Two-photon Excited Fluorescence Research Articles

Multimodal imaging platform for optical virtual skin biopsy enabled by a fiber-based two-color ultrafast laser source.

We demonstrate multimodal label-free nonlinear optical microscopy in human skin enabled by a fiber-based two-color ultrafast source. Energetic femtosecond pulses at 775 nm and 1250 nm are simultaneously generated by an Er-fiber laser source employing frequency doubling and self-phase modulation enabled spectral selection. The integrated nonlinear optical microscope driven by such a two-color femtosecond source enables the excitation of endogenous two-photon excitation fluorescence, second-harmonic generation, and third-harmonic generation in human skin. Such a 3-channel imaging platform constitutes a powerful tool for clinical application and optical virtual skin biopsy.

Efficient cell classification of mitochondrial images by using deep learning

Key challenges for affected cells, evolutionary biology and precision medicine include the effect of drug and understanding viscosity and intensity of drug-treated cells. However, this is extremely difficult because the enormous cells are affected by the drug. We developed a deep learning-based framework DNCIC that can accurately predict normal mitochondria and drug-affected cells that are rare or not observed. For optimization, we used a convolutional neural network and trained using a dataset of mitochondrial images, which were collected through the confocal microscope. The obtained algorithm was validated on the normal and affected cell images. We have trained CNN that can classify (normal and affected cells) two-photon excited fluorescence probe images. The proposed model has classified images and videos with 98% accuracy. Our results provided a foundation for drug-affected cell diagnosis.

Plasmon-enhanced two-photon excitation fluorescence of rhodamine 6G and an Eu-diketonate complex by a picosecond diode laser.

Two-photon excited fluorescence (TPEF) of rhodamine 6G (Rh6G) and tris(dibenzoylmethane) mono(5-aminophenanthroline) europium (Eu-TDPA) was measured using a pulsed diode laser head (<45 mW, 975 nm, 90 ps pulse width, 40 MHz). Fluorophores were cast on a glass slide modified with triangular silver nanoprisms. A photon-counting photomultiplier detected the TPEF of Rh6G on a glass substrate (1361 Hz) and on the nanoprisms (6322 Hz). On the other hand, Eu-TDPA did not exhibit TPEF on a glass substrate. TPEF was only observed when the extinction of the nanoprisms on the substrates was larger than 0.1. The nanoprisms enhanced the TPEF of these two fluorophores up to the detectable level using a low-power laser diode.

Facile synthesis of homochiral compounds integrating circularly polarized luminescence and two-photon excited fluorescence.

A pair of novel chiral one-dimensional compounds with the general formula [Cd(NO3)2L2]·THF (where L is R- or S-2,2'-bis(methoxymethoxy)-6,6'-bis(4-pyridyl)-1,1'-binaphthyl) for the first time show circularly polarized luminescence, two-photon excited fluorescence, and second harmonic generation activity.

Optical Visualization of Cerebral Cortex by Label-Free Multiphoton Microscopy

Cortical structures in the central nervous system exhibit an ordered laminar organization. Defined cell layers are significant to our understanding of brain structure and function. In this work, multiphoton microscopy (MPM), based on second harmonic generation and two-photon excited fluorescence, is applied for visualizing the mouse and human cerebral cortex from fresh, unfixed, and unstained specimen. We show that label-free MPM is able to effectively visualize microscopic scale of cortical neurons, six-layer cortical lamination, and functional cortex that correlates with the corresponding stained histological images. Moreover, we further demonstrate that MPM is feasible for identifying focal cortical dysplasia (FCD) in the human cerebral cortex. The border between gray and white matter, abnormal radial cortical lamination, immature small diameter neurons, pia mater, and blood vessels are also clearly visualized as the typical histopathologic hallmark of FCD. In addition, to diagnose FCD more accurately and rapidly, an image processing method is employed to extract MPM image features for auxiliary identification of FCD specimen. These results offer additional metrics to pathologists and surgeons, which hold the translational potential to be used as a diagnostic tool into routine clinical use.

In vivo two-photon imaging/excited photothermal therapy strategy of a silver-nanohybrid.

A multi-functional nanohybrid (PyAnOH-Ag) with both a two-photon photothermal therapy (TP-PTT) effect and two-photon excited fluorescence (TPEF) imaging performance has been fabricated based on interfacial coordination interactions. The hybrid possesses a high two-photon absorption cross section (δTPA, 4638 GM) and detectable TPEF signals, which leads to excellent two-photon photothermal conversion. Upon irradiation at 840 nm, the temperature of the PyAnOH-Ag-PBS suspension reaches 42 °C in just 4 min, which results in 80% photothermal toxicity on HepG2 cells. The detectable TPEF signals can be used to monitor the cell ablation procedure. Moreover, PyAnOH-Ag exhibits a good phototherapeutic effect on tumor tissue of H22-modelled mice with almost 100% tumor growth inhibition under 840 nm irradiation for 10 min, which is superior to many reported photothermal agents. This strategy of TPEF guided TP-PTT agents can be potentially applied in a variety of therapeutic agents with monitoring ability.

Gold nanorod enhanced conjugated polymer/photosensitizer composite nanoparticles for simultaneous two-photon excitation fluorescence imaging and photodynamic therapy.

Two-photon photodynamic therapy (2P-PDT) is a novel minimal invasive cancer treatment method with advantages of deep penetration and intrinsic three-dimensionally localized activation to precisely target cancerous tissues. However, the therapeutic efficacy of 2P-PDT is limited by small two-photon absorption cross sections of conventional organic photosensitizers. In addition, traditional photosensitizers generally exhibit weak emission and lack imaging modality. In this work, conjugated polymers and gold nanorods (Au NRs) were integrated to fabricate nano-sized photosensitizers to improve the performance of molecular photosensitizers for 2P-PDT. A molecular photosensitizer, tetraphenylporphyrin, was encapsulated into the conjugated polymer PFV to form conjugated polymer nanoparticles (CPNs), which were then covalently linked to silica coated Au NRs. In these integrated nanoparticles, the two-photon optical properties of tetraphenylporphyrin were first enhanced by fluorescence resonance energy transfer from PFV, then further enhanced by Au NRs through plasmon resonance. A silica shell was utilized as the spacer between Au NRs and CPNs to optimize the enhancement effects. Through the combined enhancement effects of energy transfer and plasmon resonance, two-photon excitation fluorescence and two-photon induced singlet oxygen generation of tetraphenylporphyrin were enhanced by up to 980- and 792-fold, respectively, at a silica spacer thickness of 9 nm. The application of these nanoparticles as photosensitizers for simultaneous two-photon imaging and 2P-PDT treatment have been demonstrated on HeLa cancer cells with high brightness and significantly enhanced cancer cell killing efficiency. These nanoparticles can act as promising nano-photosensitizers for 2P-PDT with simultaneous imaging modality.

Methods for Optical Skin Clearing in Molecular Optical Imaging in Dermatology.

This short review describes recent progress in using optical clearing (OC) technique in skin studies. Optical clearing is an efficient tool for enhancing the probing depth and data quality in multiphoton microscopy and Raman spectroscopy. Here, we discuss the main mechanisms of OC, its safety, advantages, and limitations. The data on the OC effect on the skin water content are presented. It was demonstrated that 70% glycerol and 100% OmnipaqueTM 300 reduce the water content in the skin. Both OC agents (OCAs) significantly affect the strongly bound and weakly bound water. However, OmnipaqueTM 300 causes considerably less skin dehydration than glycerol. In addition, the results of examination of the OC effect on autofluorescence in two-photon excitation and background fluorescence in Raman scattering at different skin depths are presented. It is shown that OmnipaqueTM 300 is a promising OCA due to its ability to reduce background fluorescence in the upper skin layers. The possibility of multimodal imaging combining optical methods and OC technique is discussed.

Gold nanorod-enhanced two-photon excitation fluorescence of conjugated oligomers for two-photon imaging guided photodynamic therapy

Plasmon enhanced two-photon fluorescence nanomaterials were developed to manipulate their optical properties for two-photon imaging guided photodynamic therapy.

A NIR-triggered gatekeeper of supramolecular conjugated unimicelles with two-photon absorption for controlled drug release.

A near-infrared (NIR)-sensitive gated assembly of supramolecular conjugated unimicelles based on robust host-guest recognition between a β-cyclodextrin-grafted hyperbranched conjugated polymer and azobenzene-functionalized poly(ethylene glycol) was constructed. Utilized as a drug carrier, these unimicelles exhibited controlled drug release through the NIR-triggered photoisomerization of azobenzene in cancer cells via a two-photon excited fluorescence resonance energy transfer (TP-FRET) approach, leading to efficient cancer therapy.

Ligand shell size effects on one- and two-photon excitation fluorescence of zwitterion functionalized gold nanoclusters.

Gold nanoclusters (Au NCs) are an emerging class of luminescent nanomaterials but still suffer from moderate photoluminescence quantum yields. Recent efforts have focused on tailoring their emission properties. Introducing zwitterionic ligands to cap the metallic kernel is an efficient approach to enhance their one-photon excitation fluorescence intensity. In this work, we extend this concept to the nonlinear optical regime, i.e., two-photon excitation fluorescence. For a proper comparison between theory and experiment, both ligand and solvent effects should be considered. The effects of ligand shell size and of aqueous solvent on the optical properties of zwitterion functionalized gold nanoclusters have been studied by performing quantum mechanics/molecular mechanics (QM/MM) simulations.

Tuning the π-bridge of quadrupolar triarylborane chromophores for one- and two-photon excited fluorescence imaging of lysosomes in live cells.

A series of tetracationic quadrupolar chromophores containing three-coordinate boron π-acceptors linked by different π-bridges, namely 4,4'-biphenyl, 2,7-pyrene, 2,7-fluorene, 3,6-carbazole and 5,5'-di(thien-2-yl)-3,6-diketopyrrolopyrrole, were synthesized. While their neutral precursors 1-5 displayed highly solvatochromic fluorescence, the water-soluble tetracationic target molecules 1M-5M, did not, but their emission colour could be tuned from blue to pink by changing the π-bridge. Compound 5M, containing the diketopyrrolopyrrole bridge, exhibits the most red-shifted absorption and emission maxima and the largest two-photon absorption cross-section (4560 GM at 740 nm in MeCN). Confocal laser scanning fluorescence microscopy studies in live cells confirm localization of the dye at the lysosome. Moreover, the low cytotoxicity, and high photostability of 5M combined with two-photon excited fluorescence imaging studies demonstrate its excellent potential for lysosomal imaging in live cells.

Label-Free Assessment of Premalignant Gastric Lesions Using Multimodal Nonlinear Optical Microscopy

In this paper, a nonlinear optical microscopy employing two-photon excited fluorescence and second-harmonic generation was used for the detection of premalignant gastric lesions. It was found that gland morphology and collagen structure in mucosa will change with the progression of gastric diseases from normal to intestinal metaplasia, to low-grade intraepithelial neoplasia, and to high-grade intraepithelial neoplasia, and this microscopy was able to directly distinguish these warning symptoms. Furthermore, two features were quantified from nonlinear optical images to demonstrate the changes of gland size and collagen content during the development process of preneoplastic lesions. These results clearly show that nonlinear optical microscopy can effectively differentiate normal and precancerous gastric tissues without contrast agents, which would be helpful for early diagnosis and treatment of gastric diseases. This study may provide the groundwork for further application of nonlinear optical microscopy in clinical practice.

Lateral Heterogeneity of Cholesterol Distribution in Cell Plasma Membrane: Investigation by Microfluorimetry, Immunofluorescence, and TOF-SIMS

Lateral heterogeneity of cholesterol distribution in cell plasma membrane was revealed by a complex study using microfluorimetry, immunofluorescence microscopy, and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Glioblastoma cells U87MG exhibit a high motility, and cell movements are accompanied by redistribution of proteins along the membrane surface. The formation of the protein caveolin-1 clusters at one of the plasma membrane edges was detected by confocal microscopy on cells labeled with antibodies against caveolin 1. Using two-photon excitation fluorescence of a membrane probe 4-dimethylaminochalcone, membrane areas of about 200 × 200 nm were examined on glioblastoma cells. Simultaneous detection of the decay kinetics and the fluorescence spectrum revealed the presence of regions with an increased cholesterol concentration in the membrane at the poles of the live migrating cell. TOF-SIMS provided direct data with high spatial resolution indicating colocalization of cholesterol and caveolin 1 and confirmed previously published data on the association of caveolin 1 complexes with cholesterol clusters. Thus, three independent methods of cell membrane analysis testify that localization of cholesterol-enriched membrane regions correlates with morphological features of moving glioblastoma cells.

Increased nicotinamide adenine dinucleotide pool promotes colon cancer progression by suppressing reactive oxygen species level.

Nicotinamide adenine dinucleotide (NAD) exists in an oxidized form (NAD+) and a reduced form (NADH). NAD + plays crucial roles in cancer metabolism, including in cellular signaling, energy production and redox regulation. However, it remains unclear whether NAD(H) pool size (NAD + and NADH) could be used as biomarker for colon cancer progression. Here, we showed that the NAD(H) pool size and NAD +/NADH ratio both increased during colorectal cancer (CRC) progression due to activation of the NAD + salvage pathway mediated by nicotinamide phosphoribosyltransferase (NAMPT). The NAMPT expression was upregulated in adenoma and adenocarcinoma tissues from CRC patients. The NADH fluorescence intensity measured by two‐photon excitation fluorescence (TPEF) microscopy was consistently increased in CRC cell lines, azoxymethane/dextran sodium sulfate (AOM/DSS)‐induced CRC tissues and tumor tissues from CRC patients. The increases in the NAD(H) pool inhibited the accumulation of excessive reactive oxygen species (ROS) levels and FK866, a specific inhibitor of NAMPT, treatment decreased the CRC nodule size by increasing ROS levels in AOM/DSS mice. Collectively, our results suggest that NAMPT‐mediated upregulation of the NAD(H) pool protects cancer cells against detrimental oxidative stress and that detecting NADH fluorescence by TPEF microscopy could be a potential method for monitoring CRC progression.

Quasi-simultaneous multiplane calcium imaging of neuronal circuits.

Two-photon excitation fluorescence microscopy is widely used to study the activity of neuronal circuits. However, the fast imaging is typically constrained to a single lateral plane for a standard microscope design. Given that cortical neuronal networks in a mouse brain are complex three-dimensional structures organised in six histologically defined layers which extend over many hundreds of micrometres, there is a strong demand for microscope systems that can record neuronal signalling in volumes. Henceforth, we developed a quasi-simultaneous multiplane imaging technique combining an acousto-optic deflector and static remote focusing to provide fast imaging of neurons from different axial positions inside the cortical layers without the need for mechanical disturbance of either the objective lens or the specimen. The hardware and the software are easily adaptable to existing two-photon microscopes. Here, we demonstrated that our imaging method can record, at high speed and high image contrast, the calcium dynamics of neurons in two different imaging planes separated axially with the in-focus and the refocused planes 120 µm and 250 µm below the brain surface respectively.

Selective S Cone Damage and Retinal Remodeling Following Intense Ultrashort Pulse Laser Exposures in the Near-Infrared.

PurposeInfrared ultrashort pulse lasers are becoming increasingly popular for applications in the living eye. However, safety standards are not yet well established. Here we investigate retinal damage close to threshold for this pulse regime in the living macaque eye.MethodsRetinal radiant exposures between 214 and 856 J/cm2 were delivered to the photoreceptor layer with an ultrashort pulse laser (730 nm, 55 fs, 80 MHz) through a two-photon adaptive optics scanning light ophthalmoscope. Retinal exposures were followed up immediately after and over several weeks with high-resolution reflectance and two-photon excited fluorescence ophthalmoscopy, providing structural and functional information.ResultsRetinal radiant exposures of 856 J/cm2 resulted in permanent S cone damage. Immediately after the exposure, the affected cones emitted about 2.6 times less two-photon excited fluorescence (TPEF) and showed an altered TPEF time course. Several weeks after the initial exposure, S cone outer and inner segments had disappeared. The space was filled by rods in the peripheral retina and cones near the fovea.ConclusionInterestingly, S cones are the receptor class with the lowest sensitivity in the near-infrared but are known to be particularly susceptible to ultraviolet and blue light. This effect of selective S cone damage after intense infrared ultrashort pulse laser exposure may be due to nonlinear absorption and distinct from pure thermal and mechanical mechanisms often associated with ultrashort pulse lasers.

Nonlinear optical spectroscopy and two-photon excited fluorescence spectroscopy reveal the excited states of fluorophores embedded in a beetle's elytra.

Upon illumination by ultraviolet light, many animal species emit light through fluorescence processes arising from fluorophores embedded within their biological tissues. Fluorescence studies in living organisms are however relatively scarce and so far limited to the linear regime. Multiphoton excitation fluorescence analyses as well as nonlinear optical techniques offer unique possibilities to investigate the effects of the local environment on the excited states of fluorophores. Herein, these techniques are applied for the first time to study of the naturally controlled fluorescence in insects. The case of the male Hoplia coerulea beetle is investigated because the scales covering the beetle's elytra are known to possess an internal photonic structure with embedded fluorophores, which controls both the beetle's coloration and the fluorescence emission. An intense two-photon excitation fluorescence signal is observed, the intensity of which changes upon contact with water. A third-harmonic generation signal is also detected, the intensity of which depends on the light polarization state. The analysis of these nonlinear optical and fluorescent responses unveils the multi-excited states character of the fluorophore molecules embedded in the beetle's elytra. The role of form anisotropy in the photonic structure, which causes additional tailoring of the beetle's optical responses, is demonstrated by circularly polarized light and nonlinear optical measurements.

Effect of aggregation behavior on linear and nonlinear photophysical properties of lipid-water amphiphilic photosensitizers

With the wide application of two-photon technology in biological field, lipid-water amphiphilic chromophores containing a large conjugation structure are required. In this study, six polyethylene glycol (PEG) modified benzylidene cycloalkanone photosensitizers (PSs) were selected as models to investigate the effect of their aggregation behaviors on their photophysical properties in deionized water. The results showed that all of these PSs could form aggregates at a suitable concentration in aqueous solution. However, the aggregation behaviors of symmetrically modified PSs (B2, X2 and M2) and asymmetrically modified ones (B3, X3 and M3) were quite different, which had significant effects on their photophysical properties, especially for two-photon excited fluorescence (2PEF). Under a high concentration (2×10−4 M), the compact aggregates of B3 and X3 exhibited enhanced 2PEF intensity versus fluorescence quenching of others. Moreover, B2, X2 and M2 presented higher polarity and lower cytotoxicity, while B2 and B3 with shorter PEG showed good performance in two-photon polymerization, indicating B2 will be a potential PS for 3D microfabrication of biosafety materials.

Fully convolutional networks in multimodal nonlinear microscopy images for automated detection of head and neck carcinoma: Pilot study.

A fully convolutional neural networks (FCN)-based automated image analysis algorithm to discriminate between head and neck cancer and noncancerous epithelium based on nonlinear microscopic images was developed. Head and neck cancer sections were used for standard histopathology and co-registered with multimodal images from the same sections using the combination of coherent anti-Stokes Raman scattering, two-photon excited fluorescence, and second harmonic generation microscopy. The images analyzed with semantic segmentation using a FCN for four classes: cancer, normal epithelium, background, and other tissue types. A total of 114 images of 12 patients were analyzed. Using a patch score aggregation, the average recognition rate and an overall recognition rate or the four classes were 88.9% and 86.7%, respectively. A total of 113 seconds were needed to process a whole-slice image in the dataset. Multimodal nonlinear microscopy in combination with automated image analysis using FCN seems to be a promising technique for objective differentiation between head and neck cancer and noncancerous epithelium.