Two-photon Fluorescence Research Articles

Fast InVivo Two-Photon Fluorescence Imaging via Lateral and Axial Resolution Restoration WithSelf-Supervised Learning.

Two-photon fluorescence (TPF) imaging opens a new avenue to achieve high resolution at extended penetration depths. However, it is difficult for conventional TPF imaging systems to simultaneously achieve high resolution and speed. In this work, we develop an innovative deep learning framework of Lateral and Axial Resolution Restoration (LARR) to break the contradiction between imaging resolution and speed. LARR employs a self-supervised training scheme to computationally restore the sparsely sampled TPF images to resolution isotropic images by 4-fold axial and 16-fold lateral resolution enhancement. The simulation studies and experimental results demonstrate the excellent performance of LARR to preserve fine structural features with improved signal-to-noise ratio and structure similarity index. Moreover, the TPF imaging system with the LARR is able to achieve 60-fold improved imaging speed and comparable resolution as compared with the conventional TPF system. The outstanding performance makes LARR a potential tool for fast TPF imaging with high resolution.

Evaluation of safe exposure time for two-photon microscopy imaging of acrylic-painted mock-ups

Abstract Nonlinear optical microscopies are widely used in the biological and biomedical fields, as they are non-invasive techniques that permit the safe structural and morphological characterisation of cells and tissues. They are increasingly being used in the Cultural Heritage field because of their ability to overcome some limits of the well-established optical techniques. However, since nonlinear optical microscopies use pulsed laser sources with high peak power, their application in Cultural Heritage raises concerns due to artworks’ unique, priceless, and delicate nature. In this paper, we present a new method for evaluating the photo-induced damage when using a near-infrared femtosecond pulsed laser to perform two-photon excited fluorescence imaging of acrylic-painted mock-ups. In particular, we explore herein several irradiation conditions, varying the exposure time and excitation power, in order to provide useful experimental indications for safely imaging acrylic paints with two-photon microscopy.

Microbial methane production from calcium carbonate at moderately alkaline pH

Carbonate minerals are considered a metastable carbon reservoir under alkaline conditions. Here we demonstrate calcium carbonate as a sole inorganic carbon source at moderately alkaline pH for a methanogenic microbial community enriched from alkaline, saline wetland soil. In reactors amended with calcium carbonate as the sole source of inorganic carbon, concentrations of methane and aqueous calcium increased concurrent with headspace hydrogen depletion. Cells were observed in association with the carbonate mineral matrix via coherent anti-Stokes Raman scattering and two-photon excitation fluorescence microscopy. Genome-resolved metagenomics of the enrichment community confirmed the presence of a methanogen (Methanobacterium sp.) with the metabolic potential for hydrogenotrophic methanogenesis in addition to five bacterial community members. These results demonstrate that carbonate minerals can serve as an inorganic carbon source for hydrogenotrophic methanogenesis, leading to mineral dissolution under stable alkaline conditions. As such, hydrogenotrophic methanogens may impact carbon biogeochemistry and carbonate mineral stability in environmental systems.

Two-photon excited fluorescence with shaped laser pulses for refractive beams

We report two-photon excited fluorescence of dyes for refracted laser beams by utilizing tailored laser pulses. A fluorescence contrast difference due to phase shaping could be achieved between different coumarin dyes. Particularly, an increased contrast difference is obtained for configurations close to the Brewster angle. Furthermore, by using a subsequent deformable phase plate for spatial shaping it was possible to precisely adjust the laser beam for controlled refraction at the liquid dye surface. A polarization-dependent refraction was observed when directing the shaped laser beam on the curved liquid adhesion meniscus close to the cuvette wall. This results in a refraction-dependent contrast difference. The presented method could be utilized for surface-sensitive biophotonic imaging applications.Graphic abstract

FRET-based mesoporous organosilica nanoplatforms for in vitro and in vivo anticancer two-photon photodynamic therapy.

We report the synthesis of multifunctional periodic mesoporous organosilica nanoparticles (PMO NPs) with substantial two-photon absorption properties and targeting capability for two-photon excitation fluorescence (TPEF) and photodynamic therapy (TPE-PDT). Prepared using an adapted sol-gel synthesis, the nanoplatforms integrated two silylated chromophores in their three-dimensional matrix to maximize non-radiative Förster resonance energy transfer from a high two-photon absorption fluorophore donor to a porphyrin derivative acceptor, leading to an enhanced generation of reactive oxygen species. Combinations of biodegradable and non-biodegradable bis(triethoxysilyl)alkoxysilanes were employed for the synthesis of the NPs, and the corresponding photophysical studies revealed high efficiency levels of FRET. Next, the cellular uptake and toxicities of pristine and functionalized NPs were evaluated on breast cancer cell lines upon TPEF and TPE-PDT. Notably, the use of TPE-PDT treatment led to high levels of phototoxicity on MCF-7 and MDA-MB-231 cancer cells with substantial effects when compared to one-photon excitation (OPE)-PDT treatment. Preliminary in vivo data on selective and biodegradable NPs showed a significant phototoxicity towards MDA-MB-231 on zebrafish xenograft embryos, making these advanced nanoplatforms promising candidates for future TPE-PDT-based cancer treatments.

Knock-out mouse models and single particle ICP-MS reveal that SP-D and SP-A deficiency reduces agglomeration of inhaled gold nanoparticles in vivo without significant changes to overall lung clearance

The role of surfactant proteins A and D (SP-A and SP-D) in lung clearance and translocation to secondary organs of inhaled nanoparticles was investigated by exposing SP-A and SP-D knockout (AKO and DKO) and wild type (WT) mice nose-only for 3 hours to an aerosol of 20 nm gold nanoparticles (AuNPs). Animals were euthanised at 0-, 1-, 7- and 28-days post-exposure. Analysis by inductively coupled plasma mass spectrometry (ICP-MS) of the liver and kidneys showed that extrapulmonary translocation was below the limits of detection. Imaging of the lungs by laser ablation ICP-MS confirmed the homogenous distribution of AuNPs. Coherent anti-Stokes Raman Scattering, Second Harmonic Generation and Two-Photon Fluorescence imaging were applied for semi-quantitative analysis of the uptake of AuNPs by alveolar macrophages and found uptake increased with time post-exposure, peaking after 7 days, and with the largest increase in uptake being in WT mice. Single particle ICP-MS allowed particle counting and sizing of AuNPs in the lungs showing that particle agglomeration following deposition within the lung was greater for the wildtype than the knockout models, indicating a role for SP-A and SP-D in agglomeration, however, any effect of this on overall lung clearance was minimal. For all groups, the Au (mass) lung burden initial clearance half-time was approximately 20–25 d, however, the AuNP (particle number) lung burden clearance half-time was shorter at approximately 10 days. In general terms, differences between the results for the three models were limited, indicating the preferential clearance of smaller particles from the lung.

Active axial motion compensation in multiphoton-excited fluorescence microscopy.

In living organisms, the natural motion caused by heartbeat, breathing, or muscle movements leads to the deformation of tissue caused by translation and stretching of the tissue structure. This effect results in the displacement or deformation of the plane of observation for intravital microscopy and causes motion-induced aberrations of the resulting image data. This, in turn, places severe limitations on the time during which specific events can be observed in intravital imaging experiments. These limitations can be overcome if the tissue motion can be compensated such that the plane of observation remains steady. We have developed a mathematical shape space model that can predict the periodic motion of a cylindrical tissue phantom resembling blood vessels. This model is then used to rapidly calculate the future position of the plane of observation of a two-photon laser scanning fluorescence microscope. The focal plane is continuously adjusted to the calculated position with a piezo-actuated objective lens holder. We demonstrate active motion compensation for non-harmonic axial displacements of the vessel phantom with a field of view up to 400 µm × 400 µm, vertical amplitudes of more than 100 µm, and at a rate of 0.5 Hz.

Evaluation of safe exposure time for two-photon microscopy imaging of acrylic-painted mock-ups

Abstract Nonlinear optical microscopies are widely used in the biological and biomedical fields, as they are non-invasive techniques that permit the safe structural and morphological characterisation of cells and tissues. They are increasingly being used in the Cultural Heritage field because of their ability to overcome some limits of the well-established optical techniques. However, since nonlinear optical microscopies use pulsed laser sources with high peak power, their application in Cultural Heritage raises concerns due to artworks' unique, priceless, and delicate nature. In this paper, we present a new method for evaluating the photo-induced damage when using a near-infrared femtosecond pulsed laser to perform two-photon excited fluorescence imaging of acrylic-painted mock-ups. In particular, we explore herein several irradiation conditions, varying the exposure time and excitation power, in order to provide useful experimental indications for safely imaging acrylic paints with two-photon microscopy.

Boosting One- and Two-Photon Excited Fluorescence of Interpenetrated Tetraphenylethene-Based Metal-Organic Frameworks (TPE-MOFs) by Linker Installation.

Immobilizing organic chromophores within the rigid framework of metal-organic frameworks (MOFs) augments fluorescence by effectively curtailing molecular motions. Yet, the substantial interspaces and free volumes inherent to MOFs can undermine photoluminescence efficiency, as they partially constrain intramolecular dynamics. In this study, we achieved optimization of both one- and two-photon excited fluorescence by incorporating linkers into an interpenetrated tetraphenylethene-based MOF (TPE-MOF). This linker installation strategy enables fine-tuning of both crystal packing density and ligand conformations. Strikingly, the desolvated MOFs exhibit remarkable two-photon absorption (TPA) cross-sections, reaching an impressive 8801 GM. Consequently, these materials demonstrate exceptional performance in one- and two-photon excited cellular imaging of HepG2 cells. Our work introduces an innovative approach to enhancing two-photon excited fluorescence (TPEF) and broadens the scope of research into one- and two-photon excited fluorescence (1/2 PEF).

Transferrin Modified Gold Nanoclusters-Based Biosensing Nanoplatform for High-Precision Multimodal Bioimaging of Tumor Cells.

Bioimaging technology has been broadly used in biomedicine, and the growth of multimodal imaging technology based on synergistic advantages can overcome the shortcomings of traditional single-modal bioimaging methods and attain high specificity and sensitivity in the fields of bioimaging and biosensing. The analysis of low-abundance microRNAs (miRNAs) in complex organisms is of high importance for early-stage diagnosis and clinical treatment of tumors. In our current study, a biosensing nanoplatform based on Tf-AuNCs and MnO2 nanosheets was developed for multimodal imaging of tumor cells. First, oxidizable MnO2 nanosheets provided a smart tool for use as nanocarriers and contrast agents for intracellular glutathione (GSH)-activated magnetic resonance imaging (MRI). Then, MnO2 nanosheets delivered Tf-AuNC-based biosensing nanoplatforms into cells through endocytosis. Endogenous GSH degraded MnO2 nanosheets into Mn2+, and the released functional nucleic acid probes can perform specific biosensing responses to miR-21 exhibiting multimodal imaging including two-photon near-infrared fluorescence imaging (TP-NIRFI), fluorescence lifetime imaging (FLIM), and MRI. Finally, the biosensing nanoplatform achieved satisfactory results in tumor cells and tissues by TP-NIRFI (300.0 μm penetration depth), FLIM (τ ≈ 50.0 ns), and MRI. Therefore, biosensing nanoplatforms based on Tf-AuNCs and MnO2 nanosheets show great potential for multimodal detection and imaging in tumor cells.

FAST: Fast, free, consistent, and unsupervised oligodendrocyte segmentation and tracking system.

To develop reparative therapies for neurological disorders like multiple sclerosis (MS), we need to better understand the physiology of loss and replacement of oligodendrocytes, the cells that make myelin and are the target of damage in MS. In vivo two-photon fluorescence microscopy allows direct visualization of oligodendrocytes in the intact brain of transgenic mouse models, promising a deeper understanding of the longitudinal dynamics of replacing oligodendrocytes after damage. However, the task of tracking the fate of individual oligodendrocytes requires extensive effort for manual annotation and is especially challenging in three-dimensional images. While several models exist for annotating cells in two-dimensional images, few models exist to annotate cells in three-dimensional images and even fewer are designed for tracking cells in longitudinal imaging. Notably, existing options often come with a substantial financial investment, being predominantly commercial or confined to proprietary software. Furthermore, the complexity of processes and myelin formed by individual oligodendrocytes can result in the failure of algorithms that are specifically designed for tracking cell bodies alone. Here, we propose a fast, free, consistent, and unsupervised beta-mixture oligodendrocyte segmentation system (FAST) that is written in open-source software, and can segment and track oligodendrocytes in three-dimensional images over time with minimal human input. We showed that the FAST model can segment and track oligodendrocytes similarly to a blinded human observer. Although FAST was developed to apply to our studies on oligodendrocytes, we anticipate that it can be modified to study four-dimensional in vivo data of any brain cell with associated complex processes.Significance Statement We have developed "FAST: Fast, free, consistent, and unsupervised oligodendrocyte segmentation and tracking system" to solve our challenge of quantification of four-dimensional data acquired from longitudinal in vivo imaging. Although it was developed for oligodendrocytes, we will make the code entirely open source and user-friendly, and expect that it will be useful for segmentation for any cell body from a complex cell amenable to longitudinal in vivo imaging.

Modal focal adaptive optics for Bessel-focus two-photon fluorescence microscopy.

Adaptive optics (AO) improves the spatial resolution of microscopy by correcting optical aberrations. While its application has been well established in microscopy modalities utilizing a circular pupil, its adaptation to systems with non-circular pupils, such as Bessel-focus two-photon fluorescence microscopy (2PFM) with an annular pupil, remains relatively uncharted. Herein, we present a modal focal AO (MFAO) method for Bessel-focus 2PFM. Measuring and correcting aberration using a spatial light modulator placed in conjugation with the focal plane of the microscope objective, MFAO employs Zernike annular polynomials - a first in AO implementation - to achieve performance on par with a previous zonal AO method, but with a notably simplified optical configuration. We validated the performance of MFAO in correcting artificial and sample-induced aberrations, as well as in in vivo imaging of zebrafish larvae and mouse brains. By expanding the application of modal AO to annular pupils as well as aberration measurement and correction to a wavefront modulator at the objective focal plane, MFAO represents a notable advancement in the implementation of AO in microscopy.

Multiphoton and Harmonic Imaging of Microarchitected Materials.

Microadditive manufacturing has revolutionized the production of complex, nano- to microscale components across various fields. This work investigates two-photon (2P) and three-photon (3P) fluorescence imaging, as well as third-harmonic generation (THG) microscopy, to examine periodic microarchitected lattice structures fabricated using multiphoton lithography (MPL). By immersing the structures in refractive index matching fluids, we demonstrate high-fidelity 3D reconstructions of both fluorescent structures using 2P and 3P microscopy as well as low-fluorescence structures using THG microscopy. These results show that multiphoton fluorescence (MPF) imaging offers reduced signal decay with respect to depth compared to single-photon techniques in the examined structures. We further demonstrate the ability to nondestructively identify intentional internal modifications of the structure that are not immediately visible with scanning electron microscope (SEM) images and compression-induced fractures, highlighting the potential of these techniques for quality control and defect detection in microadditively manufactured components.

3D real-time single particle tracking using two-photon fluorescence from bright dye-based organic nanoparticles.

This paper addresses the use of ultrabright dye-based fluorescent organic nanoparticles in a 3D single-particle tracking two-photon microscopy setup. The nanoparticles consist of an assembly of quadrupolar dyes, presenting a large two-photon absorption cross-section. They exhibit low photobleaching, crucial for long-term tracking, and their high brightness allows nanometer localization precision. Their small size compared to previously used nonlinear inorganic nanocrystals, stable structure at physiological temperature, and adjustable optical properties make them promising tools for further biological research. This study highlights their potential to track dynamic processes with precision and stability, paving the way for exploring cellular processes at the nanoscale.

Rational engineering of a recognition group to construct a two-photon reaction-based fluorescent probe for rapid and selective sensing of cysteine.

It is highly required to rationally design fluorescent probes via a molecular engineering strategy with desired analytical performance for applications in sensing and imaging. Reaction-based fluorescent probes for highly selective sensing of cysteine (Cys) are mainly based on the participation of Cys in reactions such as, addition-cyclization with acrylates, cyclization with aldehydes, coordination displacement, Michael addition reactions, and cleavage reactions. Cys-triggered reactions with the O atoms of ether bonds has also been used to construct reaction-based fluorescent probes based on the substitution of the ether with the nucleophilic thiolate of Cys. However, many of the developed probes still suffer from long response times, interference from homocysteine (Hcy) and glutathione (GSH), high background fluorescence, and a lack of two-photon absorption (TPA) properties. Herein, we successfully design a Cys-sensitive two-photon fluorescent probe (F-BTD) using a donor-acceptor-donor (D-A-D) type π-extended benzothiadiazole framework as the fluorophore, with nitrobenzofuran (NBD) as the recognition unit. The proposed F-BTD probe displays some advantages over other probes including a rapid response time, high selectivity, low background fluorescence, and two-photon imaging capability. The F-BTD probe is applied to the two-photon fluorescence imaging of endogenous and exogenous Cys in HeLa cells with satisfactory results. For comparison, commonly used biothiol recognition groups including 2,4-dinitrobezensulfonyl and 2,4-dinitrophenyl are also used to construct S-BTD and N-BTD probes, respectively. The response mechanism of F-BTD to Cys is studied in detail through kinetic studies and transition-state analysis. This study may provide an example of how to design fluorescent probes with desired analytical performance by considering the recognition group as an important index of the design.

Advancements in organic fluorescent materials: unveiling the potential of peripheral group modification in dithienyl-diketopyrrolopyrrole derivatives for one- and two-photon bioimaging.

The quest for novel organic fluorescent materials capable of two-photon absorption (2PA) has intensified in recent years due to their promising applications in biological imaging. Two-photon fluorescence microscopy (2PFM) offers high spatial-temporal resolution, reduced photodamage, and deeper tissue penetration compared to conventional techniques. However, the development of bright two-photon molecular markers remains a challenge, necessitating compounds with high fluorescence quantum yield and 2PA cross-section (σ2PA). Strategies such as increasing π-conjugation have shown promise but are hindered by synthesis complexities and limited biocompatibility. Alternatively, incorporating electron-donating (ED) or electron-withdrawing (EW) peripheral groups in a main structure has emerged as a viable approach, leading to significant enhancements in σ2PA. This study highlights the advantages and challenges of these strategies, emphasizing the importance of exploring new organic compounds and evaluating the efficacy of peripheral groups for advanced two-photon bioimaging applications.

Harnessing Raman spectroscopy and multimodal imaging of cartilage for osteoarthritis diagnosis

Osteoarthritis (OA) is a complex disease of cartilage characterised by joint pain, functional limitation, and reduced quality of life with affected joint movement leading to pain and limited mobility. Current methods to diagnose OA are predominantly limited to X-ray, MRI and invasive joint fluid analysis, all of which lack chemical or molecular specificity and are limited to detection of the disease at later stages. A rapid minimally invasive and non-destructive approach to disease diagnosis is a critical unmet need. Label-free techniques such as Raman Spectroscopy (RS), Coherent anti-Stokes Raman scattering (CARS), Second Harmonic Generation (SHG) and Two Photon Fluorescence (TPF) are increasingly being used to characterise cartilage tissue. However, current studies are based on whole tissue analysis and do not consider the different and structurally distinct layers in cartilage. In this work, we use Raman spectroscopy to obtain signatures from the superficial (top) and deep (bottom) layer of healthy and osteoarthritic cartilage samples from 64 patients (19 control and 45 OA). Spectra were acquired both in the ‘fingerprint’ region from 700 to 1720 cm− 1 and high-frequency stretching region from 2500 to 3300 cm− 1. Principal component and linear discriminant analysis was used to identify the peaks that contributed significantly to classification accuracy of the different samples. The most pronounced differences were observed at the proline (855 cm− 1 and 921 cm− 1) and hydroxyproline (877 cm− 1 and 938 cm− 1), sulphated glycosaminoglycan (sGAG) (1064 cm− 1 and 1380 cm− 1) frequencies for both control and OA as well as the 1245 cm− 1 and 1272 cm− 1, 1320 cm− 1 and 1345 cm− 1, 1451 cm− 1 collagen modes were altered in OA samples, consistent with expected collagen structural changes. Classification accuracy based on Raman fingerprint spectral analysis of superficial and deep layer cartilage for controls was found to be 97% and 93% on using individual/all spectra and, 100% and 95% on using mean spectra per patient, respectively. OA diseased cartilage was classified with an accuracy of 88% and 84% for individual/all spectra, and 96% and 95% for mean spectra per patient based on analysis of the superficial and the deep layers, respectively. Raman spectra from the C-H stretching region (2500–3300 cm− 1) resulted in high classification accuracy for identification of different layers and OA diseased cartilage but low accuracy for controls. Differential changes in superficial and deep layer cartilage signatures were observed with age (under 60 and over 60 years), in contrast, less significant differences were observed with gender. Prominent chemical changes in the different layers of cartilage were preliminarily imaged using CARS, SHG and TPF. Cell clustering was observed in OA together with differences in pericellular matrix and collagen structure in the superficial and the deep layers correlating with the Raman spectral analysis. The current study demonstrates the potential of Raman Spectroscopy and multimodal imaging to interrogate cartilage tissue and provides insight into the chemical and structural composition of its different layers with significant implications for OA diagnosis for an increasing aging demographic.

Detecting Early Degradation of Wood Ultrastructure with Nonlinear Optical Imaging and Fluorescence Lifetime Analysis.

Understanding the deterioration processes in wooden artefacts is essential for accurately assessing their conservation status and developing effective preservation strategies. Advanced imaging techniques are currently being explored to study the impact of chemical changes on the structural and mechanical properties of wood. Nonlinear optical modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), combined with fluorescence lifetime imaging microscopy (FLIM), offer a promising non-destructive diagnostic method for evaluating lignocellulose-based materials. In this study, we employed a nonlinear multimodal approach to examine the effects of artificially induced delignification on samples of Norway spruce (Picea abies) and European beech (Fagus sylvatica) subjected to increasing treatment durations. The integration of SHG/TPEF imaging and multi-component fluorescence lifetime analysis enabled the detection of localized variations in nonlinear signals and τ-phase of key biopolymers within wood cell walls. This methodology provides a powerful tool for early detection of wood deterioration, facilitating proactive conservation efforts of wooden artefacts.

Photon-Based Innovations in Oncology: Precise Diagnostic Techniques and Advanced Therapies

In diagnostics, photons are used in basic methods such as computed tomography (CT) and positron emission tomography (PET), which are pivotal tools for high-resolution, non-invasive tumor detection, offering insights into tumor staging and progression. Mentioned techniques facilitate early diagnosis and the planning of therapeutic strategies. However, new methods are emerging, enhancing the precision and detail of diagnostics, such as ultra-weak photon emission (UPE) imagining, two-photon fluorescence imaging, photo acoustic imaging, and others. Therapeutically, external beam radiation therapy (EBRT) uses photons to target cancer cells while minimizing harm to healthy tissue. Photodynamic therapy (PDT), which uses light-sensitive compounds activated by specific wavelengths, represents a photon-based treatment applicable to certain malignancies. Other treatments include photo thermal therapy (PTT), radio dynamic therapy (RDT), intensity-modulated radiation therapy (IMRT), volumetric modulated arc therapy (VMAT), and more. These constantly evolving photon-driven technologies can be used to treat a broad spectrum of cancers, such as pancreatic, prostate, breast, and skin cancers. This review article discusses the latest photon-based methods in oncology, focusing on new possibilities, solutions, perspectives, and the potential disadvantages of these approaches.

Multiphoton Microscopy Assessment of Healing From Tendon Laceration and Microthermal Coagula in a Rat Model.

To study the healing response of rat Achilles tendon when lacerated or treated with intense therapeutic ultrasound (ITU) via utilization of multiphoton microscopy (MPM) imaging and histology. The right Achilles tendon of each Sprague Dawley rat within a cohort was partially lacerated. 1 to 2 days post-surgery, each rat received ITU treatment of the Achilles tendon on either the right or left leg. Rats were euthanized in groups at 1, 3, 7, 14, or 28 days posttreatment and their tendons were explanted, formalin fixed, paraffin embedded, sectioned, and placed on slides for imaging. Slides from each time point were imaged using a laboratory built MPM with a 780 nm Ti:Sapphire laser. The resulting second harmonic generation (SHG) and two-photon excited fluorescence (2PEF) signals were captured, assessed, and compared to brightfield microscopy images of the same section subsequently stained with hematoxylin and eosin. At early timepoints, 2PEF images show the presence of red blood cells, infiltration of inflammatory cells and formation of a fibrin clot at laceration sites, and attraction of fibroblasts to ITU coagula. SHG images indicate an absence of organized collagen in both types of lesions. At later timepoints, new organized collagen can be seen at the laceration sites, and the concentration of inflammatory cells has noticeably decreased. Automated detection of red blood cells and infiltrative cells, as well as analysis of SHG signal intensity and homogeneity was performed at laceration locations. Results show that all quantities except SHG signal intensity approach normal values by day 28. Thus, combined analysis of 2PEF and SHG images elucidates tendon healing processes that align with and complement histological findings. These results indicate that multiphoton imaging can effectively visualize the healing response to mechanical (laceration) and thermal (ITU) injury, including the organization of new collagen which is more difficult to visualize with histology.