Three-photon Excited Fluorescence Research Articles

Three-photon excited fluorescence imaging in neuroscience: From principles to applications.

The development of three-photon microscopy (3PM) has greatly expanded the capability of imaging deep within biological tissues, enabling neuroscientists to visualize the structure and activity of neuronal populations with greater depth than two-photon imaging. In this review, we outline the history and physical principles of 3PM technology. We cover the current techniques for improving the performance of 3PM. Furthermore, we summarize the imaging applications of 3PM for various brain regions and species. Finally, we discuss the future of 3PM applications for neuroscience.

Control the Single-, Two-, and Three-Photon Excited Fluorescence of Atomically Precise Metal Nanoclusters.

It remains challenging to control the single-, two-, and three-photon excited fluorescence of metal nanoclusters. In this work, the control over the non-linear optics of metal nanoclusters as single-, two-, and three-photon excited fluorescence has been accomplished via exploiting the solvent effect. An emissive nanocluster, Au9 Ag6 (SPht OMe)4 (DPPOE)3 Cl3 , was synthesized and structurally determined. The solvent effect can not only control the fluorescence of this nanocluster, but more significantly, it can also regulate the photoluminescence nature of the cluster as single-, two-, and three-photon excited fluorescence. We concluded that the increased solution polarity, improved dipole moment, enlarged HOMO-LUMO energy gap, and reduced solution viscosity of the cluster in solutions endow them with excellent high-order multiphoton excited fluorescence. The results provide an intriguing cluster template that enables us to manipulate the linear and nonlinear optics at the atomic level.

A potent luminogen with NIR-IIb excitable AIE features for ultradeep brain vascular and hemodynamic three-photon imaging

Three-photon excited fluorescence microscopy (3PEFM) has emerged as a promising protocol for visualizing deep-brain vasculature and hemodynamics. However, the current situation is still far from satisfactory, due to small excitation action cross-section and short excitation wavelength of those previously reported 3PEFM luminogens. Herein, we manipulated molecular engineering by subtly regulating structural planarization/twisting to achieve ingenious integration of large three-photon absorption cross-section, high fluorescence quantum yield, ultralong near-infrared IIb excitation, and aggregation-induced emission features. The resulting molecule, namely DPCZ-BT, exhibited as high as 50.6% of fluorescence quantum yield and as large as 2.0 × 10−81 cm6s2/photon2 of three-photon absorption cross-section, which can be excited by 1665 nm fs laser and presents a recorded penetration depth of 1860 μm for deep-brain vascular structural imaging with high spatiotemporal resolution and signal-to-background ratio. Moreover, DPCZ-BT having good photostability and excellent biocompatibility is capable of impressively approaching 1600 μm depth in monitoring red blood cells flow velocity with extraordinary clarity for hemodynamics.

Three-dimensional characterization of collagen remodeling in cell-seeded collagen scaffolds via polarization second harmonic generation.

In this study, we use non-linear imaging microscopy to characterize the structural properties of porous collagen-GAG scaffolds (CGS) seeded with human umbilical vein endothelial cells (HUVECs), as well as human mesenchymal stem cells (hMSCs), a co-culture previously reported to form vessel-like structures inside CGS. The evolution of the resulting tissue construct was monitored over 10 days via simultaneous two- and three-photon excited fluorescence microscopy. Time-lapsed 2- and 3-photon excited fluorescence imaging was utilized to monitor the temporal evolution of the vascular-like structures up to 100 µm inside the scaffold up to 10 days post-seeding. 3D polarization-dependent second harmonic generation (PSHG) was utilized to monitor collagen-based scaffold remodeling and determine collagen fibril orientation up to 200 µm inside the scaffold. We demonstrate that polarization-dependent second harmonic generation can provide a novel way to quantify the reorganization of the collagen architecture in CGS simultaneously with key biomechanical interactions between seeded cells and CGS that regulate the formation of vessel-like structures inside 3D tissue constructs. A comparison between samples at different days in vitro revealed that gradually, the scaffolds developed an orthogonal net-like architecture, previously found in real skin.

Chemical complexity of the retina addressed by novel phasor analysis of unstained multimodal microscopy

Abstract Unstained multimodal microscopy is capable of non-invasively obtaining chemically specific information with sub-micron spatial resolution; however, spectral overlap makes quantitative analysis difficult. Here, multimodal images that include second-harmonic generation, third-harmonic generation, and two- and three-photon excited fluorescence signals from unstained retinas are analyzed. The composition from each layer of the retina is determined using a novel variation of phasor analysis that is not limited to three components. The super-phasor unmixing (SPU) method is compared with fully constrained linear spectral unmixing. The seven spectroscopic signals in the spectral range 300–690 nm enable the quantitative unmixing of sub-micron pixel spectra even in the presence of noise. The performance of SPU was found to be significantly superior to linear unmixing especially in regions of high spectral overlap. The analysis being presented represents an important step in addressing chemical complexity in congested spaces with applicability to biomedical imaging and unmixing of hyperspectral images.

Protein-crystal detection with a compact multimodal multiphoton microscope

There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future.

Pressure-Induced Multiphoton Excited Fluorochromic Metal-Organic Frameworks for Improving MPEF Properties.

In multiphoton excited fluorescence (MPEF), high-energy upconversion emission is obtained from low-energy excitation by absorbance of two or more photons simultaneously. In a pressure-induced fluorochromic process, the emission energy is switched by outer pressure stimuli. Now, five metal-organic frameworks containing the same ligand with simultaneous multiphoton absorption and pressure-induced fluorochromic attributes were studied. One-, two-, and three-photon excited fluorescence (1/2/3PEF) can be achieved in the frameworks, which exhibit pressure-induced blue-to-yellow fluorochromism. The performances are closely dependent with the topologies, flexibilities, and packing states of the frameworks and chromophores therein. The multiphoton upconversion performance can be intensified by pressure-related structural contraction. Over ten-fold increment in the 2PA active cross-section up to 2217 GM is achieved in pressed LIFM-114 compared with the 210 GM for pristine sample at 780 nm.

Tunable Aggregation-Induced Emission Nanoparticles by Varying Isolation Groups in Perylene Diimide Derivatives and Application in Three-Photon Fluorescence Bioimaging.

The development of fluorogens with deep-red emission is one of the hottest topics of investigation in the field of bio/chemosensors and bioimaging. Herein, the tunable fluorescence of perylene diimide (PDI) derivatives was achieved by the incorporation of varied isolation groups linked on the PDI core. With the enlarged sizes of isolation groups, the conversion from aggregation caused quenching to aggregation-induced emission was obtained in their fluorescence variations from solutions to nanoparticles, as the result of the efficient inhibition of π-π stacking by the larger isolation groups. Accordingly, DCzPDI bearing 1,3-di(9H-carbazol-9-yl)benzene as the biggest isolation group exhibited the bright deep-red emission in the aggregated state with a quantum yield of 12.3%. Combined with the three-photon excited fluorescence microscopy (3PFM) technology, through-skull 3PFM imaging of mouse cerebral vasculature can be realized by DCzPDI nanoparticles with good biocompatibility, and the penetration depth can be as deep as 450 μm.

Enhanced three-photon absorption and excited up-conversion fluorescence of phenanthroimidazole derivatives

Two novel phenanthroimidazole derivatives were synthesized for efficient three-photon excited fluorescence and absorption. By introducing spirobi [9H-fluorene] to replace fluorene, the three-photon-excited fluorescence and cross-section are significantly enhanced to 3.56 ± 0.15 × 10−78 cm6 s2 photon−2, which is among the highest reported values for organic molecules. Our results supply a useful method to develop novel materials for applications of three-photon excited fluorescence and absorption.

Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood.

Red blood cells (RBC) in two-photon excited fluorescence (TPEF) microscopy usually appear as dark disks because of their low fluorescent signal. Here we use 15fs 800nm pulses for TPEF, 45fs 1060nm pulses for three-photon excited fluorescence, and third harmonic generation (THG) imaging. We find sufficient fluorescent signal that we attribute to hemoglobin fluorescence after comparing time and wavelength resolved spectra of other expected RBC endogenous fluorophores: NADH, FAD, biliverdin, and bilirubin. We find that both TPEF and THG microscopy can be used to examine erythrocyte morphology non-invasively without breaching a blood storage bag.

Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging.

We study the optimal conditions for high throughput two-photon excited fluorescence (2PEF) and three-photon excited fluorescence (3PEF) imaging using femtosecond lasers. We derive relations that allow maximization of the rate of imaging depending on the average power, pulse repetition rate, and noise characteristics of the laser, as well as on the size and structure of the sample. We perform our analysis using ~100 MHz, ~1 MHz and 1 kHz pulse rates and using both a tightly-focused illumination beam with diffraction-limited image resolution, as well loosely focused illumination with a relatively low image resolution, where the latter utilizes separate illumination and fluorescence detection beam paths. Our theoretical estimates agree with the experiments, which makes our approach especially useful for optimizing high throughput imaging of large samples with a field-of-view up to 10x10 cm2.

Superior optical nonlinearity of an exceptional fluorescent stilbene dye

Strong multiphoton absorption and harmonic generation in organic fluorescent chromophores are, respectively, significant in many fields of research. However, most of fluorescent chromophores fall short of the full potential due to the absence of the combination of such different nonlinear upconversion behaviors. Here, we demonstrate that an exceptional fluorescent stilbene dye could exhibit efficient two- and three-photon absorption under the excitation of femtosecond pulses in solution phase. Benefiting from its biocompatibility and strong excited state absorption behavior, in vitro two-photon bioimaging and superior optical limiting have been exploited, respectively. Simultaneously, the chromophore could generate efficient three-photon excited fluorescence and third-harmonic generation (THG) when dispersed into PMMA film, circumventing the limitations of classical fluorescent chromophores. Such chromophore may find application in the production of coherent light sources of higher photon energy. Moreover, the combination of three-photon excited fluorescence and THG can be used in tandem to provide complementary information in biomedical studies.

Synthesis and efficient three-photon excited green fluorescence of pyridine–triphenylamine conjugated dyes

Pyridine-end-capped triphenylamine derivatives (Py2-TPA)n (n = 1,2,4) were synthesized using palladium-catalyzed Heck reaction and characterized by 1H NMR, 13C NMR, and HRMS-MALDI-TOF. Three-photon absorption and three-photon excited fluorescence properties of chromophores (Py2-TPA)n were studied using a femtosecond Ti:Sapphire laser system. These chromophores exhibit efficient three-photon excited green fluorescence peaking at 540–590 nm. The three-photon absorption cross-section of σ3 = 6.99 × 10−79 cm6 s2 photon−2 in the femtosecond regime has been obtained from (Py2-TPA)4. The ratio of 3PA cross-sections in CH2Cl2 solution σ3 (Py2-TPA)4:σ3 (Py2-TPA)2:σ3 (Py2-TPA)1 is 6.0:4.2:1. The ratio of conjugated π-electron numbers in (Py2-TPA)n is 4.4(n = 4):2.6(n = 2):1(n = 1). The increasing the push–pull units of (Py2-TPA)n and π-electron conjugation length or increasing the extent of charge transfer from the ends to the middle aryl core results in an increase of the 3PA cross-section. The results indicate that large aromatic conjugated chromophores (Py2-TPA)n containing pyridine–triphenylamine D–A pairs and a conjugated aryl core provide a new opportunity for 3PA-materials and three-photon fluorophores for practical application.

Three-photon excited fluorescence imaging of unstained tissue using a GRIN lens endoscope

We present a compact and portable three-photon gradient index (GRIN) lens endoscope system suitable for imaging of unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The lateral and axial resolution in water is 1.0 μm and 9.5 μm, respectively. The ~200 μm diameter field of view is imaged at 2 frames/s using a fiber-based excitation source at 1040 nm. Ex vivo imaging is demonstrated with unstained mouse lung at 5.9 mW average power. These results demonstrate the feasibility of three-photon GRIN lens endoscopy for optical biopsy.

Synthesis and efficient solid-state emission of conjugated donor–acceptor–donor triphenylamine chromophores

Two symmetrical donor–acceptor–donor chromophores built by connecting two triphenylamine donors to a central electron acceptor 2,5-diphenyl-1,3,4-oxadiazole or anthracene core have been synthesized and characterized. The aggregation-induced emission phenomenon of these compounds with bright and deep emission from orange to green in the solid state can be obsreved. The chromophores based on the triphenylamine unit exhibit efficient solid-state emission. The results indicate that D–A–D conjugation bridging provides a new opportunity for AIE-materials. The multiphoton induced fluorescence spectra of these chromophores were measured using a femtosecond Ti:sapphire. The three-photon excited fluorescence spectra are in the green region with peaks at 554 nm for An-BIPAS and 504 nm for Ox-BIASP in THF, respectively. These AIE-active compounds are potential materials to design sensitive and selective fluorescent sensors or bioprobes.

Non-linear fluorescence lifetime imaging of biological tissues

In recent years fluorescence microscopy has become a widely used tool for tissue imaging and spectroscopy. Optical techniques, based on both linear and non-linear excitation, have been broadly applied to imaging and characterization of biological tissues. Among fluorescence techniques used in tissue imaging applications, in recent years two and three-photon excited fluorescence have gained increased importance because of their high-resolution deep tissue imaging capability inside optically turbid samples. The main limitation of steady-state fluorescence imaging techniques consists in providing only morphological information; functional information is not detectable without technical improvements. A spectroscopic approach, based on lifetime measurement of tissue fluorescence, can provide functional information about tissue conditions, including its environment, red-ox state, and pH, and hence physiological characterization of the tissue under investigation. Measurement of the fluorescence lifetime is a very important issue for characterizing a biological tissue. Deviation of this property from a control value can be taken as an indicator of disorder and/or malignancy in diseased tissues. Even if much work on this topic has still to be done, including the interpretation of fluorescence lifetime data, we believe that this methodology will gain increasing importance in the field of biophotonics. In this paper, we review methodologies, potentials and results obtained by using fluorescence lifetime imaging microscopy for the investigation of biological tissues.

Enhanced Three-Photon Absorption by Symmetric TwistedIntramolecular Charge Transfer

We report on a novel organic chromophore with symmetric twistedintramolecular charge transfer (TICT) state on excitation. Theproperties of nonlinear transmission induced by three-photon absorption(3 PA) are demonstrated pumped with nanosecond laser pulse. Large 3 PAcross sections as high as the order of 10−74 cm6s2 havebeen obtained for nanosecond and picosecond laser pulses at 1064 nmfrom intensity-dependent transmission measurements. Similar two emissivebehaviours from one-photon and three-photon excited fluorescence spectraindicate that the linear and nonlinear fluorescences share the same TICTrelaxation process from the excited states. The intensity dependence ofupconversion fluorescence on the incident intensity obeys the cubic lawthat characterizes the three-photon absorption.

Two- and three-photon excited fluorescence in Y-shaped molecules

In this work, we have studied the two- and three-photon excited fluorescence on a new series of Y-shaped chromophores, using pulses at 750 and 1400 nm from an optical parametric amplifier pumped by 150 fs pulses from a Ti:sapphire chirped pulse amplified system. The measured two- and three-photon absorption cross-sections are in the order of 1000 × 10 −50 cm 4 s and 5 × 10 −78 cm 6 s 2, respectively. Besides, the two-photon excited fluorescence signal, achieved using low energy pulses (Ti:sapphire modelocked oscillator), were used in an evolutionary strategy to optimize either the laser pulse or the two-photon excited fluorescence.

Second-harmonic imaging of plant polysaccharides

The application of second-harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterization of biologically and commercially important plant polysaccharides. We find that imaging of cellulose requires rather high laser powers, which are above optimal values for live cell imaging. Starch, however, is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong three-photon excited fluorescence, which may be enhanced by resonance effects.

Electric-field-induced dynamics in radial liquid crystal droplets studied by multiphoton-excited fluorescence microscopy

Time-resolved multiphoton-excited fluorescence microscopy is used to study electric-field-induced reorientation dynamics in single liquid crystal (LC) droplets within polymer-dispersed liquid crystal films. Films comprised of nematic LC dispersed in a poly(isobutyl methacrylate) matrix are characterized. An electric field is applied laterally across each droplet, using two parallel copper wires embedded in the film. Three-photon excited fluorescence images are recorded with 200 μs time resolution as the field is modulated on and off. Dramatic spatial variations in the time scales for orientational relaxation within individual droplets are observed. These effects are attributed to polymer/LC interfacial interactions and relaxation of the LC through a transient, metastable organizational state.