Two-photon Excited Fluorescence Research Articles

Crystal structures, one- and two-photon excited fluorescence of two complexes based on D-π-A structural 2,2-bipyridine derivative

By coordination of an electron donor-π-bridge-electron acceptor (D-π-A) optical functional organic ligand (L) with ZnBr2 and HgBr2, two fluorescent complexes ZnBr2L (1) and HgBr2L (2) were obtained. The structures of them were solved by single crystal X-ray diffraction analysis. Study of linear and nonlinear optical properties has shown that the complexes exhibit excellent one- and two-photon excited fluorescence and large two-photon absorption cross-section values. Dye 1 shows the potential application in fluorescence microscopy bioimaging.

Label-Free Imaging of Blood Vessels in Human Normal Breast and Breast Tumor Tissue Using Multiphoton Microscopy.

Blood vessels are the important components of the circulatory systems that transport blood throughout the human body and maintain the homeostasis of physiological tissues. Pathologically, blood vessels are often affected by diseases, leading to the formation of unstable, irregular, and hyperpermeable blood vessels. In the tumor microenvironment, abnormal leakage of tumor blood vessels is related to the histological grade and malignant potential of tumors and may also facilitate metastasis of cancer. Visual diagnosis of blood vessels is very important for us to understand the occurrence and development of diseases. Multiphoton microscopy (MPM) is a potential label-free diagnostic tool based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). MPM can effectively observe the morphological changes of biological tissues at the molecular and cellular levels. In this work, we demonstrate that label-free MPM can be used to visualize the microstructure of blood vessels in human normal breast and breast tumor tissue. Moreover, MPM can monitor the changes of blood vessels in tumor microenvironment. These results show that the MPM will become a promising technique for clinicians to study the properties of the microstructure of the blood vessels.

Elucidation of the mechanism and energy barrier for anesthetic triggered membrane fusion in model membranes

Membrane fusion is vital for cellular function and is generally mediated via fusogenic proteins and peptides. The mechanistic details and subsequently the transition state dynamics of membrane fusion will be dependent on the type of the fusogenic agent. We have previously established the potential of general anesthetics as a new class of fusion triggering agents in model membranes. We employed two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to report on vesicle association kinetics and steady-state fluorescence dequenching assays to monitor lipid mixing kinetics. Using halothane to trigger fusion in 110 nm diameter dioleoylphosphatidylcholine (DOPC) liposomes, we found that lipid rearrangement towards the formation of the fusion stalk was rate limiting. The activation barrier for halothane induced membrane fusion in 110 nm vesicles was found to be ∼40 kJ mol−1. We calculated the enthalpy and entropy of the transition state to be ∼40 kJ mol−1and ∼180 J mol−1K−1, respectively. We have found that the addition of halothane effectively lowers the energy barrier for membrane fusion in less curved vesicles largely due to entropic advantages.

Automatic and label-free identification of blood vessels in gliomas using the combination of multiphoton microscopy and image analysis.

Currently, the targeted treatment of tumor based on the tumor microenvironment is newly developed. Blood vessels are the key parts in the tumor microenvironment, which is taken as a new visible target for tumor therapy. Multiphoton microscopy (MPM), based on the second harmonic generation and two-photon excited fluorescence, is available to make the label-free analysis on the blood vessels in human gliomas. MPM can reveal the vascular morphological characteristics in gliomas, including vascular malformation, intense vascular proliferation, perivascular collagen deposition, perivascular lymphocytes aggregation and microvascular proliferation. In addition, the image analysis algorithms were developed to automatically calculate the perivascular collagen content, vascular cavity area, lumen area, wall area and vessel number. Thus, the vascular morphology, the perivascular collagen deposition and intense vascular proliferation degree can be further quantitatively characterized. Compared with the pathological analysis, the combination of MPM and image analysis has potential advantages in making a quantitative and qualitative analyzing on vascular morphology in glioma microenvironment. As micro-endoscope and two-photon fiberscope are technologically improved, this combined method will be a useful imaging way to make the real-time research on the targeting tumor microenvironment in gliomas.

Determination of Two-Photon Absorption Cross-Sections in Selected Diketo-Pyrrolo-Pyrroles

The paper deals with determination of two-photon absorption cross-sections of selected representatives of diketo-pyrrolo-pyrrole. The materials were optimized in order to test the influence of various substitutions on the diketo-pyrrolo-pyrrole basic molecule. The two-photon absorption cross-sections were determined using two-photon excited fluorescence technique. A considerably high two-photon absorption cross-section in order of 1000 GM was found for non-symmetrically substituted derivative with donor-acceptor structure. All of the derivatives showed strong fluorescence in solid state visible by naked eye. These results demonstrate that this class of materials can be used in e.g. two-photon fluorescence microscopy in the form of nanoparticles.

Optical second harmonic generation microscopy: application to the sensitive detection of cell membrane damage.

Optical second harmonic generation (SHG) is a nonlinear optical process which is sensitive to the symmetry of media. SHG microscopy allows for selective probing of a non-centrosymmetric area of sample. This type of nonlinear optical microscope was first used to observe ferroelectric domains and has been applied to various specimens including the biological samples to date. Imaging of the endogenous SHG of biological tissue has been utilized for the selective observation of filament systems in tissues such as collagen, myosin, and microtubules, which exhibit a polar structure. The cellular membrane can be selectively observed by the SHG microscope through membrane staining with amphiphilic polar dye molecules. It has been reported that, by imaging exogenous SHG of the membrane, sensitive detection of membrane damage could be realized using the SHG microscope. Because the staining dye is fluorescent, both SHG and two-photon excited fluorescence (TPF) images can be obtained simultaneously. How the SHG intensity depends on the molecular alignment of the polar dye molecules that reflects the ordering of lipid molecules in the plasma membrane and the necessity of the normalization of the SHG intensity by the TPF intensity is discussed. Furthermore, the assessment of the membrane damage induced by exposing polycation to HeLa cells has been compared with the conventional cytotoxicity and cell viability tests to demonstrate the higher sensitivity of the present SHG-based assay.

Improved quantitative assessment of HBV-associated liver fibrosis using second-harmonic generation microscopy with feature selection

Quantitative assessments of liver fibrosis using second-harmonic generation/two-photon excited fluorescence microscopy provide greater sensitivity and accuracy than collagen proportionate area while eliminating operator-dependent variation in the staining process. In conjunction with sophisticated image analysis algorithms and feature selection, we might reduce the computation cost in future and narrow down the candidates for further clinical studies. We sampled a total of 244 liver specimens from patients with hepatitis B viral infections who underwent liver biopsy or liver resection at the National Taiwan University Hospital. The samples were then imaged using a Genesis (HistoIndex Pte. Ltd, Singapore) system, wherein second-harmonic generation microscopy was used to visualize collagen, and two-photon excited fluorescence microscopy was used to visualize other cell structures. We used 100 morphological features extracted from the images to assess correlations with METAVIR fibrosis scores. Out of 100 quantitative measurements, 76 showed significant correlation with METAVIR scoring, thereby enabling the statistical discrimination of patients in various stages of the disease. These 76 features were also narrowed down by the nonlinear test to 10 candidate measurements, which can be further investigated in detail. Our experimental results showed that the model with 10 selected features can beat the one with second-harmonic generation only, and performed equivalently well compared the model with 76 features, especially for early-stage discrimination. Features presenting significant correlation were used to fit a single combined index in order to predict pathological staging, thereby making it possible to reveal incremental progress during treatment.

Depth resolved label-free multimodal optical imaging platform to study morpho-molecular composition of tissue

Multimodal imaging platforms offer a vast array of tissue information in a single image acquisition by combining complementary imaging techniques. By merging different systems, better tissue characterization can be achieved than is possible by the constituent imaging modalities alone. The combination of optical coherence tomography (OCT) with non-linear optical imaging (NLOI) techniques such as two-photon excited fluorescence (TPEF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) provides access to detailed information of tissue structure and molecular composition in a fast, label-free and non-invasive manner. We introduce a multimodal label-free approach for morphomolecular imaging and spectroscopy and validate the system in mouse skin demonstrating the potential of the system for colocalized acquisition of OCT and NLOI signals.

Tuning the pKa of two-photon bis-chromophoric probes for ratiometric fluorescence imaging of acidic pH in lysosomes

Lysosomes are organelles containing many hydrolytic enzymes responsible for degrading macromolecules. Abnormal lysosomal pH changes are known to associate with dysfunction of cells linking to various diseases such as cancer and neurodegenerative disorders. Thus, it is of paramount importance to monitor lysosomal pH changes in order to investigate the pathological conditions. We report herein two novel, highly sensitive and fast responsive bis-chromophoric ratiometric two-photon fluorescent probes with different emission wavelengths, namely VP and VL for acidic pH sensing in live cells. Importantly, by adopting bis-chromophoric approach, the VP and VL probes bearing pyridyl and quinolyl as acid sensing sites exhibit pKa values of 4.62 and 5.26, respectively, which are ideal for quantitative analysis of lysosomal pH changes in live cells. These two biocompatible probes are not only highly lysosomal targeting, sensitive towards pH change with distinct emission color shifting but also highly two-photon active in cells with excellent photostability and reversibility. These probes were successfully applied to ratiometrically track and image pH fluctuation in lysosomes of HeLa cells by one- and two-photon excited fluorescence microscopy. For the first time, we have demonstrated here that the bis-chromophoric strategy is a useful tool to effectively modify and tune the pKa of a fluorescent probe.

Fluorescence enhancement of small squaraine dye and its two-photon excited fluorescence in long-term near-infrared I&II bioimaging.

Two-photon excited fluorescence (TPEF) plays an important role in bioimaging, the longer excitation wavelength improves its imaging depths, which gives us deeper biological information. Here, we reported the two-photon absorption of a small squaraine dye (SD), and we found that the TPEF of the small SD can be enhanced significantly using albumin, the TPEF of SD in water was enhanced 17.7 times by adding bull serum albumin (BSA) in the solution. Meanwhile, the cell imaging results indicated that the SD can enter cell effectively in less than 30 min and emit bright TPEF. Furthermore, the SD showed excellent stability against photobleaching in near-infrared II (1200 nm). The cytotoxicity experiment showed that the cytotoxicity of SD is relatively low. Our work demonstrates the excellent two-photon effect of SD in cells, potential application value of SD in two-photon bioimaging, protein detection and near infrared sensing.

Polarization-insensitive micro-metalens for high-resolution and miniaturized all-fiber two-photon microendoscopic fluorescence imaging

A high transmission efficiency and polarization-insensitive micro-metalens with a 100 μm diameter has been designed for two-photon excited microendoscopic fluorescence imaging. We theoretically calculate the vector field near the focal region of a high NA objective lens to confirm the effectiveness and feasibility of the micro-metalens. The simulation results of the designed micro-metalens are essentially in agreement with that of an objective lens under linearly or radially polarized hollow Gaussian beam (HGB) illumination. A tightly focused spot with an FWHM of 0.56 μm and a beam quality of 72.54% is obtained when the designed micro-metalens is illuminated by radially polarized 5-order HGB at wavelength 915 nm and the corresponding lateral resolution of the two-photon fluorescence signal is 0.39 μm. Moreover, this ultrathin micro-metalens can be fabricated onto a fiber tip and directly penetrate into tissues. This miniaturized imaging system can provide a compact all-fiber two-photon excitation microendoscopic imaging system.

Multimodal multiphoton imaging for label-free monitoring of early gastric cancer

BackgroundEarly gastric cancer is associated with a much better prognosis than advanced disease, and strategies to improve prognosis is strictly dependent on earlier detection and accurate diagnosis. Therefore, a label-free, non-invasive imaging technique that allows the precise identification of morphologic changes in early gastric cancer would be of considerable clinical interest.MethodsIn this study, multiphoton microscopy (MPM) using two-photon excited fluorescence combined with second-harmonic generation was used for the identification of early gastric cancer.ResultsThis microscope was able to directly reveal improved cellular detail and stromal changes during the development of early gastric cancer. Furthermore, two features were quantified from MPM images to assess the cell change in size and stromal collagen change as gastric lesion developed from normal to early cancer.ConclusionsThese results clearly show that multiphoton microscopy can be used to examine early gastric cancer at the cellular level without the need for exogenous contrast agents. This study would be helpful for early diagnosis and treatment of gastric cancer, and may provide the groundwork for further exploration into the application of multiphoton microscopy in clinical practice.

Automated classification of hepatocellular carcinoma differentiation using multiphoton microscopy and deep learning.

In the case of hepatocellular carcinoma (HCC) samples, classification of differentiation is crucial for determining prognosis and treatment strategy decisions. However, a label-free and automated classification system for HCC grading has not been yet developed. Hence, in this study, we demonstrate the fusion of multiphoton microscopy and a deep-learning algorithm for classifying HCC differentiation to produce an innovative computer-aided diagnostic method. Convolutional neural networks based on the VGG-16 framework were trained using 217 combined two-photon excitation fluorescence and second-harmonic generation images; the resulting classification accuracy of the HCC differentiation grade was over 90%. Our results suggest that a combination of multiphoton microscopy and deep learning can realize label-free, automated methods for various tissues, diseases and other related classification problems.

Ultrashort pulse Kagome hollow-core photonic crystal fiber delivery for nonlinear optical imaging.

We report ultrashort pulse delivery through a hypocycloid-core inhibited-coupling Kagome hollow-core photonic crystal fiber (HC-PCF). Undistorted 10 fs and 6.6 nJ pulses were launched through 1 m long fiber without fiber dispersion pre-compensation and 80% efficiency. The performance of this technology for biomedical imaging is demonstrated on a biological sample by incorporating the fiber into a two-photon excited fluorescence (TPEF) laser scanning microscope (LSM) achieving a pulse width of 15 fs at the sample location. To the best of our knowledge, this is the first report on undistorted TPEF imaging in a LSM with 15 fs pulses delivered through a 1 m long Kagome HC-PCF with high throughput.

Simulation design of wide-field temporal-focusing multiphoton excitation with a tunable excitation wavelength

The optical parameters of temporal-focusing multiphoton excitation microscopy (TFMPEM), which is capable of achieving varying wavelength excitation for multiple fluorophore measurement, was systematically examined to have good excitation performance. For this purpose, the approaches were adopted to quantitatively evaluate the grating groove density, focal length of the collimating lens and objective, and different excitation wavelengths. A grating with a groove density of 830 lines/mm enables the TFMPEM system to achieve a wavelength range of 700–1000 nm by adjusting the incident angle of the ultrafast laser on the grating; a diffraction efficiency of 81 ± 3% was obtained at this wavelength range. By using the 830 lines/mm grating, a collimating lens with a 500 mm focal length and a 60× water immersion objective, we achieved a large excitation area and a better filling effect of the spectrum band of the pulse on the back focal plane of the objective; these parameters ensure high optical sectioning and small variation in the illumination power density within this wavelength range. The variation ranges of the excitation area, optical sectioning, and illumination power density of 4272 ± 798 μm2, 2.6 ± 0.3 μm, and 71 ± 29%, respectively, were obtained in two-photon excitation fluorescence imaging at 700–1000 nm excitation wavelengths.

Prediction of the consistency of pituitary adenomas based on multiphoton microscopy

Consistency of pituitary adenomas is an important variable in the surgical resection of these lesions. Preoperative evaluation of tumor consistency will be useful in guiding the operative approach. In this study, multiphoton microscopy (MPM) was used to evaluate the consistency of pituitary adenomas by combining qualitative and quantitative approaches. Three types of pituitary adenomas with different consistencies were first observed through the MPM imaging system to provide the cellular micro-structural details from fresh and unstained tissue samples. The morphologies of cell and collagen were clearly discerned by the endogenous second harmonic generation (SHG) signal from collagen and two-photon excited fluorescence (TPEF) signal from nicotinamide adenine dinucleotide and flavin adenine dinucleotide. Furthermore, RSHG/TPEF (the ratio of the maximum SHG signal over the maximum TPEF signal) and collagen content were calculated as quantitative parameters, which would provide an objective standard for evaluation of the tumor consistency. With the development of multiphoton endoscopy for in vivo imaging, MPM would provide the accurate estimation of the adenoma consistency and help neurosurgeons select appropriate surgical methods during surgery.

Quantification of hepatic steatosis in chronic liver disease using novel automated method of second harmonic generation and two-photon excited fluorescence

The presence of hepatic steatosis (HS) is an important histological feature in a variety of liver disease. It is critical to assess HS accurately, particularly where it plays an integral part in defining the disease. Conventional methods of quantifying HS remain semi-quantitative, with potential limitations in precision, accuracy and subjectivity. Second Harmonic Generation (SHG) microscopy is a novel technology using multiphoton imaging techniques with applicability in histological tissue assessment. Using an automated algorithm based on signature SHG parameters, we explored the utility and application of SHG for the diagnosis and quantification of HS. SHG microscopy analysis using GENESIS (HistoIndex, Singapore) was applied on 86 archived liver biopsy samples. Reliability was correlated with 3 liver histopathologists. Data analysis was performed using SPSS. There was minimal inter-observer variability between the 3 liver histopathologists, with an intraclass correlation of 0.92 (95% CI 0.89–0.95; p < 0.001). Good correlation was observed between the histopathologists and automated SHG microscopy assessment of HS with Pearson correlation of 0.93: p < 0.001. SHG microscopy provides a valuable tool for objective, more precise measure of HS using an automated approach. Our study reflects proof of concept evidence for potential future refinement to current conventional histological assessment.

Cobalt oxyhydroxide modified with poly-β-cyclodextrin and a cyanine dye as a nanoplatform for two-photon imaging of ascorbic acid in living cells and tissue.

This article describes the development of several nanoconjugates composed of cobalt (III) oxyhydroxide and DEASPI/βCDP, where DEASPI stands for the dye trans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium, and βCDP stands for β-cyclodextrin. The material enables sensitive fluorometric detection and 3D imaging of ascorbic acid (AA) in biological samples. A nanomicelle composed of DEASPI and βCDP was prepared to act as a two-photon absorbance (TPA) nanofluorophore with desirable two-photon-sensitized fluorescence, high penetration depth, and excellent cell-permeability). The CoOOH nanoflakes were placed on the surface of the nanomicelle to act as both a quencher of fluorescence and as the recognition unit for AA. In the presence of AA, the CoOOH nanoflakes are reduced to Co (II), and this triggers the recovery of fluorescence. This new nanoprobe exhibits amplified two-photon fluorescence (excitation at 840nm; emission at 565nm), high sensitivity, and good selectivity. In-vitro imaging of endogenous AA was demonstrated in living HeLa cells. It was also employed to 3D imaging of exogenous AA in tissue by two-photon excitation microscopy to a depth of up to 320μm. In our perception, this nanoprobe represents a valuable tool for elucidating the roles of AA in biochemical and clinical studies. Graphical abstract Schematic presentation of the preparation of a novel Poly β-Cyclodextrin/TPdye conjugated with cobalt oxyhydroxide nanoplatform and its application for high sensitive and two-photon 3D imaging of ascorbic acid (AA) in living cells and deep tissues.

Digital staining through the application of deep neural networks to multi-modal multi-photon microscopy.

Deep neural networks have been used to map multi-modal, multi-photon microscopy measurements of a label-free tissue sample to its corresponding histologically stained brightfield microscope colour image. It is shown that the extra structural and functional contrasts provided by using two source modes, namely two-photon excitation microscopy and fluorescence lifetime imaging, result in a more faithful reconstruction of the target haematoxylin and eosin stained mode. This modal mapping procedure can aid histopathologists, since it provides access to unobserved imaging modalities, and translates the high-dimensional numerical data generated by multi-modal, multi-photon microscopy into traditionally accepted visual forms. Furthermore, by combining the strengths of traditional chemical staining and modern multi-photon microscopy techniques, modal mapping enables label-free, non-invasive studies of in vivo tissue samples or intravital microscopic imaging inside living animals. The results show that modal co-registration and the inclusion of spatial variations increase the visual accuracy of the mapped results.

Multiple surface plasmon resonances enhanced nonlinear optical microscopy

Abstract The nonlinear optical microscopies of coherent two-photon excited fluorescence and anti-Stokes Raman scattering are strongly enhanced by multiple surface plasmon resonances (MSPRs). The Au@Ag nanorods presented strong MSPRs peaks at 800 and 400 nm, and can enhance nonlinear optical microscopy at fundamental and double frequencies, respectively. A two-dimensional (2D) material of g-C3N4 is employed to study the plasmon-enhanced nonlinear optical microscopy by the femtosecond laser. The electric analysis reveals that the MSPRs of the Au@Ag nanorod can significantly enhance the signals of two-photon excited fluorescence and anti-Stokes Raman scattering by up to the orders of 104 and 1016, respectively. The results demonstrate the great advantages of plasmon-enhanced nonlinear optical microscopy for the optical analysis on 2D materials, thus providing a new adventure for increasing the optical resolutions of nonlinear optical microscopy.