emission under 980 nm laser excitation, and the emission intensity could be significantly enhanced by coating a NaMnF3 or NaMnF3: 20% Yb3+ shell layer on their surfaces. Moreover, NaMnF3: 20% Yb3+, 2% Er3+ nanoparticles shelled with NaMnF3: 20% Nd3+ or NaMnF3: 20% Yb3+, 20% Nd3+ were synthesized for the first time, and displayed pure red emission when excited by an 808 nm laser. The core/shell structured NaMnF3: Yb/Er@NaMnF3: Yb or NaMnF3: Yb/Er@NaMnF3: Yb/Nd nanoparticles were applied to bioimaging. The NaMnF3 based UCNPs exhibited good photostability and biocompatibility in HeLa cells. The obtained images indicated that the UCNPs could mainly exist in the cytoplasmic regions in the cells. Besides, images from the same region exposed to laser irradiations at 808 nm and 980 nm were found to be comparable. This result indicated that, for NaMnF3: 20% Yb3+, 2% Er3+@NaMnF3: 20% Yb3+, 20% Nd3+ nanoparticles, laser excitation at 808 nm was as efficient as that at 980 nm for bioimaging.

Optical Coherence Tomography (OCT) is a valuable technology that has been used to obtain microstructure images of tissue, and has several advantages, though its applications are limited in high-scattering tissues. Therefore, semiconducting polymer nanoparticles (SPNs) that possess strong absorption characteristics are applied to decrease light scattering in tissues and used as exogenous contrast agents for enhancing the contrast of OCT imaging detection. In this paper, we prepared two kinds of SPNs, termed PIDT-TBZ SPNs and PBDT-TBZ SPNs, as the contrast agents for OCT detection to enhance the signal. Firstly, we proved that they were good contrast agents for OCT imaging in agar–TiO2. After that, the contrast effects of these two SPNs were quantitatively analyzed, and then cerebral blood vessels were monitored by a home-made SD-OCT system. Finally, we created OCT images in vitro and in vivo with these two probes and performed quantitative analysis using the images. The results indicated that these SPNs created a clear contrast enhancement of small vessels in the OCT imaging process, which provides a basis for the application of SPNs as contrast agents for bioimaging studies.

Living organisms are generally composed of complex cellular processes which persist only within their native environments. To enhance our understanding of the biological processes lying within complex milieus, various techniques have been developed. Specifically, the emergence of super-resolution microscopy has generated a renaissance in cell biology by redefining the existing dogma towards nanoscale cell dynamics, single synaptic vesicles, and other complex bioprocesses by overcoming the diffraction-imposed resolution barrier that is associated with conventional microscopy techniques. Besides the typical technical reliance on the optical framework and computational algorithm, super-resolution imaging microscopy resorts largely to fluorescent materials with special photophysical properties, including fluorescent proteins, organic fluorophores and nanomaterials. In this tutorial review article, with the emphasis on cell biology, we summarize the recent developments in fluorescent materials being utilized in various super-resolution techniques with successful integration into bio-imaging applications. Fluorescent proteins (FP) applied in super-resolution microscopy will not be covered herein as it has already been well summarized; additionally, we demonstrate the breadth of opportunities offered from a future perspective.

The ability to control the local surface plasmonic resonance (LSPR) absorption peaks of silver nanoparticles will greatly broaden the scope of their practical application. Conventional methods tune the LSPR peaks by modifying the shape or size of the silver nanoparticles. Here, we present a novel method to tune the LSPR band by controlling the particle corner sharpness. A modified photochemical method was used to prepare silver nanoparticles. It was found that the nanoparticles irradiated using light-emitting diodes (LEDs) with a wavelength of 455 nm were decahedral, although the reaction temperature was different. However, the in-plane dipole LSPR peak of the as-prepared silver nanodecahedra exhibited an evident red shift from 460 nm to 500 nm during the synthesis process, and the wavelength of the LSPR peak increased linearly as the reaction time increased. A numerical simulation conducted to investigate the mechanism behind the shift revealed that the red shift of the LSPR peak was mainly induced by the evolution of the corner sharpness of the silver nanodecahedra. These results demonstrated the effectiveness of the method in precisely tuning the LSPR peak by controlling the reaction time. By turning off the irradiation light, the photochemical process could be immediately terminated, and the LSPR peak of the silver nanoparticles remained constant. Compared with conventional methods, the present tuning precision can reach 1 nm.

•We develop an sp-ECR method to measure the acceptor-to-donor extinction coefficient ratio.•We provide a novel notion to correct the cross excitation of FRET quantification.•sp-ECR is applicable to monitoring the dynamic biochemical processes in single living cells.•Caspase-3 activation in single cells is a rapid process within 20 min.This report presents a simple method named as sp-ECR to determine the molar extinction coefficient ratio (γ(λex)) of acceptor-to-donor in living cells at excitation wavelength λex, which is closely associated with the acceptor cross-excitation, the hardest issue of FRET quantification. sp-ECR determines γ(λex) by spectrally unmixing the emission spectrum of a donor–acceptor tandem construct under λex excitation without any additional references, such that this method can be performed under optimal imaging condition. We used sp-ECR to measure the γ(458) of Venus/Cerulean in living HepG2 cells on a confocal microscope, and the measured values were consistent with those obtained by lux-FRET method. We also used sp-ECR to measure the γ(458) values of Venus/Cerulean and YFP/CFP as well as YFP/GFP, the commonly used FRET FPs pairs in other two kinds of cancer cell lines on the confocal microscope, and found that the extinction coefficients of FPs depended on cell lines. After predetermining the γ(458) of Venus to ECFP, we used sp-ECR method to monitor the staurosporine (STS)-induced dynamical caspase-3 activation in single live A549 cells expressing SCAT3 by spectrally resolving the absolute FRET efficiency of SCAT3, and found that STS-induced caspase-3 activation in single cells is a very rapid process within 20 min.

In this paper, we propose a method to significantly enhance the local-field of a gap plasmonic system by placing a metallic nanoparticle in close proximity to a substrate covered with a thin film using a gain material (∼100 nm thickness). Compared with a conventional dielectric substrate, the thin gain film can contribute to several, or dozens, of times more intense local electric fields in the gap between the particle and the substrate. We use the finite difference time domain method to numerically analyze the influences of the gain coefficient of the film and of the other parameters on the field enhancement. The numerical results show that there is an optimal refractive index of the gain film that enables us to achieve a maximal field enhancement for a given NP radius. Moreover, the optimal refractive index of the gain film can be incorporated into any available materials using metal nanoparticles with an appropriate radius.

Near-infrared (NIR) fluorescent probes are increasingly popular in biological imaging and sensing, as long-wavelength (650–900 nm) excitation and emission have the advantages of minimum photodamage, deep tissue penetration, and minimum interference from autofluorescence in living systems. Here, a series of long-wavelength BODIPY dyes SPC, DC-SPC, DPC, and DC-DPC are synthesized conveniently and efficiently. They exhibit excellent photophysical properties in far red to near-infrared region, including large extinction coefficients, high fluorescence quantum yields, good photostability, and reasonable two-photon absorption cross section. Comparison of single-molecular imaging confirms that DPC is a much more efficient and more photostable NIR fluorophore than the commonly used Cy5. Also importantly, two kinds of convenient functionalization sites have been reserved: the aryl iodide for organometallic couplings and the terminal alkyne groups for click reactions. Further derivatives DC-SPC-PPh3 exhibit specificity to localize in mitochondria. The introduction of triphenylphosphonium (TPP) moieties mediates its hydrophilic–lipophilic balance and makes DC-SPC-PPh3 appropriate for cell labeling. Their long-wavelength emission at ∼650 nm can efficiently avoid the spectral crosstalk with other probes emitting in the visible light region. Superior photostability, low cytotoxicity, and two-photon excitable properties demonstrate its utility as a standard colocalizing agent to estimate the other probes’ local distribution.

A novel star-shaped chromophore, Tr–PBI, was constructed by fusing three perylenebisimide branches and a truxene core. Tr–PBI exhibits high photostability and excellent two-photon properties: the maximum of δTPA is 11000 GM at 990 nm and fluorescence quantum efficiency Φ is 0.40 in THF.

The fluorescence lifetime of fluorescent proteins is affected by the concentration of solutes in a medium, in inverse correlation with local refractive index. In this paper, we introduce the concept of using this dependence to probe cellular molecular environment and its transformation during cellular processes. We employ the fluorescence lifetime of Green Fluorescent Protein and tdTomato Fluorescent Protein expressed in cultured cells and probe the changes in the local molecular environment during the cell cycle progression. We report that the longest fluorescence lifetimes occurred during mitosis. Following the cell division, the fluorescence lifetimes of these proteins were rapidly shortened. Furthermore the fluorescence lifetime of tdTomato in the nucleoplasm gradually increased throughout the span of S-phase and remained constantly long until the end of interphase. We interpret the observed fluorescence lifetime changes to be derived from changes in concentration of macromolecular solutes in the cell interior throughout cell cycle progression.

•The working mechanisms and practical applications of SERS-based ultrasensitive detection are illustrated.•A systematic classification on the recent multiple strategies for ultra-high SERS detection sensitivity is presented.•A detailed analysis on the up-to-date applications in vitro and in vivo is outlined.•Optimal reference guidelines for specific detection or monitoring of analytes in highly diluted solutions are highlighted.Ultrasensitive detection of chemicals and biological analytes in trace or single molecular level is highly desirable in both scientific and technological fields, e.g., analytical chemistry, life science, materials science, biomedical diagnostics, and forensic science, etc. With high sensitivity, high specificity, narrow line-widths, and multiplexed non-destructive testing capabilities, surface enhanced Raman scattering (SERS)-based sensing is the most promising approach to monitor targeted analytes in the vicinity of nanostructured surface. An insight into the recent advances of SERS-based ultrasensitive sensing platform can provide an effective reference guideline to develop an optimal detection approach for arising real-world applications. Many SERS-based review articles mainly focus on the fundamental theory of SERS, nanostructured plasmonic SERS sensors, and single molecule SERS detection. However, no comprehensive review article targeting SERS-based ultrasensitive detection strategies, their working mechanisms and illustrative practical applications has been reported yet. Hence, it is important to project the latest SERS-based ultrasensitive detection research in a review, which will present a reference guideline to develop an optimal detection approach for specific detection or monitoring of analytes in highly diluted solutions. We present a systematic classification and discussion on the recent multiple strategies to achieve ultra-high SERS detection sensitivity. We also outline a detailed analysis on the up-to-date applications in vitro and in vivo. Finally, we also discuss a new trend in SERS-based ultrasensitive sensing applications.

A novel star-shaped chromophore, Tr–PBI, was constructed by fusing three perylenebisimide branches and a truxene core. Tr–PBI exhibits high photostability and excellent two-photon properties: the maximum of δTPA is 11000 GM at 990 nm and fluorescence quantum efficiency Φ is 0.40 in THF.

Living organisms are generally composed of complex cellular processes which persist only within their native environments. To enhance our understanding of the biological processes lying within complex milieus, various techniques have been developed. Specifically, the emergence of super-resolution microscopy has generated a renaissance in cell biology by redefining the existing dogma towards nanoscale cell dynamics, single synaptic vesicles, and other complex bioprocesses by overcoming the diffraction-imposed resolution barrier that is associated with conventional microscopy techniques. Besides the typical technical reliance on the optical framework and computational algorithm, super-resolution imaging microscopy resorts largely to fluorescent materials with special photophysical properties, including fluorescent proteins, organic fluorophores and nanomaterials. In this tutorial review article, with the emphasis on cell biology, we summarize the recent developments in fluorescent materials being utilized in various super-resolution techniques with successful integration into bio-imaging applications. Fluorescent proteins (FP) applied in super-resolution microscopy will not be covered herein as it has already been well summarized; additionally, we demonstrate the breadth of opportunities offered from a future perspective.