- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Slide-free imaging of hematoxylin-eosin stained whole-mount tissues using combined third-harmonic generation and three-photon fluorescence microscopy
摘要： Intraoperative margin assessment of surgical tissues during cancer surgery is clinically important, especially in the case of tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is considered crucial. Frozen pathology is the gold standard of assessing excised tissues for signs of remaining cancerous lesions. The current protocol, however, is time-consuming and labor-intensive. Instead of the complex frozen sectioning, staining, and traditional white light microscopy imaging protocol, optically-sectioned histopathological imaging of hematoxylin-eosin stained whole-mount skin tissues with a sub-femtoliter resolution is demonstrated by using nonlinear microscopy in this study. With our proposed method, the reagents of staining and the contrast of imaging are fully consistent with the current clinical standard of frozen pathology, thus facilitating rapid intraoperative assessment of surgical tissues for future applications.
关键词： hematoxylin-eosin,three photon microscopy,third harmonic generation microscopy,surgical border,margin assessment
更新于2025-09-23 15:23:52
-
Editorial: Advances in Label Free Tissue Imaging With Laser Scanning Microscopy Techniques
摘要： Significant efforts are being spent at the time being for transferring various laser scanning microscopy (LSM) techniques to the realm of tissue characterization, because of their potential to circumvent some of the most important disadvantages of traditional histopathology approaches based on excisional biopsy and tissue staining. Although conventional histopathology is currently regarded as a golden standard for the diagnosing pathologies that reflect in tissular modification (e.g., cancers), limitations such as long diagnosis time, invasiveness, artifacts, sampling error, time consumption, high costs, and interpretive variability make such approaches to be impractical in many scenarios, while also placing considerable pressure on the sustainability of healthcare systems around the world. The potential of LSM techniques to contribute to overcoming these aspects derives from their “non-invasive” character. They can exploit various endogenous optical signals generated by tissues upon interaction with a laser beam and are able to provide optical sections (virtual biopsies) that reflect the tissular architecture at controlled depths. Many studies reported to date showed that LSM techniques can provide label-free information of similar pathologic relevance to the information collected for characterization/confirmation purposes with traditional histopathology approaches. These techniques are thus capable of probing optical properties of tissues with deep implications for resolving important anatomical and physiological aspects which represent hallmarks for disease predisposition and progression. To date techniques such as Confocal Laser Scanning Microscopy (CLSM) [1], Fluorescence Lifetime Imaging (FLIM) [2], Two-Photon Excited Fluorescence Microscopy (TPEF) [2–6], Second Harmonic Generation Microscopy (SHG) [5, 6], Third Harmonic Generation Microscopy (THG) [4], Coherent Anti-Stokes Raman Scattering Microscopy (CARS) [3, 7], as well as other LSM variants such as the Brillouin Microscopy [8] have already been demonstrated to be powerful tools for investigating tissue morphology, functionality, and biochemical composition with high spatial and temporal resolution. In the opinion of many, these techniques, together with investigations approaches based on their combined use, will soon become the central element of the default tissue characterization frameworks for both ex vivo and in vivo assays. Furthermore, emerging LSM techniques exploiting various ingenious strategies to achieve superresolved images in a label-free manner [9–12] are also likely to be transferred soon toward applications addressing tissue imaging.
关键词： tissue imaging,two photon excitation fluorescence microscopy,Brillouin microspectroscopy,laser scanning microscopy,second harmonic generation microscopy
更新于2025-09-23 15:19:57
-
[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
-
Optical coupling between resonant dielectric nanoparticles and dielectric nanowires probed by third harmonic generation microscopy
摘要： Localized electromagnetic modes and negligible Ohmic losses dictate the growing interest to subwavelength all-dielectric nanoparticles. Although an exhaustive volume of literature dealt with interaction of all-dielectric nanostructures with free-space electromagnetic fields, they received little attention as integrated photonic elements. We present an experimental and numerical study of optical coupling between a resonant subwavelength silicon nanodisk and a silicon nanowire, as probed by third harmonic generation microscopy and full-wave simulations. First, by placing the nanodisks at different distances from the nanowire, we observed third harmonic intensity modulation by a factor of up to 4.5. This modulation is assigned to changes in the local field enhancement within the nanodisks caused by their coupling to the nanowires and subsequent shifting and broadening of their magnetic-type resonances. Interestingly, although the nanowire presents an additional loss channel for the nanodisk, we observed an increase in the local field strength within the nanodisk, as verified by rigorous full-wave simulations. Inversely, for the gap sizes that are smaller than ≈ 200 nm, we observe the influence of the nanoparticles on the propagation properties of the fundamental waveguide modes of the nanowire. The better understanding of the mutual influence of the Mie-resonant nanoparticles and waveguiding structures heralds integration of the former on photonic chips.
关键词： dielectric nanowire,third harmonic generation microscopy,silicon nanoparticles,Optical coupling
更新于2025-09-04 15:30:14