Intravital Two-Photon Microscopy of the Transplanted Mouse Lung.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in naïve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Multiline orthogonal scanning temporal focusing (mosTF) microscopy for scattering reduction in in vivo brain imaging.

Temporal focusing two-photon microscopy has been utilized for high-resolution imaging of neuronal and synaptic structures across volumes spanning hundreds of microns in vivo. However, a limitation of temporal focusing is the rapid degradation of the signal-to-background ratio and resolution with increasing imaging depth. This degradation is due to scattered emission photons being widely distributed, resulting in a strong background. To overcome this challenge, we have developed multiline orthogonal scanning temporal focusing (mosTF) microscopy. mosTF captures a sequence of images at each scan location of the excitation line. A reconstruction algorithm then reassigns scattered photons back to their correct scan positions. We demonstrate the effectiveness of mosTF by acquiring neuronal images of mice in vivo. Our results show remarkable improvements in in vivo brain imaging with mosTF, while maintaining its speed advantage.

Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo.

Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

Prognostic significance of collagen signatures in pancreatic ductal adenocarcinoma obtained from second-harmonic generation imaging.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) ranks among the deadliest types of cancer, and it will be meaningful to search for new biomarkers with prognostic value to help clinicians tailor therapeutic strategies. METHODS: Here we tried to use an advanced optical imaging technique, multiphoton microscopy (MPM) combining second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) imaging, for the label-free detection of PDAC tissues from a cohort of 149 patients. An automated image processing method was used to extract collagen features from SHG images and the Kaplan-Meier survival analysis and Cox proportional hazards regression were used to assess the prognostic value of collagen signatures. RESULTS: SHG images clearly show the different characteristics of collagen fibers in tumor microenvironment. We gained eight collagen morphological features, and a Feature-score was derived for each patient by the combination of these features using ridge regression. Statistical analyses reveal that Feature-score is an independent factor, and can predict the overall survival of PDAC patients as well as provide well risk stratification. CONCLUSIONS: SHG imaging technique can potentially be a tool for the accurate diagnosis of PDAC, and this optical biomarker (Feature-score) may help clinicians make more approximate treatment decisions.

Pan-cortical 2-photon mesoscopic imaging and neurobehavioral alignment in awake, behaving mice.

The flow of neural activity across the neocortex during active sensory discrimination is constrained by task-specific cognitive demands, movements, and internal states. During behavior, the brain appears to sample from a broad repertoire of activation motifs. Understanding how these patterns of local and global activity are selected in relation to both spontaneous and task-dependent behavior requires in-depth study of densely sampled activity at single neuron resolution across large regions of cortex. In a significant advance toward this goal, we developed procedures to record mesoscale 2-photon Ca2+ imaging data from two novel in vivo preparations that, between them, allow for simultaneous access to nearly all 0f the mouse dorsal and lateral neocortex. As a proof of principle, we aligned neural activity with both behavioral primitives and high-level motifs to reveal the existence of large populations of neurons that coordinated their activity across cortical areas with spontaneous changes in movement and/or arousal. The methods we detail here facilitate the identification and exploration of widespread, spatially heterogeneous neural ensembles whose activity is related to diverse aspects of behavior.

Two spirobifluene-based turn-on fluorescent probes for highly selective detection of Cysteine and the applications in cells two-photon fluorescence imaging.

Two spirobifluene-based fluorescent probes SPF1 and SPF2, were designed and synthesized. The probes displayed "turn-on" fluorescence response for Cysteine. One of the challenges in developing a Cysteine probe is to secure high selectivity. SPF1/SPF2 can discriminate Cysteine from GSH as well as Hcy, and showed high substrate selectivity. The detection limit of SPF1 is 36 nM, which is excellent comparing with other optical sensors for Cysteine. The sensing mechanism of SPF1/SPF2 was verified by experimental data and theoretical calculations. There was a good linear relationship between the fluorescence intensity of SPF1/SPF2 and the concentration of Cysteine. The MTT tests indicated that SPF1/SPF2 had low cytotoxicity and good biocompatibility. Theoretical calculations demonstrated that SPF1, SPF2, and their related reaction products with Cysteine exhibited good two-photon absorption properties. Finally, SPF1/SPF2 had been successfully applied to the imaging of Cysteine in living cells under two-photon excitation.

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.

Comapping Cellular Content and Extracellular Matrix with Hemodynamics in Intact Arterial Tissues Using Scanning Immunofluorescent Multiphoton Microscopy.

Deviation of blood flow from an optimal range is known to be associated with the initiation and progression of vascular pathologies. Important open questions remain about how the abnormal flow drives specific wall changes in pathologies such as cerebral aneurysms where the flow is highly heterogeneous and complex. This knowledge gap precludes the clinical use of readily available flow data to predict outcomes and improve treatment of these diseases. As both flow and the pathological wall changes are spatially heterogeneous, a crucial requirement for progress in this area is a methodology for acquiring and comapping local vascular wall biology data with local hemodynamic data. Here, we developed an imaging pipeline to address this pressing need. A protocol that employs scanning multiphoton microscopy was developed to obtain three-dimensional (3D) datasets for smooth muscle actin, collagen, and elastin in intact vascular specimens. A cluster analysis was introduced to objectively categorize the smooth muscle cells (SMC) across the vascular specimen based on SMC actin density. Finally, direct quantitative comparison of local flow and wall biology in 3D intact specimens was achieved by comapping both heterogeneous SMC data and wall thickness to patient-specific hemodynamic results.

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.

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.

Cancer Imaging by Intravital Microscopy: The Dorsal Window Chamber Model.

Intravital microscopy allows a direct visualization of cells' behavior in their environment in a living organism with all its complexity. With appropriated models, longitudinal studies of structural and functional changes can be followed in the same animal on long period. In the field of cancer, the dorsal window chamber model is the model of choice for tumor events such as cells migration, vessels growth, and their permeability or interactions between cells and vessels. Coupled with wide-field, confocal, or multiphoton fluorescence microscopes, high spatial and temporal resolutions of the cellular events can be analyzed in vivo.

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.

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.

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.

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.