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Multiphoton imaging for morphometry of the sandwich-beam structure of the human stapedial annular ligament
摘要： Background: The annular ligament of the human stapes constitutes a compliant connection between the stapes footplate and the peripheral cochlear wall at the oval window. The cross section of the human annular ligament is characterized by a three-layered structure, which resembles a sandwich-shaped composite structure. As accurate and precise descriptions of the middle-ear behavior are constrained by lack of information on the complex geometry of the annular ligament, this study aims to obtain comprehensive geometrical data of the annular ligament via multiphoton imaging. Methods: The region of interest containing the stapes and annular ligament was harvested from a fresh-frozen human temporal bone of a 46-years old female. Multiphoton imaging of the unstained sample was performed by detecting the second-harmonic generation of collagen and the autofluorescence of elastin, which are constituents of the annular ligament. The multiphoton scans were conducted on the middle-ear side and cochlear side of the annular ligament to obtain accurate images of the face layers on both sides. The face layers of the annular ligament were manually segmented on both multiphoton scans, and then registered to high-resolution mCT images. Results: Multiphoton scans of the annular ligament revealed 1) relatively large thickness of the core layer compared to the face layers, 2) asymmetric geometry of the face layers between the middle-ear side and cochlear side, and variation of their thickness and width along the footplate boundary, 3) divergent relative alignment of the two face layers, and 4) different fiber composition of the face layers along the boundary with a collagen-reinforcement near the anterior pole on the middle-ear side. Conclusion and outlook: Multiphoton microscopy is a feasible approach to obtain the detailed three-dimensional features of the human stapedial annular ligament along its full boundary. The detailed description of the sandwich-shaped structures of the annular ligament is expected to contribute to modeling of the human middle ear for precise simulation of middle-ear behavior. Further, established methodology in this study may be applicable to imaging of other middle-ear structures.
关键词： Stapes,Two-photon microscopy,Multiphoton microscopy,Core layer,Face layer,Annular ligament
更新于2025-09-23 15:23:52
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In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing
摘要： Chronic wounds are difficult to diagnose and characterize due to a lack of quantitative biomarkers. Label-free multiphoton microscopy has emerged as a useful imaging modality capable of quantifying changes in cellular metabolism using an optical redox ratio of FAD/(NADH+FAD) autofluorescence. However, the utility of an optical redox ratio for long-term in vivo monitoring of tissue metabolism has not been robustly evaluated. In this study, we demonstrate how multiphoton microscopy can be used to monitor changes in the metabolism of individual full-thickness skin wounds in vivo. 3D optical redox ratio maps and NADH fluorescence lifetime images identify differences between diabetic and control mice during the re-epithelialization of wounds. These metabolic changes are associated with a transient increase in keratinocyte proliferation at the wound edge. Our study demonstrates that high-resolution, non-invasive autofluorescence imaging can be performed in vivo and that optical redox ratios can serve as quantitative optical biomarkers of impaired wound healing.
关键词： metabolism,optical redox ratio,autofluorescence,multiphoton microscopy,in vivo imaging,diabetes,FAD,NADH,wound healing
更新于2025-09-23 15:23:52
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Monitoring dynamic collagen reorganization during skin stretching with fast polarization-resolved SHG imaging
摘要： The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization-resolved SHG microscope is implemented to map collagen orientation during mechanical assays. This system is based on line-to-line switching of polarization using an electro-optical modulator and works in epidetection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.
关键词： biomechanics,multiphoton microscopy,skin,polarization,collagen
更新于2025-09-23 15:23:52
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[IEEE 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Munich, Germany (2019.6.23-2019.6.27)] 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Label-Free Multiphoton Microscopy in Human Tissue Enabled by an Er:Fiber-Laser Based Tunable Source
摘要： Multiphoton microscopy (MPM) is an important bio-imaging tool. Different modalities can serve as a contrast agent, such as second-/third-harmonic generation (SHG/THG) and two-/three-photon excitation fluorescence (2PEF/3PEF). Ultrafast lasers with flexible wavelength tunability are crucial for driving MPM bio-imaging, and the conventional solution relies on ultrafast Ti:sapphire lasers plus an optical parametric oscillator/amplifier. Recently, we have demonstrated that ultrafast fiber lasers are a potential solution to implementing compact, robust, and wavelength tunable femtosecond sources for driving MPM. To realize wavelength tunability we employ self-phase modulation (SPM) in optical fibers to broaden a narrowband input spectrum of Yb-/Er-doped fiber lasers (YDFLs/EDFLs) up to >400-nm wide with well-isolated spectral lobes; filtering the leftmost/rightmost lobes leads to nearly transform-limited pulses [1–6]. Such a SPM-enabled spectral selection (SESS) allows us to obtain wavelength widely tunable femtosecond pulses for MPM [2,5,6]. In this submission, we representatively demonstrate label-free harmonic generation microscopy (HGM) in human skin and brain tissues. Figure 1(a) depicts a scanning microscope driven by an EDFL-based SESS source. The EDFL operates at 31-MHz repetition rate and generates 290-fs pulses centered at 1550 nm. The narrowband EDFL [blue curve in Fig. 1(a)] is coupled into 9-cm optical fiber (10-μm mode-field diameter and -10 fs2/mm group-velocity dispersion at 1550 nm). The output spectrum is shown as the red curve in Fig. 1(b) under 85-nJ coupled pulse energy. We use optical filters to select the leftmost spectral lobe peaking at 1250 nm, which leads to 11.7-nJ, 47-fs pulses. Then we employ these pulses to drive a scanning microscope and conduct HGM in human skin and brain tissues. Figure 1(c) shows the dermal papilla at the junction of epidermis and upper dermis in human skin. Basal cells are visualized by THG (cyan hot) due to optical inhomogeneity at the interface (e.g., cell membrane); SHG (red hot) originates from the non-centrosymmetric structure of collagen fibers. In Fig. 1(d), neural network and brain vasculature in human brain tissue can be visualized by THG and SHG, respectively [Fig. 1(d)]. THG contrast inside the vasculature shows also red blood cells. In conclusion, we report on MPM deep-tissue imaging enabled by an EDFL-based SESS source. It is noteworthy that besides HGM excited by 1250-nm femtosecond pulses demonstrated here, the SESS source also supports 1300-/1700-nm illumination for 3PEF of green/red fluorescent protein (GFP/RFP) [7,8]. Such a fiber-based solution can be applied to many important applications, such as histopathology, morphology, and neuroscience.
关键词： ultrafast fiber lasers,Multiphoton microscopy,self-phase modulation,bio-imaging,harmonic generation microscopy
更新于2025-09-12 10:27:22
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Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy
摘要： A protocol is presented to build a custom low-cost yet high-performance femtosecond (fs) fiber laser. This all-normal-dispersion (ANDi) ytterbium-doped fiber laser is built completely using commercially available parts, including $8,000 in fiber optic and pump laser components, plus $4,800 in standard optical components and extra-cavity accessories. Researchers new to fiber optic device fabrication may also consider investing in basic fiber splicing and laser pulse characterization equipment (~$63,000). Important for optimal laser operation, methods to verify true versus apparent (partial or noise-like) mode-locked performance are presented. This system achieves 70 fs pulse duration with a center wavelength of approximately 1,070 nm and a pulse repetition rate of 31 MHz. This fiber laser exhibits the peak performance that may be obtained for an easily assembled fiber laser system, which makes this design ideal for research laboratories aiming to develop compact and portable fs laser technologies that enable new implementations of clinical multiphoton microscopy and fs surgery.
关键词： fiber laser,multiphoton microscopy,low-cost,mode-locking,Issue 153,femtosecond pulsed laser,Engineering,custom fabrication
更新于2025-09-12 10:27:22
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Fast repetition rate fs pulsed lasers for advanced PLIM microscopy
摘要： Simultaneous metabolic and oxygen imaging is promising to follow up therapy response, disease development and to determine prognostic factors. FLIM of metabolic coenzymes is now widely accepted to be the most reliable method to determine cellular bioenergetics. Also, oxygen consumption has to be taken into account to understand treatment responses. The phosphorescence lifetime of oxygen sensors is able to indicate local oxygen changes. For phosphorescence lifetime imaging (PLIM) dyes based on ruthenium (II) coordination complexes are useful, in detail TLD1433 which possesses a variety of different triplet states, enables complex photochemistry and redox reactions. PLIM is usually reached by two photon excitation of the drug with a femtosecond (fs) pulsed Ti:Sapphire laser working at 80 MHz repetition rate and (time-correlated single photon counting) (TCSPC) detection electronics. The interesting question was whether it is possible to follow up PLIM using faster repetition rates. Faster repetition rates could be advantageous for the induction of specific photochemical reactions because of similar light doses used normally in standard CW light treatments. For this, a default 2p-FLIM–PLIM system was expanded by adding a second fs pulsed laser ("helixx") which provides 50 fs pulses at a repetition rate of 250 MHz, more than 2.3 W average power and tunable from 720 nm to 920 nm. The laser beam was coupled into the AOM instead of the default 80 MHz laser. We demonstrated successful applications of the 250 MHz laser for PLIM which correlates well with measurements done by excitation with the conventional 80 MHz laser source.
关键词： oxygen consumption,Ru(II) complexes,phosphorescence lifetime microscopy,Multiphoton microscopy,fast repetition rate excitation,short laser pulses
更新于2025-09-11 14:15:04
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Optoacoustic microscopy at multiple discrete frequencies
摘要： Optoacoustic (photoacoustic) sensing employs illumination of transient energy and is typically implemented in the time domain using nanosecond photon pulses. However, the generation of high-energy short photon pulses requires complex laser technology that imposes a low pulse repetition frequency (PRF) and limits the number of wavelengths that are concurrently available for spectral imaging. To avoid the limitations of working in the time domain, we have developed frequency-domain optoacoustic microscopy (FDOM), in which light intensity is modulated at multiple discrete frequencies. We integrated FDOM into a hybrid system with multiphoton microscopy, and we examine the relationship between image formation and modulation frequency, showcase high-fidelity images with increasing numbers of modulation frequencies from phantoms and in vivo, and identify a redundancy in optoacoustic measurements performed at multiple frequencies. We demonstrate that due to high repetition rates, FDOM achieves signal-to-noise ratios similar to those obtained by time-domain methods, using commonly available laser diodes. Moreover, we experimentally confirm various advantages of the frequency-domain implementation at discrete modulation frequencies, including concurrent illumination at two wavelengths that are carried out at different modulation frequencies as well as flow measurements in microfluidic chips and in vivo based on the optoacoustic Doppler effect. Furthermore, we discuss how FDOM redefines possibilities for optoacoustic imaging by capitalizing on the advantages of working in the frequency domain.
关键词： Doppler effect,Frequency-domain,Multiphoton microscopy,Optoacoustic microscopy,Microcirculatory blood flow
更新于2025-09-04 15:30:14
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Label-free detection of brain invasion in meningiomas by multiphoton microscopy
摘要： Brain invasion in meningiomas relates to the poor prognosis. Therefore, accurate detection and treatment of brain invasion are of particular importance. In this study, multiphoton microscopy (MPM) was introduced for label-free detection of brain invasion in meningiomas by the combination of the two-photon excited fluorescence imaging, second-harmonic generation imaging and spectral imaging. Results show that MPM has the ability to morphologically and spectrally identify brain invasion in meningiomas. What is more, the intensity ratios of the cerebral cortex over meningiomas at 475 nm, 511 nm and 540 nm were extracted as quantitative indicators for differentiating meningioma from surrounding normal cerebral cortex tissue. Together with custom-developed image processing algorithms, MPM could further identify the tumor boundary. With miniaturization and optimization of probes and optical fiber for in vivo imaging, combined with a laser ablation system, MPM would be used in an operating room for both the diagnosis and treatment of brain invasion in meningiomas.
关键词： brain invasion,two-photon excited fluorescence,multiphoton microscopy,second harmonic generation
更新于2025-09-04 15:30:14