Multiphoton Excitation Research Articles

Effect of cigarette smoke exposure and cessation on regional diffusion properties in rat intervertebral discs.

Cigarette smoking is a recognized risk factor for orthopedic disorders, particularly intervertebral disc (IVD) degenerative disease. However, the IVD pathophysiology, especially the spatial-temporal remodeling progression in the context of cigarette smoking, remains unclear. This study aimed to address this knowledge gap through a quantitative assessment of IVD structural composition and diffusion properties using a Sprague-Dawley rat model. Twenty-four rats were divided into control and smoke exposure cohorts, each with two sub-groups of six rats. One smoke exposure sub-group was sacrificed after 2 months of daily cigarette smoke exposure in a custom smoking apparatus, while the other was sacrificed after an additional 5 months of smoke cessation. The control groups were age-matched to the smoke exposure groups. A fluorescent recovery after photobleaching (FRAP) technique was used to determine solute diffusivities and multi-photon excitation (MPE) imaging was performed to characterize structural changes in the annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP). A decrease in diffusivity was observed in the CEP and the AF (radial direction only) after 2 months of smoke exposure. MPE imaging showed aberrant CEP calcification and reduced AF radial collagen fiber bundle diameter, suggesting that the IVD exhibits regionally dependent structural remodeling due to smoke exposure. Furthermore, the smoke cessation group showed deteriorating alterations of structure and diffusivities in all three-disc regions, including the NP, indicating that five-month smoke cessation alone didn't reverse the progression of IVD degenerative remodeling during aging. This study advances the understanding of IVD pathophysiology in the context of cigarette smoke exposure and cessation, laying the groundwork for potential earlier diagnosis and optimized interventions.

Effects of multiple reflections on nonlinear absorption measurements.

Accurate measurement of nonlinear absorption coefficients by transmission-based techniques, such as Z-scan and its modifications, remains challenging due to high measurement errors. In this Letter, we claim that one of the reasons for that could lie in ignoring multiple reflections of light in plane-parallel samples in the presence of multi-photon excitation. Closed-form formulations for transmittance and reflectance coefficients, considering both linear and multiphoton absorption, are obtained in the geometrical optics approximation. The contributions of multiple reflections to the total transmittance are calculated for several II-VI materials reported in the literature. The results show a nonlinear dependence on incident intensity, ranging from 15.7% down to 7.9% for CdS, from 10.3% down to 5.8% for CdSe, and from 18.1% down to 9.1% for ZnSe. The dependence of the contribution of multiple reflections on the sample thickness is shown to exhibit a near exponential decay.

Energy transfer-mediated multiphoton synergistic excitation for selective C(sp3)–H functionalization with coordination polymer

Activation and selective oxidation of inert C(sp3)–H bonds remain one of the most challenging tasks in current synthetic chemistry due to the inherent inertness of C(sp3)–H bonds. In this study, inspired by natural monooxygenases, we developed a coordination polymer with naphthalenediimide (NDI)-based ligands and binuclear iron nodes. The mixed-valence FeIIIFeII species and chlorine radicals (Cl•) are generated via ligand-to-metal charge transfer (LMCT) between FeIII and chlorine ions. These Cl• radicals abstract a hydrogen atom from the inert C(sp3)–H bond of alkanes via hydrogen atom transfer (HAT). In addition, NDI converts oxygen to 1O2 via energy transfer (EnT), which then coordinates to FeII, forming an FeIV = O intermediate for the selective oxidation of C(sp3)–H bonds. This synthetic platform, which combines photoinduced EnT, LMCT and HAT, provides a EnT-mediated parallel multiphoton excitation strategy with kinetic synergy effect for selective C(sp3)–H oxidation under mild conditions and a blueprint for designing coordination polymer-based photocatalysts for C(sp3)–H bond oxidation.

Effects of oil-water interfacial properties on protein structuring and droplet deformation in high moisture meat analogues containing oil

Adding oil to high moisture meat analogues (HMMA) can increase juiciness, and can be achieved by incorporating emulsion droplets during extrusion. Since these droplets can coalesce when subjected to high shear, selecting appropriate emulsion stabilisers is important. For several commercial plant-protein emulsion stabilisers, it was investigated how oil-water interfacial mechanical properties affect droplet deformation and protein structuring in extrusion of HMMAs. Emulsions with 10 wt% or 15 wt% oil, stabilised by potato protein isolates (POPI-1 (Rich in patatin) and POPI-2 (rich in protease inhibitor)) and pea protein isolate PPI, were used to make extrudates with 5.7 wt% and 8.5 wt% oil, respectively. In 8.5 wt%-extrudates, POPI-2 had the most oil leakage from the cooling die, while PPI had the smallest amount despite having softer and more stretchable interfaces. Blade-cutting tests showed the highest maximum force for 8.5 wt%-extrudates with POPI-1, likely because POPI-1 formed the stiffest interfaces. Tensile stress testing showed the largest fracture strain in 8.5% wt%-extrudates with PPI, corresponding to its longer wedge length. Multiphoton excitation microscopy was used to visualise the extrudates protein structure and oil droplets. This showed that droplets near the surface of the extrudate were less deformed than droplets in the centre. There were only small differences between protein stabilisers regarding oil droplet deformation, indicating droplet deformation was dominated by deformation of the protein matrix. The O/W interfacial properties significantly affected oil leakage, cutting force, and tensile strength of extrudates. These results are important to consider when designing emulsions for HMMAs processed by extruders.

Multiphoton scaling of femtosecond laser-induced refractive index change (LIRIC) in hydrogels and rabbit cornea

To find optimal conditions for performing laser induced refractive index change (LIRIC) in living eyes with both safety and efficacy, we investigated multiphoton excitation scaling of this procedure in hydrogel and excised corneal tissue. Three distinct wavelength modalities were examined: high-repetition-rate (HRR) and low-repetition-rate (LRR) 405 nm systems, as well as 800 nm and 1035 nm systems, whose LIRIC-inducing properties are described for the first time. Of all the systems, LRR 405 nm-LIRIC was able to produce the highest phase shifts at the lowest average laser powers. Relative merits and drawbacks to each modality are discussed as they relate to future efforts towards LIRIC-based refractive error correction in humans.

A Two-Photon-Active Zr-Based Metal-Organic Framework-Based Orthogonal Nanoprobe for Recognition of Cellular Senescence.

A luminescent nanoprobe capable of orthogonal sensing of two independent events is highly significant for unbiased disease-related detection such as the detection of senescent cells. Moreover, it is invaluable that the nanoprobe possesses a two-photon excitable characteristic that is highly suitable for imaging living cells and tissues. Herein, we present a two-photon-excitable multiluminescent orthogonal-sensing nanoprobe (OS nanoprobe) capable of detecting both pH elevation and β-galactosidase (β-gal) overexpression in senescent cells. In the design, Zr-based dual-emissive metal-organic frameworks prepared from two mixed amino linkers, referred to as NH2-MU, were used as the component for the ratiometric sensing of pH; additionally, fluorogenic resorufin-β-d-galactopyranoside, linked to the NH2-MU framework, enables β-gal detection. In the OS nanoprobe, the signals for pH and β-gal sensing remain independent while maintaining high colocalization. The two-photon excitable organic linkers of NH2-MU impart the OS nanoprobe with a bioimaging capability, allowing for the differentiation of senescent human foreskin fibroblast (HFF) cells from younger HFF cells or LacZ positive cells with the 800 nm laser excitation. This study marks the first instance of achieving the multiplexed orthogonal fluorescent sensing of cellular senescence using a two-photon excitation strategy, suggesting the potential of using versatile metal-organic framework (MOFs)-based fluorophores to realize the orthogonal multiplexing of disease-related biomarkers through multiphoton excitation.

Nonlinear Absorption in 2D Ruddlesden-Popper Perovskites: Pathways to Ultrafast Optical Applications.

Owing to their distinctive photophysical properties resulting from their larger exciton binding energy and the influence of dielectric and quantum confinement effects, considerable research interest has been directed toward two-dimensional Ruddlesden-Popper halide perovskites (2D RP-HPs). Particularly, 2D RP-HPs exhibit exceptional multiphoton absorption (MPA) effects that reveal versatile applications. In this work, two-photon absorption (2PA) and three-photon absorption (3PA) in 2D RP-HPs, specifically (PEA)2PbBr4 and (BA)2PbBr4 platelets, have been demonstrated through their photoluminescence spectra under multiphoton excitation, revealing a power law relationship with excitation energy. On the other hand, polarization-dependent MPA measurements are also conducted to obtain their anisotropy properties. The excellent 2PA and 3PA effects of 2D RP-HPs have found applications in ultrafast optics to construct fringe-resolved autocorrelation traces, enabling the retrieval of pulse duration not only passively mode-locked Ti:sapphire at 800 nm but also Yb-doped fiber lasers at 1030 nm.

Multiphoton Resonance Meets Tunneling Ionization: High-Efficient Photoexcitation in Strong-Field-Dressed Ions.

Tunneling ionization, a fascinating quantum phenomenon, has played the key role in the development of attosecond physics. Upon absorption of a few tens of photons, tunneling ionization creates ions in different excited states and even enables the formation of population inversion between ionic states. However, the underlying physics is still being debated. Here, we demonstrate a significant enhancement in the efficiency of multiphoton excitation when ionization of neutral molecules and resonant excitation of ions coexist in strong laser fields. It facilitates the dramatic increase in population inversion and lasing radiation in N_{2}^{+} around 1000nm pump wavelength. Utilizing the ionization-coupling theory, we discover that the synergistic interplay between tunneling ionization and multiphoton excitation enables the ionic coherence to be maximized by phase locking of the periodically created ionic dipoles and consistently maintain an optimal phase for the follow-up photoexcitation. This Letter provides new insights into the photoexcitation mechanism of ions in strong laser fields and opens up a route for optimizing ionic lasing radiations.

Hybrid Photoexcitation in Undoped Diamond by Mid-Infrared Femtosecond Laser Pulses

Direct interband and intragap photoexcitation by intense mid-infrared (4.0 and 4.7 μm) femtosecond (fs) laser pulses was explored in ultrapure chemical-vapor deposited (CVD) diamond via acquisition of characteristic ultraviolet photoluminescence of free excitons and A-band photoluminescence of electrons anchored at deep donor–acceptor or dislocation-related traps, respectively. At lower laser intensities (<10 TW/cm2) the excitonic photoluminescence yields exhibit highly nonlinear dependences with Iλ2-scaling and power slopes N ≈ 17 (4.0 μm) and 14 (4.7 μm), still insufficient to cross over the direct bandgap (≥6.5 eV) by ≈1.2 and 2.8 eV, respectively. Similarly high slope of ≈9 (4.7 μm) for intragap (≥3.5 eV) photo-population of donor–acceptor traps is still insufficient for their direct excitation by ≈1 eV. At the intermediate Iλ2-dependent values of the Keldysh parameter γ ∼ 1 such incomplete multiphoton excitation anticipates the hybrid total “multiphoton + tunneling” photoexcitation generally predicted by the Keldysh theory, but never unambiguously experimentally demonstrated. At higher laser intensities (>10 TW/cm2) both the excitonic and A-band photoluminescence yields exhibit (sub)linear slopes, apparently, indicating formation of more strongly absorbing electron–hole plasma. These findings shed light on the hybrid multiphoton + tunneling character of Keldysh photoexcitation at intermediate values γ and pave the way to defect/impurity band engineering of intragap nonlinear optical properties in bulk dielectrics for their precise fs-laser nanomodification.

Efficient multiphoton microscopy with picosecond laser pulses.

Multiphoton microscopes employ femtosecond lasers as light sources because the high peak power of the ultrashort pulse allows for multiphoton excitation of fluorescence in the examined sample. However, such short pulses are susceptible to broadening in a microscope's highly dispersive optical elements and require careful dispersion management, otherwise decreasing excitation efficiency. Here, we have developed a 10 nJ Yb:fiber picosecond laser with an integrated pulse picker unit and evaluated its performance in multiphoton microscopy. Our results show that performance comparable to femtosecond pulses can be obtained with picosecond pulses only by reducing the pulse repetition rate and that such pulses are significantly less prone to the effect of chromatic dispersion. These findings proved that the temporal pulse compression is not always efficient, and it can be omitted by using a smaller and easier-to-use all-fiber setup.

Resolving plasmon-mediated high-order multiphoton excitation pathways in dolmen nanostructures using ultrafast nonlinear optical interferometry.

The multiphoton excitation pathways of plasmonic nanorod assemblies are described. By using dolmen structures formed from the directed assembly of three gold nanorods, plasmon-mediated three-photon excitation is resolved. These high-order multiphoton excitation channels were accessed by resonantly exciting a hybrid mode of the dolmen structure that was resonant with the 800-nm carrier wavelength of an ultrafast laser system. Rotation of the exciting field polarization to a non-resonant configuration did not generate third-order responses. Hence, the multiphoton excitation and resultant non-equilibrium electron distributions were generated by structure- and mode-selective excitation. Correlation between high-order and resonant plasmon excitation was achieved through sub-cycle time-resolved interferometric detection of incoherent nonlinear emission signals. The results illustrate the advantages of nonlinear optical interferometry and Fourier analysis for distinguishing plasmon-mediated processes from those that do not require plasmon excitation.

Visible and near-infrared light-induced photoclick reactions.

Photoclick reactions combine the advantages offered by light-driven processes, that is, non-invasive and high spatiotemporal control, with classical click chemistry and have found applications ranging from surface functionalization, polymer conjugation, photocrosslinking, protein labelling and bioimaging. Despite these advances, most photoclick reactions typically require near-ultraviolet (UV) and mid-UV light to proceed. UV light can trigger undesirable responses, including cellular apoptosis, and therefore, visible and near-infrared light-induced photoclick reaction systems are highly desirable. Shifting to a longer wavelength can also reduce degradation of the photoclick reagents and products. Several strategies have been used to induce a bathochromic shift in the wavelength of irradiation-initiating photoclick reactions. For instance, the extension of the conjugated π-system, triplet-triplet energy transfer, multi-photon excitation, upconversion technology, photocatalytic and photoinitiation approaches, and designs involving photocages have all been used to achieve this goal. Current design strategies, recent advances and the outlook for long wavelength-driven photoclick reactions are presented.

Modifying Parallel Excitations into One Framework for C(sp3)─H Bond Activation with Energy Combined More Than Two Photons.

Natural photosynthesis enzymesutilize energies of several photons for challenging oxidation of water, whereas artificial photo-catalysis typically involves only single-photon excitation. Herein, a multiphoton excitation strategy is reported that combines parallel photo-excitations with a photoinduced electron transfer process for the activation of C(sp3)─H bonds, including methane. The metal-organic framework Fe3-MOF is designed to consolidate 4,4',4″-nitrilotrisbenzoic units for the photoactivation of dioxygen and trinuclear iron clusters as the HAT precursor for photoactivating alkanes. Under visible light irradiation, the dyes and iron clusters absorbed parallel photons simultaneously to reach their excited states, respectively, generating 1O2 via energy transfer and chlorine radical via ligand-to-metal charge transfer. The further excitation of organic dyes leads to the reduction of 1O2 into O2 •- through a photoinduced electron transfer, guaranteeing an extra multiphoton oxygen activation manner. The chlorine radical abstracts a hydrogen atom from alkanes, generating the carbon radical for further oxidation transformation. Accordingly, the total oxidation conversion of alkane utilizing three photoexcitation processes combines the energies of more than two photons. This new platform synergistically combines a consecutive excited photoredox organic dye and a HAT catalyst to combine the energies of more than two photons, providing a promising multiphoton catalysis strategy under energy saving, and high efficiency.

Photoluminescence of CaGa2S4: Nd, Yb compound at room temperature

In this paper, CaGa2S4:3%Nd,5%Yb; CaGa2S4:5%Yb; CaGa2S4:3%Nd compounds were synthesized, and their photoluminescence (PL) spectra, PL excitation (PLE) spectra and PL decay properties were studied at room temperature under one-photon and multi-photon excitation in a wide range of excitation intensities. The synthesis of the CaGa2S4 compound was carried out by a solid-phase reaction of a stoichiometric mixture of CaS and Ga2S3 powders in a quartz ampoule. Yb and Nd doping was carried out during the synthesis process using YbF3 and NdF3. PLE spectra were studied in the wavelength range from 250 to 850 nm, and PL spectra in the range of 400–1500 nm. It has been shown that after introducing Yb3+ ions into thiogallate crystals with Nd3+ ions, a new band near 488 nm is formed and the PL intensity increases by 1–2 orders of magnitude. The anti-Stokes PL spectra of CaGa2S4: Nd, Yb crystals in the visible region of the spectrum (400–700 nm) were studied when excited by continuous wave laser radiation with a wavelength of 975 nm. The kinetics of PL decay of all types of compounds at different wavelengths of single- and multi-photon excitation were determined. Depending on the PLE conditions, wavelength and intensity of the exciting radiation, monoexponential and non-monoexponential decay kinetics were observed with decay times in the range of 20–90 μs. Possible mechanisms of PLE and PL of CaGa2S4 crystals doped with neodymium and ytterbium ions are discussed.

Non-invasive stratigraphic analyzes of gelatine-based modern painting materials with linear and nonlinear optical methods

Stratigraphic analyzes of polychrome surfaces, such as paintings, often need samples to offer consistent results regarding the sequence and composition of the layers. Non-invasive methodologies based on linear and nonlinear optical techniques limit material removal from the objects. Recently, optical coherence tomography (OCT) has become the preferred choice of heritage scientists because it is a safe and fast alternative for studying transparent or semi-transparent layers. Yet, nonlinear optical microscopy (NLOM)) technique in its modality of multiphoton excitation fluorescence (MPEF) has emerged as a promising tool for the same purpose. Here, we explored linear (OCT and confocal Raman microspectroscopy (CRM)) and nonlinear (NLOM-MPEF) optical methods’ capability to investigate gelatine-based layers in mock-up samples and a painting dated 1939 by an artist from the Surrealistic entourage. The optical behavior of mock-up samples that imitate the painting stratigraphy and of six painting fragments detached from the support was also investigated with fiber optics reflectance spectroscopy and laser induced fluorescence (LIF). Thickness values from the mock-ups obtained with OCT, CRM, and MPEF have provided evidence of the complementarity, from a millimetric to a micrometric scale, and the limitations (e.g. strong fluorescence emission in CRM) of the methods. Moreover, the presence of gelatine was ascertained by LIF spectroscopy applied to the painting fragments and NLOM-MPEF confirmed its suitability as a non-invasive technique for investigating gelatine-based stratigraphic systems.

Multiphoton simultaneous multislice imaging.

To develop multiphoton excitation techniques for simultaneous multislice (SMS) imaging and evaluate their performance and specific absorption rate (SAR) benefit. To improve multiphoton SMS reconstruction quality with a novel CAIPIRINHA (controlled aliasing in parallel imaging results in higher acceleration) design. When a conventional single-slice RF field is applied together with an oscillating gradient field, the two can combine to generate multiphoton excitation at multiple discrete spatial locations. Because the conventional RF is reused at multiple spatial locations, multiphoton excitation offers reduced SAR for SMS applications. CAIPIRINHA shifts are often used to improve parallel-imaging acceleration. Interestingly, CAIPIRINHA-type shifts can be obtained for multiphoton SMS by updating the oscillating gradient phase at every phase encode. In this work, both a gradient-echo and a spin-echo sequence with multiphoton CAIPIRINHA-SMS excitation pulses are implemented for in vivo human imaging at 3 T. For three slices, multiphoton SMS provides a 51% reduction in SAR compared with conventional superposition SMS, whereas for five slices, SAR is reduced by 66%. Multiphoton SMS outperforms PINS (power independent of number of slices) and MultiPINS in terms of SAR reduction especially when the pulse duration is short, slices are thin, and/or the slice spacing is large. A custom CAIPIRINHA phase-encoding design for multiphoton SMS significantly improves reconstruction quality. Multiphoton SMS excitation can be obtained by combining conventional single-slice RF pulses with an oscillating gradient and offers significant SAR benefits compared with conventional superposition SMS. A novel CAIPIRINHA design allows higher multiband factors for multiphoton SMS imaging.

Application of near-infrared-to-blue upconversion luminescence for the polymerization of resin cements through zirconia discs

ObjectivesTo investigate a near-infrared-to-blue luminescence upconversion curing method for polymerizing resin cements under zirconia discs. MethodsLava zirconia discs of different thicknesses (0.5–2.0 mm) were manufactured. First, the transmittances of the NIR and two blue lights (BLs) (LED and halogen lights) through these discs were measured. Second, NaYF4:Yb3+/Tm3+ upconversion phosphor (UP) powder was milled into 0.5-μm particle sizes. A light-curable resin cement VariolinkII base was chosen as the control (UP0), and an experimental cement (UP5) was prepared by adding 5 % UPs. These two cements were examined using multiphoton excitation microscopy for particle distribution. UP5 and UP0 were polymerized with or without zirconia shielding then subjected to a microhardness test. A multifold analysis was performed to examine the effects of zirconia thickness, curing protocols (pure BL or combined BL and NIR curing), and cement type. ResultsThe transmittance of NIR was superior to that of BL through zirconia discs of all thicknesses. UP particles were homogeneously distributed in UP5 and emitted blue luminescence under 980-nm NIR excitation. UP5 showed higher microhardness values than UP0 under any curing protocol or zirconia shielding condition. The combination of 20-s BL and 40-s NIR curing yielded the highest microhardness in uncovered UP5. However, combining 40-s BL and 20-s NIR curing surpassed the other groups when the zirconia discs were thicker than 0.5 mm. SignificanceNIR exhibits higher transmission through zirconia than BL. UP particles work as strengthen fillers and photosensitizers in cements. NIR upconversion curing could be a new strategy for polymerizing resin cements under thick zirconia restorations.

Picosecond reactions of excited radical ion super-reductants

Classical photochemistry requires nanosecond excited-state lifetimes for diffusion-controlled reactions. Excited radicals with picosecond lifetimes have been implied by numerous photoredox studies, and controversy has arisen as to whether they can actually be catalytically active. We provide direct evidence for the elusive pre-association between radical ions and substrate molecules, enabling photoinduced electron transfer beyond the diffusion limit. A strategy based on two distinct light absorbers, mimicking the natural photosystems I and II, is used to generate excited radicals, unleashing extreme reduction power and activating C(sp2)―Cl and C(sp2)―F bonds. Our findings provide a long-sought mechanistic understanding for many previous synthetically-oriented works and permit more rational future photoredox reaction development. The newly developed excitation strategy pushes the current limits of reactions based on multi-photon excitation and very short-lived but highly redox active species.

Temporal‐Focusing Multiphoton Excitation Single‐Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores

AbstractSingle‐molecule localization microscopy (SMLM) based on temporal‐focusing multiphoton excitation (TFMPE) and single‐wavelength excitation is used to visualize the three‐dimensional (3D) distribution of spontaneously blinking fluorophore‐labeled subcellular structures in a thick specimen with a nanoscale‐level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out‐of‐focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single‐wavelength TFMPE achieves wide‐field and axially confined two‐photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two‐dimensional TFMPE‐SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE‐SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid‐beta peptide deposits.

Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.