Multiphoton Excitation Fluorescence Research Articles

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.

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.

Intravital observation of high-scattering and dense-labeling hepatic tissues using multi-photon fluorescence microscopy.

Achieving high-resolution and large-depth microscopic imaging in vivo under conditions characterized by high-scattering and dense-labeling, as commonly encountered in the liver, poses a formidable challenge. Here, through the optimization of multi-photon fluorescence excitation window, tailored to the unique optical properties of the liver, intravital microscopic imaging of hepatocytes and hepatic blood vessels with high spatial resolution was attained. It's worth noting that resolution degradation caused by tissue scattering of excitation light was mitigated by accounting for moderate tissue self-absorption. Leveraging high-quality multi-photon fluorescence microscopy, we discerned structural and functional alterations in hepatocytes during drug-induced acute liver failure. Furthermore, a reduction in indocyanine green metabolism rates associated with acute liver failure was observed using NIR-II fluorescence macroscopic imaging.

Available medical imaging modalities for melanoma screening

The prevalence of melanoma of the skin has seen a significant rise in recent decades, constituting approximately one-third of all diagnosed cancer cases. Melanoma, the most fatal variant among cutaneous malignancies, exhibits a 4% probability of occurrence over an individual’s lifetime. The increasing incidence and mortality rates of skin cancer impose a substantial burden on healthcare resources and the economy. In recent years, several optical modalities, including dermoscopy, reflectance confocal microscopy (RCM), optical coherence tomography, multiphoton excited fluorescence imaging, and dermatofluorescence, have been extensively studied and utilized to improve the non-invasive diagnosis of skin cancer. This review article provides an analysis of the approach employed in the recently developed optical non-invasive diagnostic technologies. It explores the clinical uses of these techniques, while also examining their respective advantages and disadvantages. Furthermore, the paper explores the possibility for additional advancements in these technologies in the future.

Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging

The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.

Abstract C020: Unconvering the hidden immunosuppressive landscape in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest forms of cancer with an extremely low survival rate. Despite the success of therapeutic T cells in hematologic cancers, many solid tumors, including PDA, remain unresponsive with such therapies, partly due to its mechanically complex, dense, and immunosuppressive stroma. Therefore, understanding the dynamic interplay between therapeutic T cells and the tumor microenvironment (TME) is crucial for revealing engineering targets for next-generation immunotherapies. We approach this problem with genetically engineered KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-1-Cre) mouse models that recapitulates PDA disease progression and stromal dynamics. Within live tumor slices from primary tumors, we are defining how stromal components such as dense collagen fiber networks and tumor-associated macrophages (TAMs) orchestrate an immunosuppressive landscape. We employ multiphoton laser scanning microscopy (MPLSM) to generate multiphoton excitation of fluorescence (cell dyes and immunofluorescence) and second harmonic generation (SHG) for collagen fibers to acquire multidimensional data (x, y, z, c, t) of T cell migration and interaction within the TME. We subsequently reveal T cell migration and interaction as a function of its physical and biological ECM components using advanced computer vision and machine learning algorithms based on the Clustered Random Convolutional Kernel Transform (cROCKET), revealing a hidden landscape in the feature space. Perturbations to the TME in KPC mice with CCR2 inhibitor CCX598 (in vivo) and clodronate liposomes (ex vivo) deplete CD11b+ myeloid cells, resulting in altered behaviors of the infiltrating T cells. Anti-PD-1 monoclonal antibody neutralizes the PD-1/PD-L1 axis in T cells, resulting in enhanced migration and infiltration in tumor slices. Citation Format: Guhan Qian, Hongrong Zhang, Aine Knott, Jon Zettervall, Ingunn Stromnes, Paolo Provenzano. Unconvering the hidden immunosuppressive landscape in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C020.

S2ISM: A Comprehensive Approach for Uncompromised Super-Resolution and Optical Sectioning in Image Scanning Microscopy

Image Scanning Microscopy (ISM) enables good signal-to-noise ratio (SNR), super-resolution and high information content imaging by leveraging array detection in a laser-scanning architecture. However, the SNR is still limited by the size of the detector, which is conventionally small to avoid collecting out-of-focus light. Nonetheless, the ISM dataset inherently contains the axial information of the fluorescence emitters. We leverage this knowledge to achieve computational optical sectioning without sacrificing the conventional benefits of ISM. We invert the physical model to fuse the raw dataset into a single image with improved sampling, SNR. lateral resolution, and optical sectioning. We provide a complete theoretical framework and validate our approach with experimental images of biological samples acquired with a custom setup equipped with a single photon avalanche diode (SPAD) array detector. Furthermore, we generalize our method to other imaging techniques, such as multi-photon excitation fluorescence microscopy and fluoresce lifetime imaging. To enable this latter, we take advantage of the single-photon timing ability of SPAD arrays, accessing additional sample information. Our method outperforms conventional reconstruction techniques and opens new perspectives for exploring the unique spatio-temporal information provided by SPAD array detectors.

Fast Diffusion Characterization by Multiphoton Excited Fluorescence Recovery while Photobleaching.

Multiphoton-excited fluorescence recovery while photobleaching (FRWP) is demonstrated as a method for quantitative measurements of rapid molecular diffusion over microsecond to millisecond timescales. Diffusion measurements are crucial in assessing molecular mobility in cell biology, materials science, and pharmacology. Optical and fluorescence microscopy techniques enable non-invasive rapid analysis of molecular diffusion but can be challenging for systems with diffusion coefficients exceeding ∼100 μm2/s. As an example, fluorescence recovery after photobleaching (FRAP) operates on the implicit assumption of a comparatively fast photobleaching step prior to a relatively slow recovery and is not generally applicable for systems exhibiting substantial recovery during photobleaching. These challenges are exacerbated in multiphoton excitation by the lower excitation efficiency and competing effects from local heating. Herein, beam-scanning FRWP with patterned line-bleach illumination is introduced as a technique that addresses FRAP limitations and further extends its application range by measuring faster diffusion events. In FRWP, the recovery of fluorescence is continuously probed after each pass of a fast-scanning mirror, and the upper bound of measurable diffusion rates is, therefore, only limited by the mirror scanning frequency. A theoretical model describing transient fluctuations in fluorescence intensity arising as a result of combined contributions from photobleaching and localized photothermal effect is introduced along with a mathematical framework for quantifying fluorescence intensity temporal curves and recovering room-temperature diffusion coefficients. FRWP is then tested by characterization of normal diffusion of rhodamine-labeled bovine serum albumin, green fluorescence protein, and immunoglobulin G molecules in aqueous solutions of varying viscosity.

Angular Patterns of Nonlinear Emission in Dye Water Droplets Stimulated by a Femtosecond Laser Pulse for LiDAR Applications

Femtosecond laser-induced fluorescence (FLIF) and femtosecond laser-induced optical breakdown spectroscopy (FIBS) are important tools for remote diagnostics of atmospheric aerosols using LiDAR (Light Identification Detection and Ranging) technology. They are based on light emission excitation in disperse media via multiphoton nonlinear processes in aerosol particles induced by high-power optical pulses. To date, the main challenge restraining the large-scale application of FLIF and FIBS in atmospheric studies is the lack of a valued theory of the stimulated light emission in liquid microparticles with a sufficiently broad range of sizes. In this paper, we fill this gap and present a theoretical model of dye water droplet emission under high intensity laser exposure that adequately simulates the processes of multiphoton excited fluorescence and optical breakdown plasma emission in microparticles and gives quantitative estimates of the angular and power characteristics of nonlinear emission. The model is based on the numerical solution to the inhomogeneous Helmholtz equations for stimulating (primary) and nonlinear (secondary) waves provided by the random nature of molecule emission in particles. We show that droplet fluorescence stimulated by multiphoton absorption generally becomes more intense with increasing particle size. Moreover, far-field plasma emission from liquid particles demonstrates a larger angular diversity when changing the droplet radius in comparison with multiphoton excited fluorescence, which is mainly due to the excitation of the internal optical field resonances in spherical particles.

In-depth structural and compositional assessment of aged terpenoid varnish layers by nonlinear optical microscopy

Varnishes are employed in painted artworks for protection from atmospheric pollution and oxidation and for improving the esthetic appearance by providing an even and brilliant surface finish. Also, in some cases, colored varnishes are applied intentionally to modify the chromatic appearance of paintings. Varnishes undergo complex and differentiated structural and chemical changes over time depending on their composition and conservation conditions. The present work investigates the extent of degradation of the outermost layers of varnish coatings due to aging as a function of depth by using nonlinear optical microscopy (NLOM) in the modality of multiphoton excitation fluorescence (MPEF). This non-invasive technique has been employed for the determination of the thickness, with a microscopic axial and lateral resolution, of the affected regions of pictorial terpenoid varnish layers, such as dammar, mastic, shellac and sandarac, subjected to various types and degrees of aging, artificial, natural and a combination of both. A homemade nonlinear optical microscope, based on a tightly focused pulsed femtosecond laser emitting at 800 nm, was used for the investigation. Single-photon laser-induced fluorescence (LIF) measurements served to determine the degree of surface aging and the optimum NLOM-MPEF operating conditions and helped to interpret the results obtained by applying the latter. These results signpost the correlations between the extent of degradation, non-invasively determined by NLOM-MPEF, the type and length of aging, the nature of the varnish and the initial thickness of the varnish layer.

Laser-based techniques for the non-invasive characterisation of grisaille paints on stained-glass windows

Grisaille was the first paint applied on stained-glass panels, used in Europe since the twelfth century. Historical written sources described the use of iron and copper together with a high lead-silica base glass in the grisailles production. This project aims to study the evolution of the grisaille paint composition throughout time and the changes in the raw materials used in their production using non-destructive and non-invasive techniques. To achieve this objective, 23 grisaille samples dated from the 13th to the twentieth centuries from nine different countries (Portugal, Poland, United Kingdom, Sweden, Norway, Belgium, Low Countries, Germany, and France) were studied by means of micro particle-induced X-ray emission (μ-PIXE), micro energy dispersive X-ray fluorescence (μ-EDXRF), laser-induced breakdown spectroscopy (LIBS), laser-induced fluorescence (LIF), non-linear optical microscopy (NLOM) in the modality of multiphoton excitation fluorescence (MPEF) and optical microscopy (OM). The results showed that it was possible to identify compositional differences and patterns throughout the samples when compared with literature results. The preference for using copper in central and south-central European countries and the addition of new compounds (CoO, Cr2O3, MnO) as colouring agents since the nineteenth century was verified. The LIBS analyses allow the identification of boron on two samples, confirming the change of base glass components since the seventeenth century. The NLOM-MPEF showed the capability of this technique to measure the grisaille paint layers’ thickness. This non-invasive multi-analytical and complementary approach proves itself efficient in identifying changes in the grisaille’s composition throughout time, which can be interpreted as changes in the raw materials and manufacture used in the production of these paint materials.

Two-Photon Absorption and Multiphoton Excited Fluorescence of Acetamide-Chalcone Derivatives: The Role of Dimethylamine Group on the Nonlinear Optical and Photophysical Properties.

This work studied the effect of different electron-withdrawing and electron-donating groups on the linear and nonlinear optical properties of acetamide-chalcone derivatives. The results showed that the addition of the dimethylamine group led to a large fluorescence emission (71% of fluorescence quantum yield in DMSO solution) that can be triggered by two and three-photon excitations, which is essential for biological applications. Furthermore, dimethylamine also red-shifts the lower energy state by approximately 90 nm, increasing the two-photon absorption cross-section of the lower energy band by more than 100% compared with the other studied compounds. All compounds presented two-electronic states observed through one and two-photon absorption spectroscopy and confirmed by Quantum Chemistry Calculations (QCCs). QCC results were also used to model the experimental two-photon absorption cross-sectional spectrum by the Sum-Over-States (SOS) approach, revealing a dependence between the coupling of the ground state with the first excited state and the transition dipole moment between these states.

Dual-resonant scanning multiphoton microscope with ultrasound lens and resonant mirror for rapid volumetric imaging

A dual-resonant scanning multiphoton (DRSM) microscope incorporating a tunable acoustic gradient index of refraction lens with a resonant mirror is developed for high-speed volumetric imaging. In the proposed microscope, the pulse train signal of a femtosecond laser is used to trigger an embedded field programmable gate array to sample the multiphoton excited fluorescence signal at the rate of one pixel per laser pulse. It is shown that a frame rate of around 8000 Hz can be obtained in the x–z plane for an image region with a size of 256 × 80 pixels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 343 × 343 × 120 μm3 with an image size of 256 × 256 × 80 voxels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 256 × 256 × 80 voxels, which represents 343 × 343 × 120 μm3 in field-of-view. The rapid volumetric imaging rate eliminates the aliasing effect for observed temporal frequencies lower than 15 Hz. The practical feasibility of the DRSM microscope is demonstrated by observing the mushroom bodies of a drosophila brain and performing 3D dynamic observations of moving 10-μm fluorescent beads.

Developments and Clinical Applications of Noninvasive Optical Technologies for Skin Cancer Diagnosis.

Skin cancer has shown a sharp increase in prevalence over the past few decades and currently accounts for one-third of all cancers diagnosed. The most lethal form of skin cancer is melanoma, which develops in 4% of individuals. The rising prevalence and increased number of fatalities of skin cancer put a significant burden on healthcare resources and the economy. However, early detection and treatment greatly improve survival rates for patients with skin cancer. Since the rising rates of both the incidence and mortality have been particularly noticeable with melanoma, significant resources have been allocated to research aimed at earlier diagnosis and a deeper knowledge of the disease. Dermoscopy, reflectance confocal microscopy, optical coherence tomography, multiphoton-excited fluorescence imaging, and dermatofluorescence are only a few of the optical modalities reviewed here that have been employed to enhance noninvasive diagnosis of skin cancer in recent years. This review article discusses the methodology behind newly emerging noninvasive optical diagnostic technologies, their clinical applications, and advantages and disadvantages of these techniques, as well as the potential for their further advancement in the future.

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

AbstractIt 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, Au9Ag6(SPhtOMe)4(DPPOE)3Cl3, 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.

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.

Pulse-Picking Multimodal Nonlinear Optical Microscopy.

Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). Femtosecond lasers are typically used for MPEF and SHG due to the requirement of high peak power for excitation, while picosecond lasers are preferred for CRS due to the need for high spectral resolution. Therefore, it is challenging to integrate CRS with MPEF and SHG for chemical imaging. We develop a pulse-picking strategy based on an acousto-optic modulator that can program the duty cycle of the laser pulse train, significantly increasing the pulse peak power at low input average power. This approach offers strong enhancement of nonlinear optical signals and makes hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy compatible with MPEF and SHG for multimodal imaging at low laser average power. The pulse-picking method also enables the evaluation and comparison of phototoxicity of laser pulses at different average and peak power levels. The photo-perturbations to biological samples are evaluated using cellular dynamics and sample morphological changes, allowing the selection of optimal laser power for the best sensitivity and minimal phototoxicity.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Real-time adaptive ultrashort pulse compressor for dynamic group delay dispersion compensation.

The optical dispersion effect in ultrafast pulse laser systems broadens the laser pulse duration and reduces the theoretical peak power. The present study proposes an adaptive ultrashort pulse compressor for compensating the optical dispersion using a direct optical-dispersion estimation by spectrogram (DOES) method. The DOES has fast and accurate computation time which is suitable for real time controller design. In the proposed approach, the group delay dispersion (GDD) and its polarity are estimated directly from the delay marginal of the trace obtained from a single-shot frequency-resolved optical gating (FROG). The estimated GDD is then processed by a closed-loop controller, which generates a command signal to drive a linear deformable mirror as required to achieve the desired laser pulse compression. The dispersion analysis, control computation, and deformable mirror control processes are implemented on a single field programmable gate array (FPGA). It is shown that the DOES dispersion computation process requires just 0.5 ms to complete. Moreover, the proposed pulse compressor compensates for both static dispersion and dynamic dispersion within five time steps when closed-loop controller is performed at a frequency of 100 Hz. The experimental results show that the proposed pulse compressor yields an effective fluorescence intensity improvement in a multiphoton excited fluorescence microscope (MPEFM).

Embedding Multiphoton Active Units within Metal-Organic Frameworks for Turning on High-Order Multiphoton Excited Fluorescence for Bioimaging.

The fabrication of high-order multiphoton excited fluorescent materials for second near-infrared (NIR-II, 1000-1700 nm) light-induced fluorescence imaging has always been an intractable challenge. In this study, a reasonable strategy guided by theoretical calculations was employed to fabricate a novel high-order multiphoton excited fluorescence (H-MPEF)-responsive UiO-type metal-organic framework (MOF, ZrTc). Strategically, the functionalization of the photonic-responsive thiazolothiazole-based organic ligand gave rise to amplified H-MPEF (four-photon activity) performance and prolonged excitation wavelength (1550 nm) of ZrTcI. The results reveal that the extended π-electron system, enhanced charge transfer, improved dipole moment, and weakened π-π stacking interactions within the MOFs endow them with excellent H-MPEF performance for NIR-II light-induced fluorescence imaging. It is an extremely rare report on H-MPEF bioimaging using MOFs and provide a universal strategy for the fabrication of H-MPEF-responsive materials.