More than double the fun with two-photon excitation microscopy.

For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy users interested in adopting TPE for intravital imaging. We focus on applications in neurobiology, present current trends towards faster, wider and deeper imaging, discuss the combination with photon counting technologies for metabolic imaging and spectroscopy, as well as highlight outstanding issues and drawbacks in development and application of these methodologies.

Three-dimensional object geometry of mitochondria-associated signal: 3-D analysis pipeline for two-photon image stacks of cerebrovascular endothelial mitochondria.

Increasing evidence indicates the role of mitochondrial and vascular dysfunction in aging and aging-associated pathologies; however, the exact mechanisms and chronological processes remain enigmatic. High-energy demand organs, such as the brain, depend on the health of their mitochondria and vasculature for the maintenance of normal functions, therefore representing vulnerable targets for aging. This methodology article describes an analysis pipeline for three-dimensional (3-D) mitochondria-associated signal geometry of two-photon image stacks of brain vasculature. The analysis methods allow the quantification of mitochondria-associated signals obtained in real time in their physiological environment. In addition, signal geometry results will allow the extrapolation of fission and fusion events under normal conditions, during aging, or in the presence of different pathological conditions, therefore contributing to our understanding of the role mitochondria play in a variety of aging-associated diseases with vascular etiology.NEW & NOTEWORTHY Analysis pipeline for 3-D mitochondria-associated signal geometry of two-photon image stacks of brain vasculature.

Compact simultaneous label-free autofluorescence multi-harmonic microscopy for user-friendly photodamage-monitored imaging.

Significance: Label-free nonlinear optical microscopy has become a powerful tool for biomedical research. However, the possible photodamage risk hinders further clinical applications. Aim: To reduce these adverse effects, we constructed a new platform of simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy, featuring four-channel multimodal imaging, inline photodamage monitoring, and pulse repetition-rate tuning. Approach: Using a large-core birefringent photonic crystal fiber for spectral broadening and a prism compressor for pulse pre-chirping, this system allows users to independently adjust pulse width, repetition rate, and energy, which is useful for optimizing imaging conditions towards no/minimal photodamage. Results: It demonstrates label-free multichannel imaging at one excitation pulse per image pixel and thus paves the way for improving the imaging speed by a faster optical scanner with a low risk of nonlinear photodamage. Moreover, the system grants users the flexibility to autonomously fine-tune repetition rate, pulse width, and average power, free from interference, ensuring the discovery of optimal imaging conditions with high SNR and minimal phototoxicity across various applications. Conclusions: The combination of a stable laser source, independently tunable ultrashort pulse, photodamage monitoring features, and a compact design makes this new system a robust, powerful, and user-friendly imaging platform.

Two-Photon and Multiphoton Microscopy in Anterior Segment Diseases of the Eye.

Two-photon excitation microscopy (TPM) and multiphoton fluorescence microscopy (MPM) are advanced forms of intravital high-resolution functional microscopy techniques that allow for the imaging of dynamic molecular processes and resolve features of the biological tissues of interest. Due to the cornea's optical properties and the uniquely accessible position of the globe, it is possible to image cells and tissues longitudinally to investigate ocular surface physiology and disease. MPM can also be used for the in vitro investigation of biological processes and drug kinetics in ocular tissues. In corneal immunology, performed via the use of TPM, cells thought to be intraepithelial dendritic cells are found to resemble tissue-resident memory T cells, and reporter mice with labeled plasmacytoid dendritic cells are imaged to understand the protective antiviral defenses of the eye. In mice with limbal progenitor cells labeled by reporters, the kinetics and localization of corneal epithelial replenishment are evaluated to advance stem cell biology. In studies of the conjunctiva and sclera, the use of such imaging together with second harmonic generation allows for the delineation of matrix wound healing, especially following glaucoma surgery. In conclusion, these imaging models play a pivotal role in the progress of ocular surface science and translational research.

Three-Four Photon Transition Mn(II) Complex Monitoring Lysosome-Related ATP in Real Time via Fluorescence Lifetime Imaging.

Currently, in situ monitoring of the adenosine triphosphate (ATP) level in lysosomes is critical to understand their involvement in various biological processes, but it remains difficult due to the interferences of limited targeting and low resolution of fluorescent probes. Herein, we report a classic Mn(II) probe (FX2-MnCl2) with near-infrared (NIR) nonlinear (NLO) properties, accompanied by three-four photon transition and fivefold fluorescence enhancement in the presence of ATP. FX2-MnCl2 combines with ATP through dual recognition sites of diethoxy and manganese ions to reflect slightly fluorescence lifetime change. Through the synergy of multiphoton fluorescence imaging (MP-FI) and multiphoton fluorescence lifetime imaging microscopy (MP-FLIM), it is further demonstrated that FX2-MnCl2 displays lysosome-specific targeting behavior, which can monitor lysosome-related ATP migration under NIR laser light. This work provides a novel multiphoton transformation fluorescence complex, which might be a potential candidate as a simple and straightforward biomarker of lysosome ATP in vitro for clinical diagnosis.

Computer-Aided Multiphoton Microscopy Diagnosis of 5 Different Primary Architecture Subtypes of Meningiomas.

Meningiomas rank among the most common intracranial tumors, and surgery stands as the primary treatment modality for meningiomas. The precise subtyping and diagnosis of meningiomas, both before and during surgery, play a pivotal role in enabling neurosurgeons choose the optimal surgical program. In this study, we utilized multiphoton microscopy (MPM) based on 2-photon excited fluorescence and second-harmonic generation to identify 5 common meningioma subtypes. The morphological features of these subtypes were depicted using the MPM multichannel mode. Additionally, we developed 2 distinct programs to quantify collagen content and blood vessel density. Furthermore, the lambda mode of the MPM characterized architectural and spectral features, from which 3 quantitative indicators were extracted. Moreover, we employed machine learning to differentiate meningioma subtypes automatically, achieving high classification accuracy. These findings demonstrate the potential of MPM as a noninvasive diagnostic tool for meningioma subtyping and diagnosis, offering improved accuracy and resolution compared with traditional methods.

In vivo deep brain multiphoton fluorescence imaging emitting at NIR-I and NIR-II and excited at NIR-IV.

Multiphoton microscopy (MPM) enables deep brain imaging. Three optical windows: NIR-I, NIR-II, and NIR-III are widely used. Recently, NIR-IV (the 2200 nm window) has been demonstrated to be the last and longest window for deep tissue MPM. However, so far MPM covers only two optical windows labeled by single fluorescent probe, one for emission and one for excitation. Here we demonstrate in vivo deep brain MPM covering three optical windows, with emission at NIR-I, NIR-II, and excitation at NIR-IV, labeled by ICG. The innovations include: (1) characterizing both 3-photon excitation and emission properties of ICG emitting at both NIR-I and NIR-II, in water, plasma, and circulating blood; (2) a home-built multiphoton microscope with simultaneous dual channel detection, with which we demonstrate deep brain MPM 950 µm (NIR-I) and 850 µm (NIR-II) into the mouse brain in vivo, verifying that multi-optical window MPM is promising for deep brain imaging.

Label-free assessment of pathological changes in pancreatic intraepithelial neoplasia by biomedical multiphoton microscopy.

Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion that has the potential to progress to invasive pancreatic cancer, and early and rapid detection may offer patients a chance for treatment before the development of invasive carcinoma. Therefore, the identification of PanIN holds significant clinical importance. In this study, we first used multiphoton microscopy (MPM) combining two-photon excitation fluorescence and second-harmonic generation imaging to label-free detect PanIN and attempted to differentiate between normal pancreatic ducts and different grades of PanIN. Then, we also developed an automatic image processing strategy to extract eight morphological features of collagen fibers from MPM images to quantify the changes in collagen fibers surrounding the ducts. Experimental results demonstrate that the combination of MPM and quantitative information can accurately identify normal pancreatic ducts and different grades of PanIN. This study may contribute to the rapid diagnosis of pancreatic diseases and may lay the foundation for further clinical application of MPM.

Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities.

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

Multiphoton imaging of the monosachharide induced formation of fluorescent advanced glycation end products in tissues.

We studied the in vitro rate of fluorescent advanced glycation end products (fAGEs) formation with multiphoton microscopy in different porcine tissues (aorta, cornea, kidney, dermis, and tendon). These tissues were treated with d-glucose, d-galactose, and d-fructose, three primary monosaccharides found in human diets. We found that the use of d-fructose resulted in the highest glycation rate, followed by d-galactose and then d-glucose. Moreover, compared to non-collagen tissue constituents such as elastic fibers and cells, the rate of tissue glycation was consistently higher in collagen, suggesting that collagen is a more sensitive target for fAGE formation. However, we also found that collagen in different tissues exhibits different rates of fAGE formation, with slower rates observed in tightly packed tissues such as cornea and tendon. Our study suggests that for fAGE to be developed into a long-term glycemic biomarker, loosely organized collagen tissues located in the proximity of vasculature may be the best targets.

Compact portable higher harmonic generation microscopy for the real time assessment of unprocessed thyroid tissue.

During thyroid surgery fast and reliable intra-operative pathological feedback has the potential to avoid a two-stage procedure and significantly reduce health care costs in patients undergoing a diagnostic hemithyroidectomy (HT). We explored higher harmonic generation (HHG) microscopy, which combines second harmonic generation (SHG), third harmonic generation (THG), and multiphoton excited autofluorescence (MPEF) for this purpose. With a compact, portable HHG microscope, images of freshly excised healthy tissue, benign nodules (follicular adenoma) and malignant tissue (papillary carcinoma, follicular carcinoma and spindle cell carcinoma) were recorded. The images were generated on unprocessed tissue within minutes and show relevant morphological thyroid structures in good accordance with the histology images. The thyroid follicle architecture, cells, cell nuclei (THG), collagen organization (SHG) and the distribution of thyroglobulin and/or thyroid hormones T3 or T4 (MPEF) could be visualized. We conclude that SHG/THG/MPEF imaging is a promising tool for clinical intraoperative assessment of thyroid tissue.

The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo.

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.

Multiphoton Imaging of Maturation in Tissue Engineering.

Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.

The Effect of Diabetes Mellitus Type 1 on the Energy Metabolism of Hepatocytes: Multiphoton Microscopy and Fluorescence Lifetime Imaging.

A decrease in the regenerative potential of the liver during the development of non-alcoholic fatty liver disease (NAFLD), which is observed in the vast majority of patients with diabetes mellitus type 1, significantly increases the risk of postoperative liver failure. In this regard, it is necessary to develop new approaches for the rapid intraoperative assessment of the condition of liver tissue in the presence of concomitant liver pathology. A modern label-free approach based on multiphoton microscopy, second harmonic generation (SHG), and fluorescence lifetime imaging microscopy (FLIM) allow for the evaluation of the structure of liver tissue as well as the assessment of the metabolic state of hepatocytes, even at the cellular level. We obtained optical criteria and identified specific changes in the metabolic state of hepatocytes for a reduced liver regenerative potential in the presence of induced diabetes mellitus type 1. The obtained criteria will expand the possibilities for the express assessment of the structural and functional state of liver tissue in clinical practice.

Cascaded domain multiphoton spatial frequency modulation imaging.

Significance: Multiphoton microscopy is a powerful imaging tool for biomedical applications. A variety of techniques and respective benefits exist for multiphoton microscopy, but an enhanced resolution is especially desired. Additionally multiphoton microscopy requires ultrafast pulses for excitation, so optimization of the pulse duration at the sample is critical for strong signals. Aim: We aim to perform enhanced resolution imaging that is robust to scattering using a structured illumination technique while also providing a rapid and easily repeatable means to optimize group delay dispersion (GDD) compensation through to the sample. Approach: Spatial frequency modulation imaging (SPIFI) is used in two domains: the spatial domain (SD) and the wavelength domain (WD). The WD-SPIFI system is an in-line tool enabling GDD optimization that considers all material through to the sample. The SD-SPIFI system follows and enables enhanced resolution imaging. Results: The WD-SPIFI dispersion optimization performance is confirmed with independent pulse characterization, enabling rapid optimization of pulses for imaging with the SD-SPIFI system. The SD-SPIFI system demonstrates enhanced resolution imaging without the use of photon counting enabled by signal to noise improvements due to the WD-SPIFI system. Conclusions: Implementing SPIFI in-line in two domains enables full-path dispersion compensation optimization through to the sample for enhanced resolution multiphoton microscopy.

Two-photon microscopy for microrobotics: Visualization of micro-agents below fixed tissue.

Optical microscopy is frequently used to visualize microrobotic agents (i.e., micro-agents) and physical surroundings with a relatively high spatio-temporal resolution. However, the limited penetration depth of optical microscopy techniques used in microrobotics (in the order of 100 µm) reduces the capability of visualizing micro-agents below biological tissue. Two-photon microscopy is a technique that exploits the principle of two-photon absorption, permitting live tissue imaging with sub-micron resolution and optical penetration depths (over 500 µm). The two-photon absorption principle has been widely applied to fabricate sub-millimeter scale components via direct laser writing (DLW). Yet, its use as an imaging tool for microrobotics remains unexplored in the state-of-the-art. This study introduces and reports on two-photon microscopy as an alternative technique for visualizing micro-agents below biological tissue. In order to validate two-photon image acquisition for microrobotics, two-type micro-agents are fabricated and employed: (1) electrospun fibers stained with an exogenous fluorophore and (2) bio-inspired structure printed with autofluorescent resin via DLW. The experiments are devised and conducted to obtain three-dimensional reconstructions of both micro-agents, perform a qualitative study of laser-tissue interaction, and visualize micro-agents along with tissue using second-harmonic generation. We experimentally demonstrate two-photon microscopy of micro-agents below formalin-fixed tissue with a maximum penetration depth of 800 µm and continuous imaging of magnetic electrospun fibers with one frame per second acquisition rate (in a field of view of 135 × 135 µm2). Our results show that two-photon microscopy can be an alternative imaging technique for microrobotics by enabling visualization of micro-agents under in vitro and ex ovo conditions. Furthermore, bridging the gap between two-photon microscopy and the microrobotics field has the potential to facilitate in vivo visualization of micro-agents.

Rapid label-free detection of early-stage lung adenocarcinoma and tumor boundary via multiphoton microscopy.

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths in China. Rapid and precise evaluation of tumor tissue during lung cancer surgery can reduce operative time and improve negative-margin assessment, thus increasing disease-free and overall survival rates. This study aimed to explore the potential of label-free multiphoton microscopy (MPM) for imaging adenocarcinoma tissues, detecting histopathological features, and distinguishing between normal and cancerous lung tissues. We showed that second harmonic generation (SHG) signals exhibit significant specificity for collagen fibers, enabling the quantification of collagen features in lung adenocarcinomas. In addition, we developed a collagen score that could be used to distinguish between normal and tumor areas at the tumor boundary, showing good classification performance. Our findings demonstrate that MPM imaging technology combined with an image-based collagen feature extraction method can rapidly and accurately detect early-stage lung adenocarcinoma tissues.

Quantitative Assessment of Hepatic Steatosis Using Label-Free Multiphoton Imaging and Customized Image Processing Program.

Nonalcoholic fatty liver disease is rapidly becoming one of the most common causes of chronic liver disease worldwide and is the leading cause of liver-related morbidity and mortality. A quantitative assessment of the degree of steatosis would be more advantageous for diagnostic evaluation and exploring the patterns of disease progression. Here, multiphoton microscopy, based on the second harmonic generation and 2-photon excited fluorescence, was used to label-free image the samples of nonalcoholic fatty liver. Imaging results confirm that multiphoton microscopy is capable of directly visualizing important pathologic features such as normal hepatocytes, hepatic steatosis, Mallory bodies, necrosis, inflammation, collagen deposition, microvessel, and so on and is a reliable auxiliary tool for the diagnosis of nonalcoholic fatty liver disease. Furthermore, we developed an image segmentation algorithm to simultaneously assess hepatic steatosis and fibrotic changes, and quantitative results reveal that there is a correlation between the degree of steatosis and collagen content. We also developed a feature extraction program to precisely display the spatial distribution of hepatocyte steatosis in tissues. These studies may be beneficial for a better clinical understanding of the process of steatosis as well as for exploring possible therapeutic targets.

Differentiating morphea from lichen Sclerosus by using multiphoton microscopy combined with U-Net model for elastic fiber segmentation.

This paper describes a methodology to differentiate morphea from lichen sclerosus based on examination with multiphoton microscopy (MPM) composed of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Subcellular-resolution images were acquired by MPM from unstained lesion tissues then process spectral analysis to quantify the TPEF and SHG signals. Moreover, U-Net was employed to segment elastic fiber in TPEF images to combine with collagen fiber in SHG images for precise fiber quantification. Predictions of segmentation showed excellent performance on several evaluation indicators. The mIoU, mPA, and F1 score reach 0.8516, 0.9281, and 0.941. The quantitative analysis demonstrated the increase of collagen fibers in morphea compared to that in lichen sclerosus cases. Meanwhile, the great diminution of elastic fiber in the dermis of lichen sclerosus was depicted based on MPM imaging. Thus, MPM was comparable to the histopathological examination and our experimental results accurately distinguish between morphea and lichen sclerosus.