Two-photon Autofluorescence Research Articles

In vivo and in situ robust quantitative optical biopsy of hepatocellular carcinoma and metastasis based on two-photon autofluorescence imaging.

Two-photon autofluorescence (TPAF) imaging is able to offer precise cellular metabolic information with high spatiotemporal resolution, making it a promising biopsy tool. The technique is greatly hampered by the complexity of either the optical system or data processing. Here, the excitation wavelength was optimized to simultaneously excite both flavin adenine dinucleotide and nicotinamide adenine dinucleotide and eliminate the unexpected TPAF. The optical redox ratio (ORR) images were robustly achieved without additional calibration under the optimized single-wavelength excitation. The in vitro, ex vivo, and in vivo biopsy by the TPAF method were systematically studied and compared using hepato-cellular carcinoma and metastasis as examples. It was demonstrated that the proposed TPAF method simplified the optical system, improved the robustness of ORR, and enabled early-stage cancer diagnosis, showing distinguished advantages as compared with previous methods.

Multimodal Multiphoton Tomography with a Compact Femtosecond Fiber Laser

Multiphoton tomography (MPT) based on near infrared (NIR) femtosecond laser technology has become a versatile high-resolution clinical and research imaging tool. We report on multimodal MPT with the air-cooled fiber laser tomograph MPTcompact. The ultracompact passively mode-locked erbium-doped 50/80 MHz laser operating at 780 nm is directly integrated into the 360° imaging head mounted on a flexible mechanical arm. The tomograph provides optical biopsies with subcellular resolution and optical metabolic imaging capability based on two-photon autofluorescence (AF), second harmonic generation, fluorescence lifetime imaging (FLIM) by time-correlated single photon counting with 250 ps temporal resolution, reflectance confocal microscopy, and white LED light CMOS camera imaging. For the first time, NIR femtosecond laser pulses have been used on human skin to realize simultaneous one-photon confocal imaging and two-photon imaging. Most useful information is provided by the two-photon excited intratissue AF of the coenzymes NAD(P)H and flavins. The signals of both types of coenzymes can be separated by FLIM. Furthermore, the free and protein-bound forms can be distinguished by time-resolved AF detection because the protein-bound NADH has one order higher AF lifetime than free (non-bound) NADH. The tomograph contains onboard storage batteries so that it can operate for up to a few hours independently from external power supply. Applications include in vivo cancer detection and in situ evaluation of anti-aging drugs and pharmaceuticals. The tomograph MPTcompact has been successfully tested in a clinical multicenter study for the diagnosis of malignant melanoma on 100 patients with suspicious pigmented lesions. MPT has the potential to realize non-invasive high-resolution label-free in vivo histology within minutes and, therefore, to reduce the number of physically taken biopsies.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

Graphene quantum dot composite with multiphoton excitation as near infrared-II probe in bioimaging

Non-radiative traps and structures are present on the graphene quantum dots doped with nitrogen and functionalized with amino groups (amino-N-GQDs) and multiple crystalline layers because of cross-link-enhanced emission. Secondary and tertiary amines, which are potential fluorophores, have also been observed on the polyethylenimine (PEI) coating of amino-N-GQDs. Cross-linked PEI coating reduced rotation and vibration, thereby enhancing the photoluminescence quantum yield (PL QY). Introduced nitrogen atoms from N dopants, amino-functionalized groups and PEI, as well as sulfur from polystyrene sulfonate (PSS) enhanced the cooperative effect on the properties of heteroatom-doped materials among electrons captured by new surface states. This enhanced radiative recombination and subsequently enhanced PL QY, therefore, surface conjugation improved the amino-N-GQD surfaces by increasing the quantum confinement of their emissive energy, evidenced by the increased PL QY of amino-N-GQD-PSS-PEI (or amino-N-GQD-polymer composites). In some situations, the maximum available power required for delivery to the two-photon imaging plane without damaging tissues limits imaging depth but the additional brightness provided by amino-N-GQD-polymer composites in this study extended the maximum imaging depth to 240 μm. Amino-N-GQD-polymer composites had favorable two-photon properties under two-photon excitation (self-developed femtosecond Ti–sapphire laser optical system; power: 23.93 nJ pixel−1, 160 scans, approximately 1.09 s of total exposure time; excitation wavelength: 980 nm, near-infrared-II region), indicating that cells treated with amino-N-GQD-polymer composites and the anti-epidermal growth factor receptor antibody can achieve two-photon luminescence with 1/81 of the power required for similar-intensity two-photon autofluorescence (1938.33 nJ pixel−1 with 800 scans, total exposure time of ∼5.44 s). The materials can serve as contrast agents for the non-invasive detection of biological specimens and interior tissues using a two-photon excitation wavelength in the near-infrared region.

Two-photon microendoscope for label-free imaging in stereotactic neurosurgery.

We demonstrate a gradient refractive index (GRIN) microendoscope with an outer diameter of ∼1.2 mm and a length of ∼186 mm that can fit into a stereotactic surgical cannula. Two photon imaging at an excitation wavelength of 900 nm showed a field of view of ∼180 microns and a lateral and axial resolution of 0.86 microns and 9.6 microns respectively. The microendoscope was tested by imaging autofluorescence and second harmonic generation (SHG) in label-free human brain tissue. Furthermore, preliminary image analysis indicates that image classification models can predict if an image is from the subthalamic nucleus or the surrounding tissue using conventional, bench-top two-photon autofluorescence.

Medical femtosecond laser

Medical femtosecond laser devices are used in dermatology for non-linear high-resolution imaging to obtain non-invasive and label-free optical skin biopsies (multiphoton tomography) as well as in ophthalmology for refractive corneal surgery and cataract surgery. Applications of commercial certified multiphoton tomographs include early detection of skin cancer within minutes by two-photon autofluorescence imaging of coenzymes and melanin and second harmonic imaging of collagen as well as by testing the efficacy of pharmaceutical and cosmetical products. Goals are (i) to reduce the number of physically taken human skin biopsies in hospitals and research institutions, (ii) to optimize personalized medicine, and (iii) to reduce animal studies in pharmacy. Current diagnostic tools in dermatology include surface microscopy with a dermatoscope and ultrasound but have poor resolution. Optical coherence tomography and confocal reflectance microscopy have better resolution but provide limited information based on changes of the intratissue refractive index. Multiphoton tomography provides the best resolution of all clinical imaging methods and offer functional imaging such as optical metabolic imaging based on autofluorescence lifetime imaging. Goals of femtosecond laser eye treatment are (i) the replacement of mechanical microkeratomes for corneal flap generation, (ii) the replacement of the UV nanosecond excimer laser for stroma removal, and (iii) to replace, in part, the scalpel in the surgery of cataracts and other eye diseases. So far, millions of eye treatments have been conducted around the world. The major disadvantage of current certified medical femtosecond laser devices is the high price compared with the standard mechanical and optical medical devices.

Structural and Biochemical Changes in Pericardium upon Genipin Cross-Linking Investigated Using Nondestructive and Label-Free Imaging Techniques.

Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

Label-Free Multiphoton Microscopy: Much More Than Fancy Images.

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy.

Autofluorescence Imaging of 3D Tumor-Macrophage Microscale Cultures Resolves Spatial and Temporal Dynamics of Macrophage Metabolism.

Macrophages within the tumor microenvironment (TME) exhibit a spectrum of protumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon autofluorescence imaging of NAD(P)H and FAD to nondestructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours poststimulation for mouse macrophages (RAW264.7) stimulated with IFNγ or IL4 plus IL13 in 2D culture, confirming that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse polyoma-middle T virus or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor cocultures exhibited significantly different FAD mean lifetimes and greater migration than monocultures at 24, 48, and 72 hours postseeding. In cocultures with primary human cancer cells, actively migrating monocyte-derived macrophages had greater redox ratios [NAD(P)H/FAD intensity] compared with passively migrating monocytes at 24 and 48 hours postseeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumor-immune cross-talk within the 3D TME. SIGNIFICANCE: Label-free metabolic imaging and microscale culture technologies enable monitoring of single-cell macrophage metabolism, migration, and function in the 3D tumor microenvironment.

Characterizing Vocal Fold Injury Recovery in a Rabbit Model With Three-Dimensional Virtual Histology.

In animal studies of vocal fold scarring and treatment, imaging-based evaluation is most often conducted by tissue slicing and histological staining. Given variation in anatomy, injury type, severity, and sacrifice timepoints, planar histological sections provide limited spatiotemporal details of tissue repair. Three-dimensional (3D) virtual histology may provide additional contextual spatial information, enhancing objective interpretation. The study's aim was to evaluate the suitability of magnetic resonance imaging (MRI), microscale computed tomography (CT), and nonlinear laser-scanning microscopy (NM) as virtual histology approaches for rabbit studies of vocal fold scarring. A unilateral injury was created using microcup forceps in the left vocal fold of three New Zealand White rabbits. Animals were sacrificed at 3, 10, and 39 days postinjury. ex vivo imaging of excised larynges was performed with MRI, CT, and NM modalities. The MRI modality allowed visualization of injury location and morphological internal features with 100-μm spatial resolution. The CT modality provided a view of the injury defect surface with 12-μm spatial resolution. The NM modality with optical clearing resolved second-harmonic generation signal of collagen fibers and two-photon autofluorescence in vocal fold lamina propria, muscle, and surrounding cartilage structures at submicrometer spatial scales. Features of vocal fold injury and wound healing were observed with MRI, CT, and NM. The MRI and CT modalities provided contextual spatial information and dissection guidance, whereas NM resolved extracellular matrix structure. The results serve as a proof of concept to motivate incorporation of 3D virtual histology techniques in future vocal fold injury animal studies. NA Laryngoscope, 131:1578-1587, 2021.

Alterations in extracellular matrix composition during aging and photoaging of the skin

Human skin is composed of the cell-rich epidermis, the extracellular matrix (ECM) rich dermis, and the hypodermis. Within the dermis, a dense network of ECM proteins provides structural support to the skin and regulates a wide variety of signaling pathways which govern cell proliferation and other critical processes. Both intrinsic aging, which occurs steadily over time, and extrinsic aging (photoaging), which occurs as a result of external insults such as solar radiation, cause alterations to the dermal ECM. In this study, we utilized both quantitative and global proteomics, alongside single harmonic generation (SHG) and two-photon autofluorescence (TPAF) imaging, to assess changes in dermal composition during intrinsic and extrinsic aging. We find that both intrinsic and extrinsic aging result in significant decreases in ECM-supporting proteoglycans and structural ECM integrity, evidenced by decreasing collagen abundance and increasing fibril fragmentation. Intrinsic aging also produces changes distinct from those produced by photoaging, including reductions in elastic fiber and crosslinking enzyme abundance. In contrast, photoaging is primarily defined by increases in elastic fiber-associated protein and pro-inflammatory proteases. Changes associated with photoaging are evident even in young (mid 20s) sun-exposed forearm skin, indicating that proteomic evidence of photoaging is present decades prior to clinical signs of photoaging. GO term enrichment revealed that both intrinsic aging and photoaging share common features of chronic inflammation. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD015982.

Amino-Functionalized Nitrogen-Doped Graphene-Quantum-Dot-Based Nanomaterials with Nitrogen and Amino-Functionalized Group Content Dependence for Highly Efficient Two-Photon Bioimaging.

We fabricated nanomaterials comprising amino-functionalized and nitrogen-doped graphene quantum dots (amino-N-GQDs) and investigated their photostability and intrinsic luminescence in the near-infrared spectrum to determine their suitability as contrast agents in two-photon imaging (TPI). We observed that amino-N-GQDs with a higher amount of bonded nitrogen and amino-functionalized groups (6.2%) exhibited superior two-photon properties to those with a lower amount of such nitrogen and groups (4.9%). These materials were conjugated with polymers containing sulfur (polystyrene sulfonate, PSS) and nitrogen atoms (polyethylenimine, PEI), forming amino-N-GQD–PSS–PEI specimens (amino-N-GQD-polymers). The polymers exhibited a high quantum yield, remarkable stability, and notable two-photon properties and generated no reactive oxygen species, rendering them excellent two-photon contrast agents for bioimaging. An antiepidermal growth factor receptor (AbEGFR) was used for labeling to increase specificity. Two-photon imaging (TPI) of amino-N-GQD (6.2%)-polymer-AbEGFR-treated A431 cancer cells revealed remarkable brightness, intensity, and signal-to-noise ratios for each observation at a two-photon excitation power of 16.9 nJ pixel−1 under 30 scans and a three-dimensional (3D) depth of 105 µm, indicating that amino-N-GQD (6.2%)-polymer-AbEGFR-treated cells can achieve two-photon luminescence with 71 times less power required for two-photon autofluorescence (1322.8 nJ pixel−1 with 500 scans) of similar intensity. This economy can minimize photodamage to cells, rendering amino-N-GQD-polymers suitable for noninvasive 3D bioimaging.

Multimodal spectral focusing CARS and SFG microscopy with a tailored coherent continuum from a microstructured fiber

We report a technologically novel microscopy system for bioimaging based on a 100 fs titanium:sapphire (Ti:Sa) laser pumped coherent continuum from a tailored, 9-cm long, all normal dispersion (ANDi) fiber, enabling concurrent image contrast with (a) spectral focusing coherent anti-Stokes Raman scattering (SF-CARS) (spanning 900–3200 cm−1) and (b) sum frequency generation (SFG). Both modalities were efficiently excited with power levels at the microscope focus compatible with biological samples. Moreover, using the continuum, images were recorded in the back-scattering (epi-detection) geometry, without the necessity for an expensive, computer-controlled, spatial light modulator (SLM), clearly demonstrating the strong signal levels achieved. Image contrast from the multiple modalities provided greater chemical and structural insights than imaging with any single technique in isolation. Numerical simulations supported these developments in regard to both the optimum fiber length for SC generation and the achievement of high spectral resolution in SF-CARS via careful group delay dispersion matching across the pump and Stokes pulses using just an inexpensive sequence of short glass blocks inserted into the Stokes beam. We show bio-images of mouse tissue recorded concurrently via label/stain-free contrast from multiple modalities: CARS, two-photon auto-fluorescence (TPaF) and second harmonic/sum frequency generation (SHG/SFG). Overall, our approach delivers optimum performance in back-scattered (epi-) detection configuration, suited for thick samples, at reduced complexity and cost. The addition of this simple fiber add-on to lasers already widely used for TPF microscopy can thus extend the capabilities of a significant number of existing microscopy laboratories.

Two-Photon Autofluorescence Imaging of Fixed Tissues: Feasibility and Potential Values for Biomedical Applications.

The value of optical redox imaging (ORI) of cells/tissues based on the intrinsic fluorescences of NADH (nicotinamide adenine dinucleotide) and oxidized flavoproteins (containing flavin adenine dinucleotide, i.e., FAD) has been demonstrated for potential biomedical applications including diagnosis, prognosis, and determining treatment response. However, the Chance redox scanner (a 3D cryogenic tissue imager) is limited by spatial resolution (~50μm), and tissue ORI using fluorescence microscopy (single or multi-photon) is limited by the light penetration depth. Furthermore, viable or snap-frozen tissues are usually required. In this project, we aimed to study whether ORI may be achieved for unstained fixed tissue using a state-of-the-art modern Serial Two-Photon (STP) Tomography scanner that can rapidly acquire multi-plane images at micron resolution. Tissue specimens of mouse muscle, liver, and tumor xenografts were harvested and fixed in 4% paraformaldehyde (PFA) for 24h. Tissue blocks were scanned by STP Tomography under room temperature to acquire the autofluorescence signals (NADH channel: excitation 750nm, blue emission filter; FAD channel: excitation 860nm, green emission filter). We observed remarkable signals with significant intra-tissue heterogeneity in images of NADH, FAD and redox ratio (FAD/(NADH+FAD)), which are worthy of further investigation for extracting biological information.

In vivo quantitative molecular absorption of glycerol in human skin using coherent anti-Stokes Raman scattering (CARS) and two-photon auto-fluorescence

The penetration of small molecules through the human skin is a major issue for both safety and efficacy issues in cosmetics and pharmaceutic domains. To date, the quantification of active molecular compounds in human skin following a topical application uses ex vivo skin samples mounted on Franz cell diffusion set-up together with appropriate analytical methods. Coherent anti-Stokes Raman scattering (CARS) has also been used to perform active molecule quantification on ex vivo skin samples, but no quantification has been described in human skin in vivo. Here we introduce and validate a framework for imaging and quantifying the active molecule penetration into human skin in vivo. Our approach combines nonlinear imaging microscopy modalities, such as two-photon excited auto-fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), together with the use of deuterated active molecules. The imaging framework was exemplified on topically applied glycerol diluted in various vehicles such as water and xanthan gel. In vivo glycerol quantitative percutaneous penetration over time was demonstrated, showing that, contrary to water, the xanthan gel vehicle acts as a film reservoir that releases glycerol continuously over time. More generally, the proposed imaging framework provides an enabling platform for establishing functional activity of topically applied products in vivo.

Detection of Malignancy in Ocular Surface Lesions by Inverse Spectroscopic Optical Coherence Tomography and Two-Photon Autofluorescence.

PurposeAdvanced imaging is increasingly important in the diagnosis of ocular surface malignancy. Inverse spectroscopic optical coherence tomography (ISOCT) and two-photon autofluorescence microscopy (2P-AF) are emerging techniques capable of quantifying ultrastructural and metabolic changes, respectively. We aimed to detect malignancy in ocular surface lesions using ISOCT and 2P-AF.MethodsPortions of excised specimens from patients undergoing conjunctival biopsy at Boston Medical Center were imaged by ISOCT and/or 2P-AF, and submitted for histologic diagnosis. Lesions were categorized as malignant, premalignant (with dysplasia) or benign. ISOCT and 2P-AF findings were compared between categories.ResultsFourteen specimens from 13 patients were collected. The IS-OCT marker D was 2.2-fold higher in combined malignant and premalignant (4.27 ± 0.28, n = 3) versus benign (1.92 ± 0.26, n = 11) lesions (P = 9 × 10−4). ISOCT markers μs and μb were not significantly different. By 2P-AF, the redox ratio was 0.24-fold lower in premalignant (0.11 ± 0.004, n = 2) versus benign (0.45 ± 0.04, n = 9) lesions (P = 1.08 × 10−5).ConclusionsConjunctival lesions with higher malignant potential had higher D and lower redox ratios. Higher D can correlate with ultrastructural changes associated with malignancy, similar to what has been seen in cancers of the gut mucosa. Lower redox ratios can suggest the presence of the Warburg effect, which is associated with tumorigenesis.Translational RelevanceIS-OCT and 2P-AF can potentially be applied to the detection of malignancy or malignant potential in ocular surface lesions. ISOCT allows for the detection of nanoscale ultrastructural changes that are not resolvable by conventional OCT.

Tissue effects of intra-tissue refractive index shaping (IRIS): insights from two-photon autofluorescence and second harmonic generation microscopy.

Intra-tissue refractive index shaping (IRIS) is a novel, non-ablative form of vision correction by which femtosecond laser pulses are tightly focused into ocular tissues to induce localized refractive index (RI) change via nonlinear absorption. Here, we examined the effects of Blue-IRIS on corneal microstructure to gain insights into underlying mechanisms. Three-layer grating patterns were inscribed with IRIS ~180 µm below the epithelial surface of ex vivo rabbit globes using a 400 nm femtosecond laser. Keeping laser power constant at 82 mW in the focal volume, multiple patterns were written at different scan speeds. The largest RI change induced in this study was + 0.011 at 20 mm/s. After measuring the phase change profile of each inscribed pattern, two-photon excited autofluorescence (TPEF) and second harmonic generation (SHG) microscopy were used to quantify changes in stromal structure. While TPEF increased significantly with induced RI change, there was a noticeable suppression of SHG signal in IRIS treated regions. We posit that enhancement of TPEF was due to the formation of new fluorophores, while decreases in SHG were most likely due to degradation of collagen triple helices. All in all, the changes observed suggest that IRIS works by inducing a localized, photochemical change in collagen structure.

Femtosecond fiber laser at 780 nm for two-photon autofluorescence imaging

We present an Er-doped fiber (Er:fiber)-based femtosecond laser at 780 nm with 256 MHz repetition rate, 191 fs pulse duration, and over 1 W average power. Apart from the careful third-order dispersion management, we introduce moderate self-phase modulation to broaden the output spectrum of the Er:fiber amplifier and achieve 193 fs pulse duration and 2.43 W average power. Over 40% frequency doubling efficiency is obtained by a periodically poled lithium niobate crystal. Delivering through a hollow-core photonic bandgap fiber, this robust laser becomes an ideal and convenient light source for two-photon autofluorescence imaging.

Comparison of biomechanical properties in ascending aortic aneurysms of patients with congenital bicuspid aortic valve and Marfan syndrome

BackgroundIn patients with ascending aortic aneurysms (AscAA), biomechanical differences are seen among patients with congenital bicuspid aortic valves (BAV), Marfan syndrome (MFS), and tricuspid aortic valves (TAV). We examined the hemodynamic profiles and ultrastructures of aneurysmal specimens, focusing on vascular remodelling to better understand AscAA pathogenesis. MethodsA total of 795 patients with BAV (43.97 ± 0.51 years; 93.2% male), 69 with MFS (34.43 ± 1.44 years; 86.2% male), and 90 with TAV (67.27 ± 0.58 years; 60% male) were enrolled, primarily upon admission with AscAA. The biomechanical properties of the aortic root were assessed and intraoperative specimens were analyzed by light-microscopy and two-photon autofluorescence microscopy. ResultsPatients with BAV had significantly greater distension of the aortic root, irrespective of age or aneurysmal widening (R2 = 0.543, p < 0.05). This was associated with significantly increase in the size of the tunica media. Patients with MFS displayed significant stiffness in the sinuses that worsened with age (R2 = 0.752, p < 0.001), similar to patients with TAV (R2 = 0.626, p < 0.05). Patients with MFS showed significant root elasticity with aneurysmal growth (R2 = 0.596, p < 0.05) and increased medial degeneration. Patients with TAV maintained biomechanical properties, apart from aneurysmal dimensions and high levels of inflammation. ConclusionsAmong patients with AscAA, those with BAV maintain tissue elasticity in the aortic root, regardless of age. Patients with MFS demonstrate increased sinus stiffness with medial degeneration, both during aging and with aneurysmal growth. Patients with TAV and AscAA present with increased inflammation.

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging.

The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation. Human corneas were imaged after different storage times: short-term (STS), medium-term (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition. Optical discrimination between different corneal layers and sublayers based on their morphologic characteristics revealed by two-photon autofluorescence (AF) is possible. Furthermore, all layers were characterized based on AF lifetimes to gain information on metabolic activities of cells. The NAD(P)H free to protein-bound ratio (a1/a2) of epithelial cells increased significantly in both MTS and LTS corneas compared with STS corneas. In endothelial cells, NAD(P)H a1/a2 was significantly increased in MTS samples. For keratocytes, the NAD(P)H a1/a2 decreased significantly with storage time. This could indicate that the metabolic activity of the epithelial and endothelial cells reduces, whereas the activity of keratocytes increases with storage time. The analysis of the stroma SHG images indicated that the organization of collagen fibers decreases with storage time. The feasibility of measuring the endothelial cell density (ECD) using TPI was demonstrated. An ECD of 1461 ± 190 cells/mm2 was obtained for MTS samples based on TPI. TPI can provide information not accessible by current clinical methods, such as the cells' metabolic state and structural organization of the stroma, with subcellular resolution. Thus, it may improve the screening process of corneas prior to transplantation and might help to optimize the storage conditions.